CN101341118A - Compositions containing, methods involving, and uses of non-natural amino acids and polypeptides - Google Patents

Compositions containing, methods involving, and uses of non-natural amino acids and polypeptides Download PDF

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CN101341118A
CN101341118A CNA2005800443282A CN200580044328A CN101341118A CN 101341118 A CN101341118 A CN 101341118A CN A2005800443282 A CNA2005800443282 A CN A2005800443282A CN 200580044328 A CN200580044328 A CN 200580044328A CN 101341118 A CN101341118 A CN 101341118A
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substituted
group
alkylidene group
amino acid
alkyl
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苗振伟
刘俊杰
赛·诺曼
罗素·德瑞弗
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Ambrx Inc
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Ambrx Inc
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Priority to CN201810031829.6A priority Critical patent/CN108047086B/en
Priority to CN201510093838.4A priority patent/CN104803865A/en
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    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

Disclosed herein are non-natural amino acids and polypeptides that include at least one non-natural amino acid and methods for making such non-natural amino acids and polypeptides. The non-natural amino acids, by themselves or as a part of a polypeptide, can include a wide range of possible functionalities, but typical have at least one oxime, carbonyl, dicarbonyl, and/or hydroxylamine group. Also disclosed herein are non-natural amino acid polypeptides that are further modified post-translationally, methods for effecting such modifications, and methods for purifying such polypeptides. Typically, the modified non-natural amino acid polypeptides include at least one oxime, carbonyl, dicarbonyl, and/or hydroxylamine group. Further disclosed are methods for using such non-natural amino acid polypeptides and modified non-natural amino acid polypeptides, including therapeutic, diagnostic, and other biotechnology uses.

Description

The purposes that contains the composition of alpha-non-natural amino acid and polypeptide, the method that relates to alpha-non-natural amino acid and polypeptide and alpha-non-natural amino acid and polypeptide
The cross reference of related application
The application's case is advocated the U.S. Provisional Application case the 60/638th of application on December 22nd, 2004, No. 418, the U.S. Provisional Application case the 60/638th of application on December 22nd, 2004, No. 527, the U.S. Provisional Application case the 60/639th of application on December 22nd, 2004, No. 195, the U.S. Provisional Application case the 60/696th of application on July 1st, 2005, No. 210, the U.S. Provisional Application case the 60/696th of application on July 1st, 2005, the U.S. Provisional Application case the 60/696th of No. 302 and on July 1st, 2005 application, No. 068 interests, the disclosure of wherein said application case is all to be incorporated herein by reference.
Technical field
Do not have
Background technology
The ability that nongenetic coded amino acid (that is, " alpha-non-natural amino acid ") is incorporated in the protein makes permission introduce chemical functional group, and it can provide the natural functional group of existence (such as the ε-NH of Methionin 2, sulfydryl-SH of halfcystine, the imino-of Histidine etc.) valuable surrogate.Known some chemical functional group is inert to the functional group that exists in 20 kinds of common heredity coded amino acids, but with can be incorporated into the functional group's cleaning on the alpha-non-natural amino acid and react effectively to form stable binding.
Nowadays methods availalbe is optionally introduced non-existent chemical functional group in the protein, its to existing all functional groups in 20 kinds of common heredity coded amino acids for chemically inert and its can be used for and comprise that some functional group's reagent effectively and optionally reacts to form stable covalent linkage.
Summary of the invention
Describe herein be used to make, purifying, sign and use alpha-non-natural amino acid, non-natural amino acid polypeptides and method, composition, technology and the strategy of modified non-natural amino acid polypeptides.One side is method, composition, technology and the strategy of derive alpha-non-natural amino acid and/or non-natural amino acid polypeptides.In one embodiment, these methods, composition, technology and strategy relate to chemically derived, relate to biologically-derivedly in other embodiments, relate to physics in other embodiments and derive, and relate to the deutero-combination in other embodiments.In other or extra embodiment, these are derived and have regioselectivity.In other or extra embodiment, these are derived and have regiospecificity.In other or extra embodiment, these are derived at ambient temperature for rapidly.In other or extra embodiment, these are derived and take place in the aqueous solution.In other or extra embodiment, these are derived in generation under the pH value between about 4 and about 10.In other or extra embodiment, by adding promotor, in reagent that contains alpha-non-natural amino acid and derivative reagent, these are derived and are stoichiometry, stoichiometric near stoichiometry or class.In other or extra embodiment, provide by add promotor allow required group stoichiometry, near stoichiometry or class stoichiometry be incorporated into method on the non-natural amino acid polypeptides.In other or extra embodiment, provide by add promotor allow required group stoichiometry, near stoichiometry or class stoichiometry be incorporated into strategy, reaction mixture, synthesis condition on the non-natural amino acid polypeptides.
On the one hand for being used for based on chemically derived peptide of oxime key and proteinic alpha-non-natural amino acid.In other or extra embodiment, alpha-non-natural amino acid is incorporated in the polypeptide, that is, these embodiment are non-natural amino acid polypeptides.In other or extra embodiment, alpha-non-natural amino acid on its side chain through functionalized so that itself and derived molecules reaction generation oxime key.In other or extra embodiment for producing the non-natural amino acid polypeptides that contain the oxime non-natural amino acid polypeptides with the derived molecules reaction.In other or extra embodiment, alpha-non-natural amino acid be selected from have carbonyl, the amino acid of dicarbapentaborane, acetal, azanol or oxime side chain.In other or extra embodiment, alpha-non-natural amino acid is to be selected from the amino acid that has through protection or carbonyl, dicarbapentaborane, azanol or oxime side chain through sheltering.In other or extra embodiment, alpha-non-natural amino acid comprises the side chain of sheltering through oxime.In other or extra embodiment, alpha-non-natural amino acid comprises carbonyl or dicarbapentaborane side chain, and wherein carbonyl or dicarbapentaborane are to be selected from ketone or aldehyde.In another embodiment for containing the alpha-non-natural amino acid that can form the functional group of oxime after useful suitable functionalized co-reactant is handled.In another or extra embodiment, alpha-non-natural amino acid structurally is similar to natural amino acid, but contains a kind of among the aforementioned functional group.In another or other embodiment, alpha-non-natural amino acid is similar to phenylalanine or tyrosine (aromatic amino acid); And in another embodiment, alpha-non-natural amino acid is similar to L-Ala and leucine (hydrophobic amino acid).In one embodiment, alpha-non-natural amino acid has the character with those different in kinds of natural amino acid.In one embodiment, these different propertiess are the chemical reactivity of side chain, the reactive side chain experience reaction that allows alpha-non-natural amino acid of this different chemical in another embodiment, and as the unit of polypeptide, even the natural unitary side chain of amino acid that exists in the same polypeptide does not experience previous reaction yet.In another embodiment, the side chain of alpha-non-natural amino acid has and the natural orthogonal chemistry of amino acid whose side chain that exists.In another embodiment, the side chain of alpha-non-natural amino acid comprises the part that contains electrophilic reagent; In another embodiment, the part that contains electrophilic reagent on the side chain of alpha-non-natural amino acid can experience nucleophilic attack to produce oxime deutero-protein.In in this paragraph in the previous embodiment any one, alpha-non-natural amino acid can be used as the independent molecule existence and maybe can incorporate in the polypeptide of any length; If be the latter, so described polypeptide can further merge natural existence or alpha-non-natural amino acid.
On the other hand for being used to prepare molecule through the azanol replacement based on the non-natural amino acid polypeptides of deriving of oxime key.In another embodiment for be used for by derived molecules with contain carbonyl or the non-natural amino acid polypeptides of dicarbapentaborane between form the derive molecule through the azanol replacement of the non-natural amino acid polypeptides that contains carbonyl or dicarbapentaborane of oxime key.In other embodiments, the aforementioned non-natural amino acid polypeptides that contains carbonyl or dicarbapentaborane is the non-natural amino acid polypeptides that contains ketone group.In other or extra embodiment, the alpha-non-natural amino acid that contains carbonyl or dicarbapentaborane comprises the side chain that is selected from ketone or aldehyde.In other or extra embodiment, the molecule that replaces through azanol comprises the group who is selected from following each thing: mark; Dyestuff; Polymkeric substance; Water-soluble polymers; The derivative of polyoxyethylene glycol; Photocrosslinking agent; Cytotoxic compound; Medicine; Affinity marker; The photoaffinity mark; Reactive compounds; Resin; Second protein or polypeptide or polypeptide analog; Antibody or antibody fragment; Metal chelator; Cofactor; Lipid acid; Carbohydrate; Polynucleotide; DNA; RNA; Antisense polynucleotide; Carbohydrate, water-soluble branch-shape polymer, cyclodextrin, biomaterial; Nanoparticle; Spin labeling; Fluorophore, metallic part; Radioactive segment; Novel functional group; With the covalently or non-covalently interactional group of other molecules; The light cage covers part; But but the part that actinic radiation excites, ligand photoisomerization part; Vitamin H; The vitamin H analogue; Be combined with the part of heavy atom; But the group of chemical cracking; But the group of photodestruciton; The side chain that prolongs; The sugar of carbon bond connection; Redox active agent; Amino thioic acid sulfoacid; The toxicity part; Through isotope-labeled part; The biophysics probe; The phosphorescence group; Chemiluminescent groups; The electron dense group; The magnetic group; Insert group; Chromophoric group; Energy transfer agent; Biologically active agent; Detectable label; Small molecules; Inhibition Yeast Nucleic Acid, radioactive nuleus thuja acid; Neutron capture agent; The derivative of vitamin H; Quantum dot; Nanometer is transmitted plain; Radioactivity is transmitted plain; Abzyme, activation composite activating agent, virus, adjuvant, aglycone, anaphylactogen, angiostatin, hormone antagonist, antioxidant, fit, guide RNA, Saponin/TSM, shuttle vectors, macromole, plan epitope, acceptor, reverse micelle with and any combination.In other or extra embodiment, the molecule that replaces through azanol be polyoxyethylene glycol (PEG) molecule through the azanol replacement.In another embodiment, the side chain of alpha-non-natural amino acid has and the natural orthogonal chemistry of amino acid whose those side chains that exists, and it allows alpha-non-natural amino acid and the molecular selectivity reaction that replaces through azanol.In another embodiment, the side chain of alpha-non-natural amino acid comprises the part that contains electrophilic reagent of reacting with the molecular selectivity that contains azanol; In another embodiment, the part that contains electrophilic reagent on the side chain of alpha-non-natural amino acid can experience nucleophilic attack to produce oxime deutero-protein.On the other hand the modified non-natural amino acid polypeptides for by derived molecules and non-natural amino acid polypeptides reaction produce relevant with the embodiment that in this paragraph, describes.Other embodiment comprise modifying any further modification of non-natural amino acid polypeptides.
On the other hand for being used to prepare molecule through carbonyl or dicarbapentaborane replacement based on the non-natural amino acid polypeptides of deriving of oxime key.In another embodiment for be used for by derived molecules with contain the peptide of oxime or the derive molecule through carbonyl or dicarbapentaborane replacement of the non-natural amino acid polypeptides that contains oxime of the oxime permutoid reaction between the protein.In another embodiment for can in the pH value scope between about 4 and about 8, experiencing oxime and the molecule that exchanges with the non-natural amino acid polypeptides that contains oxime through carbonyl or dicarbapentaborane replacement.In another embodiment for being used for by forming the molecule that the oxime key is derived and contained oxime or contain the non-natural amino acid polypeptides of azanol with containing oxime (therefore forming new oxime key) or contain between the non-natural amino acid polypeptides of azanol through carbonyl or dicarbapentaborane replacement by the switch type reaction at derived molecules.In another embodiment, the molecule through carbonyl or dicarbapentaborane replacement is the molecule that replaces through aldehyde.In other embodiments, the molecule through carbonyl or dicarbapentaborane replacement comprises the group who is selected from following each thing: mark; Dyestuff; Polymkeric substance; Water-soluble polymers; The derivative of polyoxyethylene glycol; Photocrosslinking agent; Cytotoxic compound; Medicine; Affinity marker; The photoaffinity mark; Reactive compounds; Resin; Second protein or polypeptide or polypeptide analog; Antibody or antibody fragment; Metal chelator; Cofactor; Lipid acid; Carbohydrate; Polynucleotide; DNA; RNA; Antisense polynucleotide; Carbohydrate, water-soluble branch-shape polymer, cyclodextrin, biomaterial; Nanoparticle; Spin labeling; Fluorophore, metallic part; Radioactive segment; Novel functional group; With the covalently or non-covalently interactional group of other molecules; The light cage covers part; But but the part that actinic radiation excites, ligand photoisomerization part; Vitamin H; The vitamin H analogue; Be combined with the part of heavy atom; But the group of chemical cracking; But the group of photodestruciton; The side chain that prolongs; The sugar of carbon bond connection; Redox active agent; Amino thioic acid sulfoacid; The toxicity part; Through isotope-labeled part; The biophysics probe; The phosphorescence group; Chemiluminescent groups; The electron dense group; The magnetic group; Insert group; Chromophoric group; Energy transfer agent; Biologically active agent; Detectable label; Small molecules; Inhibition Yeast Nucleic Acid, radioactive nuleus thuja acid; Neutron capture agent; The derivative of vitamin H; Quantum dot; Nanometer is transmitted plain; Radioactivity is transmitted plain; Abzyme, activation composite activating agent, virus, adjuvant, aglycone, anaphylactogen, angiostatin, hormone antagonist, antioxidant, fit, guide RNA, Saponin/TSM, shuttle vectors, macromole, plan epitope, acceptor, reverse micelle with and any combination.In other or extra embodiment, the molecule that replaces through aldehyde be polyoxyethylene glycol (PEG) molecule through the aldehyde replacement.In another embodiment, the side chain of alpha-non-natural amino acid has and the natural orthogonal chemistry of amino acid whose those side chains that exists, and it allows alpha-non-natural amino acid and the molecular selectivity reaction that replaces through carbonyl or dicarbapentaborane.In another embodiment, the side chain of alpha-non-natural amino acid comprises the part with the molecular selectivity reaction that contains carbonyl or dicarbapentaborane, for example oximido or azanol base; In another embodiment, the part of the nucleophilic on the side chain of alpha-non-natural amino acid can experience the electrophilic attack to produce oxime deutero-protein.On the other hand the modified non-natural amino acid polypeptides for by derived molecules and non-natural amino acid polypeptides reaction produce relevant with the embodiment that in this paragraph, describes.Other embodiment comprise modifying any further modification of non-natural amino acid polypeptides.
On the other hand for being used to produce simple function, difunctionality and multifunctional connexon based on the non-natural amino acid polypeptides of deriving of oxime key.In one embodiment for can be used for connecting the non-natural amino acid polypeptides that contains carbonyl or dicarbapentaborane and the molecule connexon (difunctionality and multifunctional) of other molecules.In another embodiment for can be used for connecting the non-natural amino acid polypeptides that contains oxime or azanol and the molecule connexon (difunctionality and multifunctional) of other molecules.In another embodiment, the non-natural amino acid polypeptides that contains carbonyl or dicarbapentaborane comprises ketone and/or aldehyde side chain.Contain among the embodiment of non-natural amino acid polypeptides of oxime or azanol in utilization, the molecule connexon contains carbonyl or dicarbapentaborane at the one end; In other embodiments, carbonyl or dicarbapentaborane are to be selected from aldehyde radical or ketone group.In other or extra embodiment, the connexon molecule that replaces through azanol be polyoxyethylene glycol (PEG) the connexon molecule through the azanol replacement.In other or extra embodiment, the connexon molecule that replaces through carbonyl or dicarbapentaborane be polyoxyethylene glycol (PEG) the connexon molecule through carbonyl or dicarbapentaborane replacement.In other embodiments, phrase " other molecules " comprises (only for instance) protein, other polymkeric substance and small molecules.In other or extra embodiment, the connexon molecule that contains azanol comprises identical or equivalent group on all ends, so that after reacting with the non-natural amino acid polypeptides that contains carbonyl or dicarbapentaborane, products therefrom is the same multimerization thing that contains the non-natural amino acid polypeptides of carbonyl or dicarbapentaborane.In other embodiments, turn to same dimerization with poly.In other or extra embodiment, the molecule connexon that contains carbonyl or dicarbapentaborane comprises identical or equivalent group on all ends, so that after reacting with the non-natural amino acid polypeptides that contains oxime or azanol, products therefrom is the same multimerization thing that contains the non-natural amino acid polypeptides of oxime or azanol.In other embodiments, turn to same dimerization with poly.In another embodiment, the side chain of alpha-non-natural amino acid has and the natural orthogonal chemistry of amino acid whose those side chains that exists, and it allows alpha-non-natural amino acid and the connexon molecular selectivity reaction that replaces through azanol.In another embodiment, the side chain of alpha-non-natural amino acid has and the natural orthogonal chemistry of amino acid whose those side chains that exists, and it allows alpha-non-natural amino acid and the connexon molecular selectivity reaction that replaces through carbonyl or dicarbapentaborane.In another embodiment, the side chain of alpha-non-natural amino acid comprises the part that contains electrophilic reagent of reacting with the connexon molecular selectivity that contains azanol; In another embodiment, the part that contains electrophilic reagent on the side chain of alpha-non-natural amino acid can experience the nucleophilic attack of the connexon molecule that contains azanol to produce oxime deutero-protein.Relevant with the embodiment that describes in this paragraph is " the modified or not modified " non-natural amino acid polypeptides that is reacted the binding that produces by connexon molecule and non-natural amino acid polypeptides on the other hand.Other embodiment comprise any further modification to " modified or not modified " non-natural amino acid polypeptides of binding.
Produce the method that the oximido product comes derived protein for condensation on the one hand by carbonyl or dicarbapentaborane and azanol reaction thing.Comprise the method that is used for producing oxime deutero-protein adduct derived protein in this aspect based on reactant that contains carbonyl or dicarbapentaborane and the reactant condensation that contains azanol.In extra or other embodiment for to contain the ketone method of protein to derive through the functionalized polyoxyethylene glycol of azanol (PEG) molecule.Extra or aspect other in for the derived molecules by containing carbonyl or dicarbapentaborane and contain the peptide of oxime or the oxime permutoid reaction between the protein is derived and contained the oxime method of protein.Extra or other aspect in, the molecule that replaces through azanol can comprise protein, other polymkeric substance and small molecules.
Be chemosynthesis be used to the to derive method of the proteinic molecule that replaces through azanol that replaces through ketone on the other hand.Be chemosynthesis be used to the to derive method of the proteinic molecule that replaces through azanol that replaces through aldehyde on the other hand.In one embodiment, the molecule that replaces through azanol can comprise peptide, other polymkeric substance (not branch and branch) and small molecules.Be preparation be suitable for the deriving method of the molecule that replaces through azanol of the non-natural amino acid polypeptides (only for instance, it comprises the non-natural amino acid polypeptides that contains ketone) that contains carbonyl or dicarbapentaborane in one embodiment.In another or extra embodiment, alpha-non-natural amino acid is incorporated on proteinic in vivo translate duration site-specific ground.In another or extra embodiment, the molecule that replaces through azanol allows that by the nucleophilic attack of carbonyl or dicarbapentaborane this locus specificity that contains the alpha-non-natural amino acid of carbonyl or dicarbapentaborane derives to form the oxime polypeptides derived in the locus specificity mode.In another or extra embodiment, the method for the molecule that preparation replaces through azanol provides the derive method of polypeptide of multiple locus specificity that obtains.In another or extra embodiment, be synthetic method through the functionalized polyoxyethylene glycol of azanol (PEG) molecule.
Be chemosynthesis be used to the to derive method of the molecule that replaces through carbonyl or dicarbapentaborane of the non-natural amino acid polypeptides that replaces through oxime on the other hand.In one embodiment, the molecule through carbonyl or dicarbapentaborane replacement is the molecule that replaces through ketone.In one embodiment, the molecule through carbonyl or dicarbapentaborane replacement is the molecule that replaces through aldehyde.In another embodiment, the molecule through carbonyl or dicarbapentaborane replacement comprises protein, polymkeric substance (not branch and branch) and small molecules.In another or extra embodiment, these methods are replenished can be in the technology of proteinic in vivo translate duration site specific incorporation of non-natural amino acids.In another or extra embodiment, be suitable for and contain the general method of oxime non-natural amino acid polypeptides reaction with the molecule that replaces through carbonyl or dicarbapentaborane that locus specificity deutero-non-natural amino acid polypeptides is provided for preparation.In another or extra embodiment, be the method for synthetic polyoxyethylene glycol (PEG) molecule that replaces through carbonyl or dicarbapentaborane.
On the other hand for using the method for the chemically derived non-natural amino acid polypeptides that replaces through carbonyl or dicarbapentaborane of the difunctionality connexon contain azanol.Connect the connexon that replaces through azanol and the method for protein that replaces through carbonyl or dicarbapentaborane for produce the oxime key by condensation reaction in one embodiment.In other or extra embodiment, the alpha-non-natural amino acid that replaces through carbonyl or dicarbapentaborane be the alpha-non-natural amino acid through the ketone replacement.In other or extra embodiment, use the difunctionality connexon that contains azanol, non-natural amino acid polypeptides is derived through locus specificity ground and/or three-dimensional structure is derived precisely controlledly.In one embodiment, these methods are used for molecule connexon (including (but not limited to) simple function, difunctionality and multifunctional connexon) is connected to the non-natural amino acid polypeptides that contains carbonyl or dicarbapentaborane (including (but not limited to) containing ketone and containing aldehyde), wherein at least one connexon end contains the azanol base, and it can be by oxime key binding to the non-natural amino acid polypeptides that contains carbonyl or dicarbapentaborane.In another or extra embodiment, these connexons are used to connect non-natural amino acid polypeptides and other molecules that contains carbonyl or dicarbapentaborane, and it is including (for example) protein, other polymkeric substance (branch and not branch) and small molecules.
In certain embodiments, the non-natural amino acid polypeptides binding is to water-soluble polymers.In certain embodiments, water-soluble polymers comprises polyalkylene glycol moiety.In certain embodiments, peg molecule is the double functional copolymer.In certain embodiments, double functional copolymer's binding is to second polypeptide.In certain embodiments, second polypeptide is identical with first polypeptide, and in other embodiments, second polypeptide is different polypeptide.In certain embodiments, non-natural amino acid polypeptides comprises the amino acid of at least two kinds of bindings to the water-soluble polymers (comprising polyalkylene glycol moiety).
In certain embodiments, non-natural amino acid polypeptides comprises replacement, interpolation or the disappearance of increase non-natural amino acid polypeptides to the avidity of acceptor.In certain embodiments, non-natural amino acid polypeptides comprises replacement, interpolation or the disappearance of the stability that increases non-natural amino acid polypeptides.In certain embodiments, non-natural amino acid polypeptides comprises water miscible replacement, interpolation or the disappearance that increases non-natural amino acid polypeptides.In certain embodiments, non-natural amino acid polypeptides comprises deliquescent replacement, interpolation or the disappearance of non-natural amino acid polypeptides in host cell that increase produces.In certain embodiments, with respect to the amino acid polypeptide that does not have replacement, adds or lack, non-natural amino acid polypeptides comprises replacement, interpolation or the disappearance of regulating protease resistant, serum half-life, immunogenicity and/or expression.
In certain embodiments, non-natural amino acid polypeptides is agonist, part agonist, antagonist, partial antagonist or reverse agonist.In certain embodiments, agonist, part agonist, antagonist, partial antagonist or oppositely agonist comprise alpha-non-natural amino acid with the water-soluble polymers binding.In certain embodiments, water-soluble polymers comprises polyalkylene glycol moiety.In certain embodiments, comprise that polypeptide (for example) with the alpha-non-natural amino acid of water-soluble polymers binding can prevent the dimerization of corresponding acceptor.In certain embodiments, comprise with the polypeptides for modulating polypeptide of the alpha-non-natural amino acid of water-soluble polymers binding and combine the combination of collocation thing, ligand or acceptor.In certain embodiments, comprise one or more character or activity with the polypeptides for modulating polypeptide of the alpha-non-natural amino acid of water-soluble polymers binding.
In certain embodiments, selecting codon is to be selected from the group that is made up of amber codon (amber codon), ocher codon (ochre codon), opal codon (opal codon), unique codon (unique codon), rare codon (rare codon), non-natural codon (unnatural codon), five base codons (five-base codon) and four base codons (four-base codon).
The method of the non-natural amino acid polypeptides of manufacturing and water-soluble polymers binding is also described herein.In certain embodiments, described method comprises making and comprises contacting with comprising with the water-soluble polymers of the part of alpha-non-natural amino acid reaction through isolated polypeptide of alpha-non-natural amino acid.In certain embodiments, any in the alpha-non-natural amino acid of incorporating into pair and the 20 kinds of common amino acids do not have reactive responding property of water-soluble polymers.In certain embodiments, aqueous-based polymers comprises polyalkylene glycol moiety.The molecular weight of polymkeric substance can be in broad range, and it is including (but not limited to) between about 100Da and about 100,000Da or higher between scope.The molecular weight of polymkeric substance can be at about 100Da and about 100, and between the 000Da, it is including (but not limited to) 100,000Da, 95,000Da, 90,000Da, 85,000Da, 80,000Da, 75,000Da, 70,000Da, 65,000Da, 60,000Da, 55,000Da, 50,000Da, 45,000Da, 40,000Da, 35,000Da, 30,000Da, 25,000Da, 20,000Da, 15,000Da, 10,000Da, 9,000Da, 8,000Da, 7,000Da, 6,000Da, 5,000Da, 4,000Da, 3,000Da, 2,000Da, 1,000Da, 900Da, 800Da, 700Da, 600Da, 500Da, 400Da, 300Da, 200Da and 100Da.In certain embodiments, the molecular weight of polymkeric substance is in about 100Da and 50, between the 000Da.In certain embodiments, the molecular weight of polymkeric substance is in about 100Da and 40, between the 000Da.In certain embodiments, the molecular weight of polymkeric substance is about 1, and 000Da and 40 is between the 000Da.In certain embodiments, the molecular weight of polymkeric substance is about 5, and 000Da and 40 is between the 000Da.In certain embodiments, the molecular weight of polymkeric substance is about 10, and 000Da and 40 is between the 000Da.In certain embodiments, peg molecule is a branched polymers.The molecular weight of side chain PEG can be about 1,000Da and about 100, and between the 000Da, it is including (but not limited to) 100,000Da, 95,000Da, 90,000Da, 85,000Da, 80,000Da, 75,000Da, 70,000Da, 65,000Da, 60,000Da, 55,000Da, 50,000Da, 45,000Da, 40,000Da, 35,000Da, 30,000Da, 25,000Da, 20,000Da, 15,000Da, 10,000Da, 9,000Da, 8,000Da, 7,000Da, 6,000Da, 5,000Da, 4,000Da, 3,000Da, 2,000Da and 1,000Da.In certain embodiments, the molecular weight of side chain PEG is about 1, and 000Da and 50 is between the 000Da.In certain embodiments, the molecular weight of side chain PEG is about 1, and 000Da and 40 is between the 000Da.In certain embodiments, the molecular weight of side chain PEG is about 5, and 000Da and 40 is between the 000Da.In certain embodiments, the molecular weight of side chain PEG is about 5, and 000Da and 20 is between the 000Da.
Also describe herein and comprise the composition that comprises at least a described alpha-non-natural amino acid and pharmaceutically acceptable supporting agent herein.In certain embodiments, the alpha-non-natural amino acid binding is to water-soluble polymers.Also description comprises the pharmaceutically acceptable supporting agent and the medical composition of polypeptide herein, and wherein at least one amino acid replaces through alpha-non-natural amino acid.In certain embodiments, alpha-non-natural amino acid comprises sugar moieties.In certain embodiments, water-soluble polymers passes through the sugar moieties binding to polypeptide.The prodrug of alpha-non-natural amino acid, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides is also described herein; Further describe the composition that comprises these prodrugs and pharmaceutically acceptable supporting agent herein.The metabolite of alpha-non-natural amino acid, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides is also described herein; These metabolites can have and replenish or the collaborative active required activity of strengthening alpha-non-natural amino acid, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides.Described herein alpha-non-natural amino acid, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides provide (comprising the patient who needs this metabolite) purposes from required metabolite to organism is also described herein.
The cell that comprises coded polypeptide and comprise the polynucleotide of selecting codon is also described herein.In certain embodiments, cell comprises quadrature RNA synthetic enzyme and/or the quadrature tRNA that is used for alpha-non-natural amino acid substitution polypeptide.In certain embodiments, cell is in cell culture, and is the cell of the part of multicellular organism (comprising Amphibians, Reptilia, birds and Mammals) in other embodiments.In any one of cell embodiment, other embodiment comprise the expression of the polynucleotide that produces non-natural amino acid polypeptides.In other embodiments for utilizing described alpha-non-natural amino acid to produce the organism of non-natural amino acid polypeptides (comprising modified non-natural amino acid polypeptides) herein.In other embodiments for containing the organism of described alpha-non-natural amino acid, non-natural amino acid polypeptides and/or modified non-natural amino acid polypeptides herein.These organisms comprise unicellular organism and multicellular organism, and it comprises Amphibians, Reptilia, birds and Mammals.In certain embodiments, non-natural amino acid polypeptides is in vitro to produce.In certain embodiments, non-natural amino acid polypeptides is to produce in cell lysates.In certain embodiments, non-natural amino acid polypeptides produces by the rrna translation.
The method of making the polypeptide that comprises alpha-non-natural amino acid is also described herein.In certain embodiments, described method is included in the cell of cultivating the polynucleotide that comprises coded polypeptide, quadrature RNA synthetic enzyme and/or quadrature tRNA under the condition of allowing expression of polypeptides; With purified polypeptide from cell and/or substratum.
The storehouse of described alpha-non-natural amino acid herein or the storehouse of described non-natural amino acid polypeptides are herein also described herein, or the storehouse of described herein modified non-natural amino acid polypeptides, or its combinatorial libraries.The array contain at least a alpha-non-natural amino acid, at least a non-natural amino acid polypeptides and/or at least a modified alpha-non-natural amino acid is also described herein.The array that contains at least a coded polypeptide and comprise the polynucleotide of selecting codon is also described herein.Described herein array can be used for the generation (polynucleotide by detecting coded polypeptide transcribe or by detecting the translation of polypeptide) of non-natural amino acid polypeptides in the screening organism.
Also describe the screening required active method in described storehouse herein herein, or use the required active method in the storehouse of the described storehouse of described array screening or other compounds and/or polypeptide and/or polynucleotide herein herein.The purposes that is used to develop and find new therapeutical agent from this activity data of storehouse screening is also described herein, and therapeutical agent itself.
The method of the treatment transformation period, serum half-life or the cycling time that increase polypeptide is also described herein.In certain embodiments, method comprises replacing with at least a alpha-non-natural amino acid and naturally has the arbitrary or multiple amino acids in the polypeptide and/or make polypeptide and the water-soluble polymers coupling.
Also describe the method that needs the patient of this treatment with the treatment of the medical composition of significant quantity herein, wherein said medical composition comprises the polypeptide that comprises alpha-non-natural amino acid and pharmaceutically acceptable supporting agent.In certain embodiments, alpha-non-natural amino acid and water-soluble polymers coupling.
In other or alternate embodiment, be the method for treatment illness, symptom or disease, described method comprises the non-natural amino acid polypeptides of throwing with the treatment significant quantity that comprises at least a alpha-non-natural amino acid, and wherein said alpha-non-natural amino acid is to be selected from by containing the oxime alpha-non-natural amino acid, contain the carbonyl alpha-non-natural amino acid, contain the dicarbapentaborane alpha-non-natural amino acid and containing the group that the azanol alpha-non-natural amino acid is formed.In other embodiments, these alpha-non-natural amino acids are incorporated herein in the described polypeptide by biosynthesizing.In other or alternate embodiment, these non-natural amino acid polypeptides comprise at least a amino acid whose alpha-non-natural amino acid that is selected from formula I-XVIII, formula XXX-XXXIV (A and B) or formula XXXX-XXXXIII.
In other or alternate embodiment, be the method for treatment illness, symptom or disease, described method comprises at least a non-natural amino acid polypeptides of throwing with the treatment significant quantity that contains the oxime alpha-non-natural amino acid that comprises, and with respect to the natural amino acid polypeptide that exists of homology, the biosynthetic biological usability that contains oxime non-natural amino acid polypeptides enhancing polypeptide of gained.
In other or alternate embodiment, be the method for treatment illness, symptom or disease, described method comprises at least a non-natural amino acid polypeptides of throwing with the treatment significant quantity that contains the oxime alpha-non-natural amino acid that comprises, and with respect to the natural amino acid polypeptide that exists of homology, the biosynthetic safety profile that contains oxime non-natural amino acid polypeptides increase polypeptide of gained.
In other or alternate embodiment, be the method for treatment illness, symptom or disease, described method comprises at least a non-natural amino acid polypeptides of throwing with the treatment significant quantity that contains the oxime alpha-non-natural amino acid that comprises, and with respect to the natural amino acid polypeptide that exists of homology, the biosynthetic oxime non-natural amino acid polypeptides that contains of gained increases the water-soluble of polypeptide.
In other or alternate embodiment, be the method for treatment illness, symptom or disease, described method comprises at least a non-natural amino acid polypeptides of throwing with the treatment significant quantity that contains the oxime alpha-non-natural amino acid that comprises, and with respect to the natural amino acid polypeptide that exists of homology, the biosynthetic treatment transformation period that contains oxime non-natural amino acid polypeptides increase polypeptide of gained.
In other or alternate embodiment, be the method for treatment illness, symptom or disease, described method comprises at least a non-natural amino acid polypeptides of throwing with the treatment significant quantity that contains the oxime alpha-non-natural amino acid that comprises, and with respect to the natural amino acid polypeptide that exists of homology, the biosynthetic serum half-life that contains oxime non-natural amino acid polypeptides increase polypeptide of gained.
In other or alternate embodiment, be the method for treatment illness, symptom or disease, described method comprises at least a non-natural amino acid polypeptides of throwing with the treatment significant quantity that contains the oxime alpha-non-natural amino acid that comprises, and with respect to the natural amino acid polypeptide that exists of homology, gained is biosynthetic to contain the cycling time that the oxime non-natural amino acid polypeptides prolongs polypeptide.
In other or alternate embodiment, be the method for treatment illness, symptom or disease, described method comprises at least a non-natural amino acid polypeptides of throwing with the treatment significant quantity that contains the oxime alpha-non-natural amino acid that comprises, and with respect to the natural amino acid polypeptide that exists of homology, the biosynthetic biological activity that contains oxime non-natural amino acid polypeptides adjusting polypeptide of gained.
In other or alternate embodiment, be the method for treatment illness, symptom or disease, described method comprises at least a non-natural amino acid polypeptides of throwing with the treatment significant quantity that contains the oxime alpha-non-natural amino acid that comprises, and with respect to the natural amino acid polypeptide that exists of homology, the biosynthetic immunogenicity that contains oxime non-natural amino acid polypeptides adjusting polypeptide of gained.
Should be appreciated that described method and composition is not limited to described ad hoc approach, scheme, cell strain herein, constructs body and reagent and can change equally herein.Should be appreciated that term used herein equally only in order to describe the purpose of specific embodiment, and be not intended to limit the category of described method and composition herein, it should only be limited by the claim of enclosing.
Unless context spells out in addition, otherwise such as reach herein in the claim of enclosing use, singulative " " and " described " comprise plural number and mention.
Unless otherwise defined, otherwise all technical terms used herein and scientific terminology have the common identical meanings of understanding of those skilled in the art in the invention as described in this article.Although can be used for describing now preferred method, device and material in described enforcement of the present invention or the test herein with described those similar or equivalent any method, device and materials herein.
Open case of all that mention and patent are all to be incorporated herein by reference in order to describe and disclose the purpose of describing in (for example) open case of constructing body and method herein, and it can use together with the present invention who describes herein.The open case of Lun Shuing only provided its disclosure before the date of application of the application's case herein.Anything should not be construed as and admits that described present inventor is owing to the previous date of inventing or having no right to do sth. in advance this disclosure for any other reason herein herein.
As used herein term " affinity marker " refer to reversible or irreversibly combine with another molecule with it is modified, with its destruction or with the mark of its formation compound.For instance, affinity marker comprises enzyme and matrix thereof, or antibody and antigen thereof.
Term " alkoxyl group ", " alkylamino " and " alkylthio " are to use with its conventional meaning, and refer to respectively by Sauerstoffatom, amino, the sulphur atom binding alkyl to the molecule.
Unless otherwise noted, otherwise term " alkyl " (self or as the part of another molecule) means straight or branched, or cyclic hydrocarbon group, or its combination, it can be saturated fully, single unsaturated or polyunsaturated and can comprise have the appointment carbon atom number divalent group and the multivalence group of (being that C1-C10 means 1 to 10 carbon).The example of saturated hydrocarbyl is including (but not limited to) following group, such as the homologue and the isomer of methyl, ethyl, n-propyl, sec.-propyl, normal-butyl, the tertiary butyl, isobutyl-, sec-butyl, cyclohexyl, (cyclohexyl) methyl, cyclopropyl methyl, (for example) n-pentyl, n-hexyl, n-heptyl, n-octyl and similar group thereof.Unsaturated alkyl is for having one or more pair of key or triple-linked alkyl.The example of unsaturated alkyl is including (but not limited to) vinyl, 2-propenyl, crot(on)yl, 2-isopentene group, 2-(butadienyl), 2,4-pentadienyl, 3-(1, the 4-pentadienyl), ethynyl, 1-proyl and 3-proyl, 3-butynyl and more high-grade homologue and isomer.Unless otherwise noted, otherwise term " alkyl " also means and comprises herein more those derivatives of the alkyl of specific definition, such as " assorted alkyl ", " alkylhalide group " and " high alkyl ".
Term " alkylidene group " (self or as the part of another molecule) means by alkane deutero-divalent group, as by (CH 2-) the n illustration, wherein n can be 1 to about 24.Only for instance, these groups are including (but not limited to) having 10 or the group of carbon atom still less, such as structure-CH 2CH 2-and-CH 2CH 2CH 2CH 2-." low alkyl group " or " low-grade alkylidene " for have usually 8 or still less carbon atom than short-chain alkyl or alkylidene group.Unless otherwise noted, otherwise term " alkylidene group " also means those groups that comprise herein and be described as " inferior assorted alkyl ".
Term " amino acid " refers to natural amino acid and the alpha-non-natural amino acid of existing, and to be similar to natural amino acid analogue and the amino acid analog thing that exists amino acid whose mode to work.Natural coded amino acid is 20 kinds of common amino acids (L-Ala, arginine, l-asparagine, aspartic acid, halfcystine, glutamine, L-glutamic acid, glycine, Histidine, Isoleucine, leucine, Methionin, methionine(Met), phenylalanine, proline(Pro), Serine, Threonine, tryptophane, tyrosine and Xie Ansuan) and burnt Methionin and selenocystein.Amino acid analogue refers to have and the natural compound that has the identical basic chemical structure of amino acid (only for instance, with hydrogen, carboxyl, amino and R group bonded alpha-carbon).These analogues can have modified R group (for instance, nor-leucine) maybe can have modified peptide main chain, and still keeps and the natural identical basic chemical structure of amino acid that exists.The limiting examples of amino acid analogue comprises homoserine, nor-leucine, methionine sulfoxide, methionine(Met) methyl sulfonium.
Amino acid in this article can be by its title, its common known trigram symbol or the one-letter symbol address of being recommended by IUPAC-IUB biochemical nomenclature commission (IUPAC-IUB Biochemical Nomenclature Commission).In addition, Nucleotide can be by its single-letter code of accepting usually address.
" N-terminal modification group " refers to any molecule that can be connected on the terminal amino group.For instance, these terminal amino groups can be in the end of polymerizable molecular, and wherein these polymerizable moleculars are including (but not limited to) polypeptide, polynucleotide and polysaccharide.End modified group is including (but not limited to) various water-soluble polymerss, peptide or protein.Only for instance, end modified group comprises polyoxyethylene glycol or serum albumin.End modified group can be used for changing the treatment feature of polymerizable molecular, and it is including (but not limited to) the serum half-life that increases peptide.
" antibody fragment " means any form of the antibody except that the total length form.Antibody fragment is included as the antibody than small component that is present in the full length antibody in this article, and through the antibody of through engineering approaches design.Antibody fragment is including (but not limited to) Fv, Fc, Fab, and combination, variable region, framework region, constant region, heavy chain, light chain and the variable region of (Fab ') 2, strand Fv (scFv), double-stranded antibody, three chain antibodies, four chain antibodies, difunctional hybrid antibody, CDR1, CDR2, CDR3, CDR, and substituting skeleton non-antibody molecule, bi-specific antibody and analogue (Maynard and Georgiou, 2000, Annu.Rev.Biomed.Eng.2:339-76; Hudson, 1998, Curr.Opin.Biotechnol.9:395-402).Another functional substructure is strand Fv (scFv), it comprise by the variable region of covalently bound heavy chain immunoglobulin of peptide connexon and light chain (people such as S-z Hu, 1996, Cancer Research, 56,3055-3061).These little (Mr 25,000) protein usually keep the antigenic specificity in the single polypeptide and affinity and can be bigger antigen-specific molecule suitable construction unit is provided.Unless other specific pointing out, otherwise use the statement and the claim of term " antibody " clearly to comprise " antibody fragment ".
As used herein, term " aromatics " or " aryl " refer to and have at least one and have the ring of conjugated pi electron system and comprise isocyclic aryl and the closed-loop construct of heterocyclic aryl (or " heteroaryl " or " heteroaromatic ").Carbocyclic ring or heterocyclic aromatic group can contain 5 to 20 annular atomses.Monocycle or the condensed that described term comprises covalent bond encircles (that is the right ring of shared adjacent carbons) group more.Aromatic group can be unsubstituted or be substituted.The limiting examples of " aromatics " or " aryl " group comprises phenyl, 1-naphthyl, 2-naphthyl, 4-xenyl, anthryl and phenanthryl.The substituting group of each in above-mentioned aryl and the heteroaryl ring system is to be selected from the acceptable substituent group of describing herein.
For for simplicity, when being used in combination (including (but not limited to) aryloxy, aryl sulphur oxygen base, aralkyl) with other terms, term " aromatics " or " aryl " comprise aryl rings and heteroaryl ring as defined above.Therefore, term " aralkyl " or " alkaryl " mean and comprise aryl wherein and be connected to those groups (it is including (but not limited to) benzyl, styroyl, pyridylmethyl and similar group thereof) on the alkyl, wherein said alkyl comprises those alkyl that carbon atom (including (but not limited to) methylene radical) has wherein been replaced by heteroatoms (Sauerstoffatom only for instance).The example of these aryl is including (but not limited to) phenoxymethyl, 2-pyridyl oxygen ylmethyl, 3-(1-naphthyl oxygen base) propyl group and similar group thereof.
As used herein, term " arylidene " refers to divalent aryl.The limiting examples of " arylidene " comprises phenylene, pyridylidene, inferior pyrimidyl and inferior thienyl.The substituting group of arylidene is selected from the acceptable substituent group of describing herein.
" double functional copolymer " also is known as " difunctionality connexon ", its refer to comprise two can with the polymkeric substance of other part specific reactions with the functional group that forms covalent linkage or non covalent bond.These parts can or contain side group on the peptide of these natural amino acids or alpha-non-natural amino acid including (but not limited to) natural amino acid or alpha-non-natural amino acid.Only for instance, the difunctionality connexon can have with first peptide on the functional group of responding property of group, and with second peptide on another functional group of responding property of group, form the joiner that comprises first peptide, difunctionality connexon and second peptide by this.Become known for numerous programs and connexon molecule that multiple compound is connected with peptide.Referring to No. the 188th, 256, (for example) European patent application; United States Patent (USP) the 4th, 671, No. 958, the 4th, 659, No. 839, the 4th, 414, No. 148, the 4th, 699, No. 784; The 4th, 680, No. 338; And the 4th, 569, No. 789, it is all to be incorporated herein by reference." polyfunctional poly compound " also is known as " multifunctional connexon ", its refer to comprise two or more can with the functional group's of other partial reactions polymkeric substance.These parts can or contain side group (including (but not limited to) amino acid side group) on the peptide of these natural amino acids or alpha-non-natural amino acid to form covalent linkage or non covalent bond including (but not limited to) natural amino acid or alpha-non-natural amino acid.Double functional copolymer or polyfunctional poly compound can be any desired length or molecular weight, and can be through selected to provide specific required interval or conformation at binding between the one or more molecule of compound and its bonded molecule or compound.
As used herein, term " biological usability " refers to material or its active part from pharmaceutical dosage form send and become at site of action or in general circulation available speed and degree.The increase of biological usability refers to increases material or its active part from pharmaceutical dosage form send and become at site of action or in general circulation available speed and degree.The increase of material or its active part concentration in blood when for instance, the increase of biological usability can be designated as and compare with other materials or active part.The limiting examples of the method that the assessment biological usability increases provides in example 88-92.This method can be used for assessing the biological usability of any polypeptide.
When using in this article, term " bioactive molecules ", " biologically-active moiety " or " biologically active agent " mean any physics or the biochemical property or the interactional any material relevant with organism that can influence biosystem, path, molecule, and described organism is including (but not limited to) virus, bacterium, phage, transposon, Protein virus, insect, fungi, plant, animal and the mankind.Specifically, as used herein, bioactive molecules is including (but not limited to) the disease that is intended to be used to diagnose, cure, alleviate, treat or prevent human or other animals, or is used to strengthen the health of the mankind or animal or any material of Mental Health in addition.The example of bioactive molecules is including (but not limited to) peptide, protein, enzyme, small-molecule drug, hard drug, soft drug, carbohydrate, inorganic atoms or molecule, dyestuff, lipid, nucleosides, radioactive nuleus, oligonucleotide, toxin, cell, virus, liposome, particulate and micella.The kind that is suitable for the biologically active agent that uses with described method and composition herein is including (but not limited to) medicine, prodrug, radioactive nuleus, developer, polymkeric substance, microbiotic, mycocide, antiviral agent, antiphlogistic, antineoplastic agent, cardiovascular agents, antianxiety agent, hormone, somatomedin, steroid agent, microbe-derived toxin and analogue thereof.
" adjusting biological activity " means the reactivity that improves or reduce polypeptide, changes the selectivity of polypeptide, strengthens or reduce the substrate selective of polypeptide.Analyzing the biological activity change can be by relatively the biological activity of non-natural polypeptide and the biological activity of natural polypeptides are carried out.
As used herein, term " biomaterial " refers to the material of biogenetic derivation, and it is including (but not limited to) from bio-reactor and/or the material that obtained by recombination method and technology.
As used herein, term " biophysics probe " refers to the probe that can detect or monitor the structural changes of molecule.These molecules can be used for detecting or monitoring protein and other macromolecular interactions including (but not limited to) protein and " biophysics probe ".But the example of biophysics probe is including (but not limited to) spin labeling, fluorophore and photoactivation group.
As used herein, term " biosynthesizing " refers to any method of utilizing translation system (cell or acellular), and it comprises in the following component of use at least one: polynucleotide, codon, tRNA and rrna.For instance, alpha-non-natural amino acid can use method and the technology described in part of V III " in vivo generation comprises the polypeptide of alpha-non-natural amino acid " and the limiting examples 14 " to incorporate into by biosynthesizing " in the non-natural amino acid polypeptides.In addition, the selection method that can " incorporate " the suitable alpha-non-natural amino acid in the non-natural amino acid polypeptides by biosynthesizing into is described among the limiting examples 15-16.
As used herein, term " vitamin H analogue " (or also be known as " vitamin H stand-in ") for except that vitamin H with high-affinity and avidin and/or any molecule of streptavidin bonded.
As used herein term " carbonyl " refer on part, contain be selected from by-C (O)-,-S (O)-,-S (O) 2-and-group of the group of C (S)-composition, it is including (but not limited to) the group that contains at least one ketone group and/or at least one aldehyde radical and/or at least one ester group and/or at least one hydroxy-acid group and/or at least one thioester substrate.These carbonyls comprise ketone, aldehyde, carboxylic acid, ester and thioesters.In addition, these groups can be the part of linearity, branch or ring molecule.
Term " C-terminal modification group " refers to any molecule that can be connected on the terminal carboxyl(group).For instance, these terminal carboxyl(group)s can be in the end of polymerizable molecular, and wherein these polymerizable moleculars are including (but not limited to) polypeptide, polynucleotide and polysaccharide.End modified group is including (but not limited to) various water-soluble polymerss, peptide or protein.Only for instance, end modified group comprises polyoxyethylene glycol or serum albumin.End modified group can be used for changing the treatment feature of polymerizable molecular, and it is including (but not limited to) the serum half-life that increases peptide.
As used herein, term " but chemical cracking group " (also being known as " chemically unstable ") refers to after being exposed to acid, alkali, oxygenant, reductive agent, chemical initiator or radical initiator and decomposes or the cracked group.
As used herein, term " chemiluminescent groups " refer to not additionally under the situation of heating because chemical reaction and luminous group.Only for instance, in the presence of alkali and metal catalyst, luminol,3-aminophthalic acid cyclic hydrazide (luminol) (5-amino-2,3-dihydro-1,4-phthalazine diketone) and oxygenant are (as hydrogen peroxide (H 2O 2)) reaction generation excited state product (3-aminophthalic acid salt, 3-APA).
As used herein, term " chromophoric group " refers to the molecule of the light that absorbs visible wavelength, UV wavelength or IR wavelength.
As used herein, term " cofactor " refers to essential atom of macromolecular effect or molecule.Cofactor is including (but not limited to) some essential other factors of the activity of mineral ion, coenzyme, protein or enzyme.Example comprises the heme in the hemochrome, the magnesium in the chlorophyll, and proteinic metal ion.
As used herein, " altogether folding " refers at least two molecules interact with each other of use and makes again folding process, reaction or the method for folding or improper folding molecular conversion for suitably folding molecule.Only for instance, " altogether folding " uses at least two polypeptide interact with each other and makes folding or improper folding polypeptide change natural, suitable folding polypeptide into.These polypeptide can contain natural amino acid and/or at least one alpha-non-natural amino acid.
As used herein, " comparison window " refers to any one the section that is used for the adjoining position of the sequence of comparison similar number adjoining position and canonical sequence two sequences after the best comparison.These adjoining positions are including (but not limited to) by about 20 to about 600 groups that sequential cells is formed, and it comprises about 50 to about 200 sequential cells, and about 100 to about 150 sequential cells.Only for instance, these sequences comprise polypeptide and contain the polypeptide of alpha-non-natural amino acid, and wherein sequential cells is including (but not limited to) natural amino acid and alpha-non-natural amino acid.In addition, only for instance, these sequences comprise polynucleotide, and wherein Nucleotide is corresponding sequential cells.The sequence alignment method that is used for comparison is what know in affiliated field.Can be used for the optimal sequence comparison of comparison by following method, it is including (but not limited to) local homology's algorithm of Smith and Waterman (1970) Adv.Appl.Math.2:482c, the homology alignment algorithm of Needleman and Wunsch (1970) J.Mol.Biol.48:443, the similarity searching method of Pearson and Lipman (1988) Proc.Nat ' l.Acad.Sci.USA 85:2444, the computerization of these algorithms realizes (wisconsin genetics software bag (Wisconsin Genetics Software Package) (Genetics Computer Group, 575 Science Dr., Madison, WI) GAP in, BESTFIT, FASTA and TFASTA), or manual comparison and visual inspection (referring to people such as (for example) Ausubel, Current Protocols in Molecular Biology (1995 supplementary issue)).
For instance, the algorithm that can be used for measuring sequence identity per-cent and sequence similarity is BLAST and BLAST2.0 algorithm, it is described in people such as Altschul respectively. (1997) Nuc.Acids Res.25:3389-3402, and people such as Altschul. and among (1990) J.Mol.Biol.215:403-410.The software that carries out the BLAST analysis can obtain by national biotechnology information center (National Center for Biotechnology Information) is open.BLAST algorithm parameter W, T and X determine the sensitivity and the speed of comparison.BLASTN program (for nucleotide sequence) uses that word length (W) is 11, expected value (E) is 10, the comparison of M=5, N=-4 and two chains is as default value.For aminoacid sequence, the BLASTP program uses that word length is 3, expected value (E) is 10 and BLOSUM62 score matrix (referring to Henikoff and Henikoff (1992) Proc.Natl.Acad.Sci.USA 89:10915) comparison (B) is 50, expected value (E) is 10, the comparison of M=5, N=-4 and two chains is as default value.The BLAST algorithm carries out under the situation that " low-complexity " strainer cuts out usually.
The BLAST algorithm also carries out the statistical study (referring to (for example) Karlin and Altschul (1993) Proc.Natl.Acad.Sci.USA 90:5873-5787) of similarity between two sequences.It is minimum and probability (P (N)) that of the similarity that is provided by the BLAST algorithm measures, and it provides the indication of the probability that chance can be mated between two Nucleotide or the aminoacid sequence.For instance, if in the comparison of test nucleic acid and reference nucleic acid, minimum and probability be less than about 0.2, or less than about 0.01, or less than about 0.001, so just think that nucleic acid is similar to reference sequences.
Term " through the variant of conservative property modification " is applied to natural and alpha-non-natural amino acid sequence and natural and non-natural nucleotide sequence, and combination.About specific nucleic acid sequence, " variant of modifying through conservative property " refers to identical or the substantially the same natural and alpha-non-natural amino acid sequence of coding, or wherein natural and non-natural nucleic acid do not encode those of natural and alpha-non-natural amino acid sequence (for substantially the same sequence) natural with non-natural nucleic acid.For instance, since the degeneracy of genetic code, the identical any given protein of nucleic acid encoding on a large amount of functions.For example, the equal coded amino acid L-Ala of codon GCA, GCC, GCG and GCU.Therefore, L-Ala is by specified each position of codon therein, and codon can be changed into arbitrary corresponding codon and not change encoded polypeptide.The variation of these nucleic acid is " silent variant ", and it be a kind of in the variation of conservative property modification.Therefore the natural or non-natural nucleotide sequence of herein each natural or the non-natural polypeptide of for example encoding is also described each of natural or non-natural nucleic acid may silent variant.It will be understood by one of ordinary skill in the art that each codon in natural or the non-natural nucleic acid is (except that AUG and TGG, wherein AUG only is the codon of methionine(Met) usually, and TGG only is the codon of tryptophane usually) can be modified to produce identical molecule on the function.Therefore, each silent variant of the natural and non-natural nucleic acid of the natural and non-natural polypeptide of coding implies in each described sequence.
About aminoacid sequence, to change, add or the disappearance encoding sequence in single natural and alpha-non-natural amino acid or little per-cent natural and alpha-non-natural amino acid nucleic acid, peptide, polypeptide or protein sequence indivedual replacements, lack or be added to " variant of modifying through conservative property ", wherein change and cause amino acid whose disappearance, amino acid whose interpolation, or natural and alpha-non-natural amino acid is through similar aminoacid replacement chemically.Know the conservative replacement table that similar natural amino acid on the function is provided in the affiliated field.These through variants that conservative property is modified be different from polymorphie variant, plant between homologue and herein described method and composition allelotrope and do not get rid of these materials.Below eight groups contain each other the amino acid that replaces for conservative property separately:
1) L-Ala (A), glycine (G);
2) aspartic acid (D), L-glutamic acid (E);
3) l-asparagine (N), glutamine (Q);
4) arginine (R), Methionin (K);
5) Isoleucine (I), leucine (L), methionine(Met) (M), Xie Ansuan (V);
6) phenylalanine (F), tyrosine (Y), tryptophane (W);
7) Serine (S), Threonine (T); And
8) halfcystine (C), methionine(Met) (M)
(referring to (for example) Creighton, Proteins:Structures and Molecular Properties (W H Freeman﹠amp; Co.; The 2nd edition (in December, 1993)).
Unless otherwise noted, otherwise term " cycloalkyl " and " Heterocyclylalkyl " (self or with the combination of other terms) annular form of expression " alkyl " and " alkyl of mixing " respectively.Therefore, cycloalkyl or Heterocyclylalkyl comprise saturated, part is unsaturated and complete undersaturated ring key connection.In addition, for Heterocyclylalkyl, heteroatoms can occupy the position that heterocycle is connected to the rest part of molecule.Heteroatoms can be including (but not limited to) oxygen, nitrogen or sulphur.The example of cycloalkyl including (but not limited to) cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, suberyl with and similar group.The example of Heterocyclylalkyl is including (but not limited to) 1-(1,2,5,6-tetrahydro pyridyl), piperidino, 2-piperidyl, 3-piperidyl, 4-morpholinyl, morpholinyl, tetrahydrofuran (THF)-2-base, tetrahydrofuran (THF)-3-base, tetramethylene sulfide-2-base, tetramethylene sulfide-3-base, 1-piperazinyl, 2-piperazinyl with and similar group.In addition, the polycyclic structure contained in term, and it is including (but not limited to) double-ring ring structure and three cyclic rings structures.Similarly, term " inferior Heterocyclylalkyl " (self or as the part of another molecule) means the divalent group derived from Heterocyclylalkyl, and term " cycloalkylidene " (self or as the part of another molecule) means the divalent group derived from cycloalkyl.
As used herein, term " cyclodextrin " refers in ring constitutes by at least 6 to 8 cyclic carbohydrates that glucose molecule is formed.Water soluble group is contained in the outside of ring; The ring the center for can hold micromolecular nonpolar relatively hole.
As used herein, term " cytotoxicity " refers to the compound of injury cell.
As used herein, " denaturing agent " refers to any compound or the material that can cause the reversible polymer stretching, extension.Only for instance, " denaturing agent " can cause proteinic reversibility to stretch.The intensity of denaturing agent will be decided by the character and the concentration of specific denaturing agent.For instance, denaturing agent is including (but not limited to) chaotropic agent, stain remover, water miscible organic solvent, phosphatide or its combination.The limiting examples of chaotropic agent is including (but not limited to) urea, guanidine and Sodium Thiocyanate 99.The limiting examples of stain remover can be including (but not limited to) strong stain remover, such as sodium lauryl sulphate or Soxylat A 25-7 (for example Tween or Triton stain remover), sarcosyl (Sarkosyl); Gentle nonionic detergent (for example, digitonin (digitonin)); Gentle cationic detergent, such as N-＞2,3-(two oily alkene oxygen bases)-propyl group-N, N, N-trimethyl ammonium; Gentle ion stain remover (for example Sodium cholic acid or Sodium desoxycholate); Or zwitterionic detergent, it is including (but not limited to) sulphonic acid betaine (Zwiftergent), 3-(3-courage amidopropyl) dimethylamino-1-propane vitriol (CHAPS) and 3-(3-courage amidopropyl) dimethylamino-2-hydroxyl-1-propane sulfonate (CHAPSO).The limiting examples of water miscible organic solvent is including (but not limited to) acetonitrile, low-grade alkane alcohol (C especially 2-C 4Alkanol is such as ethanol or Virahol), or lower alkyl glycol (C 2-C 4The alkane glycol is such as ethylene glycol) can be used as denaturing agent.The limiting examples of phosphatide is including (but not limited to) naturally occurring phosphatide, such as phosphatidylethanolamine, Yelkin TTS, phosphatidylserine and phosphatidylinositols; Or synthetic phospholipid derivative or variant, such as two caproyl Yelkin TTS or two oenanthyl Yelkin TTS.
As used herein, term " detectable label " but refer to the mark that the operational analysis conceptions of technology measure, these analytical technologies are including (but not limited to) fluorescence, chemoluminescence, spectrum, ultraviolet/visible absorption spectra, mass spectrum, nucleus magnetic resonance, mr and electrochemical method.
As used herein term " dicarbapentaborane " refer to contain at least two be selected from by-C (O)-,-S (O)-,-S (O) 2-and-group of the part of the group of C (S)-composition, it is including (but not limited to) 1,2-dicarbapentaborane, 1,3-dicarbapentaborane and 1,4-dicarbapentaborane, and the group that contains at least one ketone group and/or at least one aldehyde radical and/or at least one ester group and/or at least one hydroxy-acid group and/or at least one thioester substrate.These dicarbapentaborane comprise diketone, keto-aldehyde, ketone acid, ketone ester and ketone thioesters.In addition, these groups can be the part of linearity, branch or ring molecule.Two parts in the dicarbapentaborane can be identical or different, and can comprise the substituting group that can produce (only for instance) ester, ketone, aldehyde, thioesters or acid amides at any one place of two parts.
As used herein, term " medicine " refers to any material that uses in prevention, diagnosis, alleviation, treatment or cure diseases or symptom.
As used herein, term " dyestuff " refers to and contains chromophoric solvable coloring matter.
As used herein, term " significant quantity " refer to the symptom that can alleviate one or more diseases for the treatment of or symptom to a certain extent throw and medicament or the q.s of compound.The result can be the reduction and/or the alleviation of disease, symptom or the cause of disease of disease, or any other required change of biosystem.For instance, throw and medicament or compound including (but not limited to) natural amino acid polypeptide, non-natural amino acid polypeptides, modified natural amino acid polypeptide, or modified non-amino acid polypeptide.Can throw with the composition that contains these natural amino acid polypeptides, non-natural amino acid polypeptides, modified natural amino acid polypeptide or modified non-natural amino acid polypeptides and be used for preventative, enhancing and/or therapeutic treatment.Suitable " effectively " amount under any individual cases can be used such as the technology of dosage increase research and determine.
As used herein, term " electron dense group " refers to the group of scattered electrons when shining with electron beam.These groups are including (but not limited to) ammonium molybdate, Vikaline, cadmium iodide, 99%, carbohydrazide, six Ferric Chloride Hydrateds, vulkacit H, 98.5%, anhydrous indium chloride, lanthanum nitrate, three hydration lead acetate, three hydration lead citrates, lead nitrate, Periodic acid, phospho-molybdic acid, phospho-wolframic acid, the Tripotassium iron hexacyanide, yellow prussiate of potash, ammoniated ruthenium oxychloride, Silver Nitrate, Solargentum (Ag calibrating: 8.0-8.5%) " by force ", tetraphenylporphines silver (S-TPPS), sodium chloraurate, sodium wolframate, thallium trinitrate (TTN), thiosemicarbazide (TSC), uranyl acetate, uranyl nitrate and vanadylic sulfate.
As used herein, term " energy transfer agent " refers to the molecule that can contribute or accept from the energy of another molecule.Only for instance, FRET (fluorescence resonance energy transfer) (FRET) is a dipole-dipole coupling process, transfers to the excited energy non-radiation type by this process fluorescence donor molecule and does not excite on the acceptor molecule energy that it is received with the longer wavelength fluorescent emission subsequently.
Term " enhancing " means increase or prolongs the effectiveness or the time length of required effect.For instance, the effect of " enhancing " therapeutical agent refers to increases during treatment disease, illness or symptom or the ability of the effectiveness of extended treatment agent or time length, effect.As used herein, " enhancing significant quantity " refers to the amount that is enough to strengthen the effect of therapeutical agent in treatment during disease, illness or the symptom.When being used for the patient, should depend on the seriousness of disease, illness or symptom and process, previous treatment, patient's state of health and to the reaction of medicine for the effective amount of this purposes, and attending doctor's judgement.
As used herein, term " eukaryote " refers to the organism that belongs to Eukarya on the phylogenetics (domainEucarya), and it is including (but not limited to) animal (including (but not limited to) Mammals, insect, Reptilia, birds etc.), ciliate, plant (including (but not limited to) monocotyledons, dicotyledons and algae), fungi, yeast, flagellate, microsporidium and protobiont.
As used herein, term " fatty acid " " refer to carboxylic acid with about C6 or longer hydrocarbon side chain.
As used herein, term " fluorophore " refers to ballistic phonon and and then fluorescigenic molecule after exciting.
As used herein, term " functional group ", " active part ", " activating group ", " leavings group ", " reactive site ", " chemically reactive group " and " chemical reactivity part " refer to the part or the unit of the molecule that chemical reaction takes place.These terms some synonym and being used in this article in chemical field indicate carry out some functions or active and with the part of the molecule of other responding property of molecule.
Term " halogen " comprises fluorine, chlorine, iodine and bromine.
As used herein, term " halogen acyl group " refers to the acyl group that contains halogen part, and it is including (but not limited to)-C (O) CH 3,-C (O) CF 3,-C (O) CH 2OCH 3With and similar group.
As used herein, term " alkylhalide group " refers to the alkyl that contains halogen part, and it is including (but not limited to)-CF 3With-CH 2CF 3With and similar group.
As used herein, term " assorted alkyl " refers to straight or branched or cyclic hydrocarbon group, or its combination, it is made up of the heteroatoms that alkyl and at least one are selected from the group that is made up of O, N, Si and S, and wherein nitrogen-atoms and sulphur atom can according to circumstances can be according to circumstances through quaternized through oxidation and nitrogen heteroatom.Heteroatoms O, N and S and Si can be positioned at any interior location of assorted alkyl or be positioned at the position that alkyl is connected to the rest part of molecule.Example is including (but not limited to)-CH 2-CH 2-O-CH 3,-CH 2-CH 2-NH-CH 3,-CH 2-CH 2-N (CH 3)-CH 3,-CH 2-S-CH 2-CH 3,-CH 2-CH 2,-S (O)-CH 3,-CH 2-CH 2-S (O) 2-CH 3,-CH=CH-O-CH 3,-Si (CH 3) 3,-CH 2-CH=N-OCH 3And-CH=CH-N (CH 3)-CH 3In addition, two heteroatomss can be successive at the most, such as ,-CH 2-NH-OCH 3With-CH 2-O-Si (CH 3) 3
As used herein, term " inferior assorted alkyl " refers to by assorted alkyl deutero-divalent group, as by-CH 2-CH 2-S-CH 2CH 2-and-CH 2-S-CH 2-CH 2-NH-CH 2-illustrated (but and unrestricted).For the assorted alkyl in Asia, identical or different heteroatoms also can occupy chain one or both ends (including (but not limited to) alkylidene group oxygen base, alkylenedioxy group, alkylidene amino, alkylidene group diamino, amino oxygen base alkylidene group with and similar group).Further, roll into a ball for alkylidene group and inferior assorted alkyl bond symbasis, the direction of binding group is not the direction hint by the formula of writing the binding group.For instance, formula-C (O) 2R '-expression-C (O) 2R '-with-R ' C (O) 2-both.
As used herein, term " heteroaryl " or " heteroaromatic " refer to and contain the heteroatomic aryl that at least one is selected from N, O and S; Wherein nitrogen-atoms and sulphur atom can be according to circumstances through oxidations, and nitrogen-atoms can be according to circumstances through quaternized.Heteroaryl can be substituted or be unsubstituted.Heteroaryl can be connected on the rest part of molecule by heteroatoms.The limiting examples of heteroaryl comprises the 1-pyrryl, the 2-pyrryl, the 3-pyrryl, the 3-pyrazolyl, the 2-imidazolyl, the 4-imidazolyl, pyrazinyl, the 2-oxazolyl, the 4-oxazolyl, 2-phenyl-4-oxazolyl, the 5-oxazolyl, the 3-isoxazolyl, the 4-isoxazolyl, the 5-isoxazolyl, the 2-thiazolyl, the 4-thiazolyl, the 5-thiazolyl, the 2-furyl, the 3-furyl, the 2-thienyl, the 3-thienyl, the 2-pyridyl, the 3-pyridyl, the 4-pyridyl, the 2-pyrimidyl, the 4-pyrimidyl, the 5-benzothiazolyl, purine radicals, the 2-benzimidazolyl-, the 5-indyl, the 1-isoquinolyl, the 5-isoquinolyl, the 2-quinoxalinyl, the 5-quinoxalinyl, 3-quinolyl and 6-quinolyl.
Term " high alkyl " refers to the alkyl as alkyl as used herein.
As used herein, term " unanimity " refers to identical two or more sequences or subsequence.In addition, as used herein, term " in fact consistent " refers to when through comparison window or as use comparison algorithm or by comparing and when comparing maximum correspondence, have two or more sequences of the identical sequential cells of certain percentage in the designated area of manual comparison and visual inspection measurement.Only for instance, if sequential cells is about 60% unanimity, about 65% unanimity, about 70% unanimity, about 75% unanimity, about 80% unanimity, about 85% unanimity, about 90% consistent or about 95% unanimity on the designated area, so two or more sequences can be " consistent in fact ".These per-cents are described two or more sequences " consistence per-cent ".The consistence of sequence can be present on the zone that length is at least about 75-100 sequential cells, length is on the zone of about 50 sequential cells, or on (if specifying) whole sequence.This definition also relates to the complementary sequence of cycle tests.Only for instance, when amino-acid residue is identical, two or more peptide sequences are consistent, if and amino-acid residue is about 60% unanimity, about 65% unanimity, about 70% unanimity, about 75% unanimity, about 80% unanimity, about 85% unanimity, about 90% consistent or about 95% unanimity on the designated area, so two or more peptide sequences are " consistent in fact ".Consistence can be present in that length is at least about on 75-100 the amino acid whose zone, length is on about 50 amino acid whose zones, or on the whole sequence of (if specifying) peptide sequence.In addition, only for instance, when the nucleic acid residue is identical, two or more polynucleotide sequences are consistent, if and the nucleic acid residue is about 60% unanimity, about 65% unanimity, about 70% unanimity, about 75% unanimity, about 80% unanimity, about 85% unanimity, about 90% consistent or about 95% unanimity on the designated area, so two or more polynucleotide sequences are " consistent in fact ".Consistence can be present on the zone that length is at least about 75-100 nucleic acid, length is on the zone of about 50 nucleic acid, or on the whole sequence of (if specifying) polynucleotide sequence.
About sequence relatively, a common sequence is served as the reference sequences that cycle tests compares with it.When using sequence comparison algorithm, in cycle tests and reference sequences input computer, specify the subsequence coordinate, (in case of necessity) and specified sequence algorithm routine parameter.The default program parameter can be used, maybe alternate parameter can be specified.Sequence comparison algorithm calculates the sequence identity per-cent of cycle tests with respect to reference sequences based on program parameter subsequently.
As used herein, term " immunogenicity " refers to the antibody that responds with medicine to throwing.Can use the quantitative and qualitative test that is used for the anti-non-natural amino acid polypeptides antibody of biological fluid to obtain at the immunogenicity of therapeutic non-natural amino acid polypeptides.These calibratings are including (but not limited to) radioimmunoassay (RIA), enzyme linked immunological absorption calibrating (ELISA), electrochemiluminescent immunoassay calibrating (LIA) and fluorescence immunoassay calibrating (FIA).At the immunogenic analysis of therapeutic non-natural amino acid polypeptides comprise relatively throw with the therapeutic non-natural amino acid polypeptides after antibody response and the antibody response behind throwing and the therapeutic natural amino acid polypeptide.
As used herein, term " intercalating agent " (also being known as " insertion group ") refers to the intramolecularly space that can insert molecule or the chemical in the intermolecular space between the molecule.Only for instance, intercalating agent or insertion group can be the molecule in the accumulation base of inserting the dna double spiral.
As used herein, term " separation " never refers to and to separate in the component of paying close attention to and shift out the component of being paid close attention to.Separated material can be drying or partial desiccation state, or is the solution form, and it is including (but not limited to) the aqueous solution.Separated component can be the part that homogeneous state or separated component can be the medical composition that comprises extra pharmaceutically acceptable supporting agent and/or vehicle.But purity and uniformity operational analysis chemical technology are measured, and wherein said technology is including (but not limited to) polyacrylamide gel electrophoresis or high performance liquid chromatography.In addition, when the component of being paid close attention to through separating and during for the essential substance that exists in the preparation, described component is described as in this article through purifying in fact.As used herein, term " purifying " refer at least 85% pure, at least 90% pure, at least 95% pure, at least 99% or the purer component of being paid close attention to.Only for instance, when nucleic acid or protein do not contain at least some cellular components associating with it under native state, or nucleic acid or protein simmer down to has been than it in vivo or during the high level of the concentration that in vitro produces, these nucleic acid or protein are " separated ".Equally, for example, when separating with the open reading frame of the gene that is positioned at the gene outside and coded protein but not is paid close attention to, described gene is separated.
As used herein, term " mark " refers to incorporate in the compound and be easy to and detects, and can detect and/or monitor the material of its physical distribution by this.
As used herein, term " binding " refers to key or the chemical part that functional group and the chemical reaction between another molecule by connexon form.These keys can be including (but not limited to) covalent linkage and non covalent bond, and these chemical parts can be including (but not limited to) ester, carbonic ether, imines, phosphoric acid ester, hydrazone, acetal, ortho ester, peptide bond and oligonucleotide key.To mean described binding stable in fact and do not react with water (it is including (but not limited to) under physiological condition) under the applicable pH value long-term (perhaps even indefinitely) in water for stable binding in the hydrolysis.Unstable or degradable binding means described binding and can degrade in the water or the aqueous solution (comprising for example blood) in the hydrolysis.Unstable or the degradable binding of enzymatic means described binding can be by one or more than one enzyme liberating.Only for instance, PEG and related polymer can be included in the main polymer chain or the degradable binding in the connexon group between one or more terminal functional groups of main polymer chain and polymer molecule.These degradable bindings are including (but not limited to) the ester bond that is formed by the pure radical reaction on PEG carboxylic acid or activated PEG carboxylic acid and the biologically active agent, wherein these ester groups usually under physiological condition hydrolysis with release bioactive agent.Other hydrolysis degradable bindings are including (but not limited to) carbonic acid ester bond; The imine linkage that forms by amine and aldehyde reaction; By the pure phosphoric acid ester bond that forms with phosphate-based reaction; Hydrazone key as the reaction product of hydrazides and aldehyde; As the acetal bonds of aldehyde with the reaction product of alcohol; As the original acid ester key of formate with the reaction product of alcohol; The peptide bond that forms by the carboxyl of amino (including (but not limited to) on end) and peptide such as the polymkeric substance of PEG; And the oligonucleotide key that forms by 5 ' hydroxyl of phosphoramidite base (including (but not limited to) on the end of polymkeric substance) and oligonucleotide.
As used herein, term " substratum " refers to and is used to grow and collecting cell and/or by any substratum of these cell expressings and/or excretory product.These " substratum " are including (but not limited to) solution, solid, the semi-solid rigid carrier that maybe can support or contain any host cell, described host cell is including (for example) bacterial host cell, yeast host cell, insect host cell, plant host cell, eukaryotic host cell, mammalian host cell, Chinese hamster ovary celI, prokaryotic host cell, intestinal bacteria (E.coli) or pseudomonas (Pseudomonas) host cell, and cell content.These " substratum " have grown in polypeptide including (but not limited to) host cell wherein and have secreted in substratum wherein, and it is included in the substratum before or after the propagation step.These " substratum " also including (but not limited to) damping fluid that contains the host cell lysate or reagent, for example dissolving of polypeptide that produces in the cell and host cell or division are to discharge polypeptide.
As used herein, term " metabolite " refers to the derivative of compound (for example natural amino acid polypeptide, non-natural amino acid polypeptides, modified natural amino acid polypeptide or modified non-natural amino acid polypeptides), and it is to form when compound (for example natural amino acid polypeptide, non-natural amino acid polypeptides, modified natural amino acid polypeptide or modified non-natural amino acid polypeptides) metabolism.Term " medicinal activity metabolite " or " active metabolite " refer to the biologically active derivatives of compound (for example natural amino acid polypeptide, non-natural amino acid polypeptides, modified natural amino acid polypeptide or modified non-natural amino acid polypeptides), and it is to form when this compound (for example natural amino acid polypeptide, non-natural amino acid polypeptides, modified natural amino acid polypeptide or modified non-natural amino acid polypeptides) metabolism.
As used herein, term " metabolism " refer to predetermined substance by the process of organism transform and.These processes are including (but not limited to) hydrolysis reaction with by enzymatic reaction.Can be about metabolic other information available from ThePharmacological Basis of Therapeutics, the 9th edition, McGraw-Hill (1996).Only for instance, the natural amino acid polypeptide, non-natural amino acid polypeptides, the metabolite of modified natural amino acid polypeptide or modified non-natural amino acid polypeptides can pass through the natural amino acid polypeptide, non-natural amino acid polypeptides, modified natural amino acid polypeptide or modified non-natural amino acid polypeptides throwing and host and analysis are differentiated from host's tissue samples, or are passed through the natural amino acid polypeptide, non-natural amino acid polypeptides, modified natural amino acid polypeptide or modified non-natural amino acid polypeptides are in vitro cultivated and are analyzed the gained compound and differentiate with liver cell.
As used herein, term " metal chelator " refers to the molecule that forms metal complex with metal ion.For instance, these molecules can form two or more coordinate bonds and can form ring structure with central metallic ions.
As used herein, term " containing metal part " refers to the group that contains metal ion, atom or particle.These parts are including (but not limited to) cis-platinum (cisplatin), chelated metal ions (such as nickel, iron and platinum), and metal nanoparticle (such as nickel, iron and platinum).
As used herein, term " is combined with the part of heavy atom " and refers to the ionic group that is combined with heavier than carbon usually atom.These ions or atom are including (but not limited to) silicon, tungsten, gold, lead and uranium.
As used herein, term " modified " refers to the change of existence to natural amino acid, alpha-non-natural amino acid, natural amino acid polypeptide or non-natural amino acid polypeptides.These changes or modification can be passed through the synthetic back modification of natural amino acid, alpha-non-natural amino acid, natural amino acid polypeptide or non-natural amino acid polypeptides, or obtain by the common translation modification or the posttranslational modification of natural amino acid, alpha-non-natural amino acid, natural amino acid polypeptide or non-natural amino acid polypeptides.It is modified according to circumstances that form " modified or not modified " means natural amino acid, alpha-non-natural amino acid, natural amino acid polypeptide or the non-natural amino acid polypeptides discussed, and promptly the natural amino acid of being discussed, alpha-non-natural amino acid, natural amino acid polypeptide or non-natural amino acid polypeptides can be modified or not modified.
As used herein, term " serum half-life through regulating " refers to just variation or negative change of modified bioactive molecules with respect to its non-circulating half-life without modified forms.For instance, modified bioactive molecules is including (but not limited to) natural amino acid, alpha-non-natural amino acid, natural amino acid polypeptide or non-natural amino acid polypeptides.For instance, serum half-life is to measure by the concentration that a plurality of time points behind throwing and bioactive molecules or modified bioactive molecules are got blood sample and measured the sort of molecule in each sample.The dependency of serum-concentration and time makes can calculate serum half-life.For instance, the serum half-life through regulating can be serum half-life to be increased, and it can make it possible to the dosage regimen that obtains improveing or avoid toxic effect.This serum increase can be at least about twice, at least about three times, at least about five times, or at least about ten times.The limiting examples of the method that the assessment serum half-life increases provides in example 88-92.This method can be used for assessing the serum half-life of any polypeptide.
As used herein, term " the treatment transformation period through regulating " refers to the modified bioactive molecules of treatment significant quantity with respect to its just variation or negative variation without the transformation period of modified forms.For instance, modified bioactive molecules is including (but not limited to) natural amino acid, alpha-non-natural amino acid, natural amino acid polypeptide or non-natural amino acid polypeptides.For instance, the treatment transformation period measures by the pharmacokinetics and/or the pharmacodynamic properties of a plurality of point in time measurement molecules after dispensing.The treatment transformation period that increases can make it possible to obtain specific useful dosage regimen, specific useful total dose, or avoids improper effect.For instance, the treatment transformation period that increases can or reduce by the increase of another parameter that combine increase or minimizing, not modified molecule of the effectiveness that increases, modified molecule and its target or mechanism of action, or molecule by enzyme (such as, only for instance, proteolytic enzyme) decompose increase or minimizing generation.The limiting examples of the method that the assessment treatment transformation period increases provides in example 88-92.This method can be used for assessing the treatment transformation period of any polypeptide.
As used herein, term " nanoparticle " refers to and has the particle of about 500nm to the granularity between about 1nm.
As used herein, to refer to the mol ratio of the compound that participates in chemical reaction be about 0.75 to about 1.5 to term " near stoichiometry ".
As used herein, term " non-eukaryote " refers to non-most eukaryotes.For instance, non-most eukaryotes can belong to eubacterium (Eubacteria), and (it is including (but not limited to) intestinal bacteria (Escherichia coli), thermus thermophilus (Thermus thermophilus) or bacstearothermophilus (Bacillus stearothermophilus), Pseudomonas fluorescens (Pseudomonas fluorescens), Pseudomonas aeruginosa (Pseudomonas aeruginosa), pseudomonas putida (Pseudomonas putida)), territory on the phylogenetics, or Archimycetes (Archaea), it is including (but not limited to) Methanococcus jannaschii (Methanococcus jannaschii), hot autotrophic methane bacteria (Methanobacterium thermoautotrophicum), the ancient green-ball bacterium (Archaeoglobus fulgidus) of glimmering, strong red-hot coccus (Pyrococcus furiosus), pick Yue Shi hot-bulb bacterium (Pyrococcus horikoshii), the quick hot bacterium of gas (Aeuropyrum pernix), or have a liking for salt bacillus (Halobacterium), have a liking for the richly endowed bacterium of salt (Haloferaxvolcanii) and have a liking for salt bacillus specie NRC-1 such as Wo Shi, or the territory on the phylogenetics.
" alpha-non-natural amino acid " refers to a kind of amino acid that is not in 20 kinds of common amino acids or burnt Methionin or the selenocystein.Other terms that can use with term " alpha-non-natural amino acid " synonym be " non-naturally encoded amino acids ", " non-natural amino acid ", " there is amino acid in non-natural ", with and variously have hyphen and not with the form of hyphen.Term " alpha-non-natural amino acid " is including (but not limited to) by modifying (including (but not limited to) 20 kinds of common amino acids or burnt Methionin and selenocystein) the natural existence of natural coded amino acid but be not to incorporate amino acid in the polypeptide chain of growth into by translation mixture self.Do not exist amino acid whose example including (but not limited to) N-acetyl glucose amino-L-Serine, N-acetyl glucose amino-L-Threonine and O-phosphate tyrosine for the natural of natural coding.In addition, term " alpha-non-natural amino acid " exists including (but not limited to) the preface non-natural and can maybe can pass through to modify the amino acid that alpha-non-natural amino acid obtains by synthetic acquisition.
As used herein, term " nucleic acid " refer to deoxynucleotide, deoxynucleoside, nucleosides or Nucleotide with and the polymkeric substance of strand or double chain form.Only for instance, these nucleic acid and nucleic acid polymers be including (but not limited to) the analogue of (i) natural nucleotide, and it has and similarly combines character with reference nucleic acid and to be similar to the natural mode metabolism that has Nucleotide; (ii) oligonucleotide analogs, its analogue (thiophosphatephosphorothioate, phosphoramidate, with and analogue) including (but not limited to) the DNA that uses in PNA (peptidyl nucleic acid), the antisense technology; (iii) its variant modified through conservative property (replacing) and complementary sequence and the sequence that spells out including (but not limited to) degenerate codon.For instance, degenerate codon replaces and can realize by the sequence of the 3rd position through mixing the replacement of base and/or Hypoxanthine deoxyriboside residue that produces one of them or selected more than one (or all) codons (people such as Batzer, Nucleic Acid Res.19:5081 (1991); People such as Ohtsuka, J.Biol.Chem.260:2605-2608 (1985); And people such as Rossolini, Mol.Cell.Probes 8:91-98 (1994)).
As used herein, term " oxygenant " refers to the compound or the material that can remove electronics from oxidized compound.For instance, oxygenant comprises (but being not limited to) Sleep-promoting factor B, Gelucystine, cystamine, oxidized form dithiothreitol (DTT), oxidized form erythritol (oxidized erythreitol), and oxygen.Multiple oxygenant is applicable in the method and composition of describing herein.
As used herein, term " pharmaceutically acceptable " refers to the material including (but not limited to) salt, supporting agent or thinner, it does not eliminate the biological activity or the character of compound, and it is nontoxic relatively, that is, described material can be thrown with individual and do not cause improper biological effect or be contained in any one interaction in the component of composition wherein in harmful mode with it.
As used herein, with mark its molecule with affinity is formed the mark of the group of binding after term " photoaffinity mark " refers to and has in being exposed to light.Only for instance, this binding can be covalently or non-covalently binding.
As used herein, term " the light cage covers part " refers under specific wavelength after the irradiation covalently or non-covalently the group in conjunction with other lewis' acids.
As used herein, the group of fracture after term " but photodestruciton group " refers in being exposed to light.
As used herein, term " photocrosslinking agent " refers to and comprises that two or more can react and form the covalently or non-covalently functional group's of binding compound afterwards with two or more monomers or polymerizable molecular in being exposed to light.
As used herein, term " but photoisomerization part " refers to the group that wherein becomes another kind of isomeric form after rayed from a kind of isomeric form.
As used herein, term " polyalkylene glycol " refers to linearity or branch polyhydroxyl polyether polyvalent alcohol.These polyalkylene glycols including (but not limited to) polyoxyethylene glycol, polypropylene glycol, polytetramethylene glycol with and derivative.Other exemplary embodiments are listed in (for example) commercial supplier catalogue, such as the catalogue " polyoxyethylene glycol and the derivative that is used for biomedical applications " (" Polyethylene Glycol and Derivatives forBiomedical Applications ") (2001) of Shearwater Corporation.Only for instance, these polyhydroxyl polyether polyvalent alcohols have about 0.1kDa to the molecular-weight average between about 100kDa.For instance, these polyhydroxyl polyether polyvalent alcohols are including (but not limited to) about 100Da and about 100,000Da or higher between.The molecular weight of polymkeric substance can be at about 100Da and about 100, and between the 000Da, it is including (but not limited to) 100,000Da, 95,000Da, 90,000Da, 85,000Da, 80,000Da, 75,000Da, 70,000Da, 65,000Da, 60,000Da, 55,000Da, 50,000Da, 45,000Da, 40,000Da, 35,000Da, 30,000Da, 25,000Da, 20,000Da, 15,000Da, 10,000Da, 9,000Da, 8,000Da, 7,000Da, 6,000Da, 5,000Da, 4,000Da, 3,000Da, 2,000Da, 1,000Da, 900Da, 800Da, 700Da, 600Da, 500Da, 400Da, 300Da, 200Da and 100Da.In certain embodiments, the molecular weight of polymkeric substance is in about 100Da and 50, between the 000Da.In certain embodiments, the molecular weight of polymkeric substance is in about 100Da and 40, between the 000Da.In certain embodiments, the molecular weight of polymkeric substance is about 1, and 000Da and 40 is between the 000Da.In certain embodiments, the molecular weight of polymkeric substance is about 5, and 000Da and 40 is between the 000Da.In certain embodiments, the molecular weight of polymkeric substance is about 10, and 000Da and 40 is between the 000Da.In certain embodiments, peg molecule is a branched polymers.The molecular weight of side chain PEG can be about 1,000Da and about 100, and between the 000Da, it is including (but not limited to) 100,000Da, 95,000Da, 90,000Da, 85,000Da, 80,000Da, 75,000Da, 70,000Da, 65,000Da, 60,000Da, 55,000Da, 50,000Da, 45,000Da, 40,000Da, 35,000Da, 30,000Da, 25,000Da, 20,000Da, 15,000Da, 10,000Da, 9,000Da, 8,000Da, 7,000Da, 6,000Da, 5,000Da, 4,000Da, 3,000Da, 2,000Da and 1,000Da.In certain embodiments, the molecular weight of side chain PEG is about 1, and 000Da and 50 is between the 000Da.In certain embodiments, the molecular weight of side chain PEG is about 1, and 000Da and 40 is between the 000Da.In certain embodiments, the molecular weight of side chain PEG is about 5, and 000Da and 40 is between the 000Da.In certain embodiments, the molecular weight of side chain PEG is about 5, and 000Da and 20 is between the 000Da.
As used herein, term " polymer " " refer to by repeating the molecule that subunit is formed.These molecules are including (but not limited to) polypeptide, polynucleotide or polysaccharide or polyalkylene glycol.
Term " polypeptide ", " peptide " and " protein " are used interchangeably the polymkeric substance of mentioning amino-acid residue in this article.That is, the description that is directed to polypeptide is equally applicable to the description of peptide and to proteinic description, and vice versa.These terms are applicable to that the natural aminoacid polymers and one of them or more than one amino-acid residue of existing is the aminoacid polymers of alpha-non-natural amino acid.In addition, these " polypeptide ", " peptide " and " protein " comprise the amino acid chain of any length, and it comprises full length protein, and wherein amino-acid residue is by covalency peptide bond binding.
Term " posttranslational modification " refers at natural or alpha-non-natural amino acid and has translated the described amino acid whose any modification that takes place after incorporating in the polypeptide chain.These modify including (but not limited to) translation altogether in vivo modify, translation is in vitro modified after in vitro modifying and in vivo modifying and translate after (such as in cell free translation system), the translation altogether.
As used herein, term " prodrug " or " pharmaceutically acceptable prodrug " refer to the medicament that or in vitro changes parent drug in vivo into, wherein it does not eliminate the biological activity or the character of medicine, and it is nontoxic relatively, that is, described material can be thrown with individual and do not cause improper biological effect or be contained in any one interaction in the component of composition wherein in harmful mode with it.Prodrug is generally the medicine precursor, and throwing and person under inspection and after with post-absorption, it changes into by some process (such as being changed by pathways metabolism) and has activity or have more highly active material.Some prodrugs have the chemical group that is present on the prodrug, and it makes that the prodrug activity is less and/or gives medicine dissolution or some other character.In case chemical group cracking and/or modified, active medicine just produces from prodrug.Prodrug changes active medicine in vivo by enzymatic reaction or non-enzymatic reaction.Prodrug can provide the physicochemical property of improvement, such as the pharmacological agent value of better solvability, enhanced delivery characteristics (such as selectively targeted specific cells, tissue, organ or ligand) and improvement.The benefit of these prodrugs including (but not limited to): (i) compare with parent drug, be easy to the dispensing; (ii) prodrug can pass through the oral administration medicine supplying biological utilisation, and parent cannot; And (iii) compare with parent drug, prodrug also can have the solvability in medical composition of improvement.Prodrug comprises the derivative of the nonactive or active reduction of the pharmacology of active medicine.Prodrug can be through being designed for the amount of regulating medicine or bioactive molecules, and it arrives required site of action by the character (such as physicochemical property, biological medicine character or pharmacokinetic property) of handling medicine.The example of prodrug (without stint) can be with the form of ester (" prodrug ") and throws and the non-natural amino acid polypeptides that is beneficial to pass the cytolemma transmission, wherein water-soluble harmful to mobility, but in a single day water-soluble therein is useful cell interior, and it is hydrolyzed to carboxylic acid (active entity) with regard to metabolism subsequently.Prodrug can be through being designed to reversible medicaments derivative, to transmit with the medicine that strengthens to site-specific sex organization as properties-correcting agent.
As used herein, term " prevention significant quantity " refers to the amount that prophylactically is applied to the composition that contains at least a non-natural amino acid polypeptides or at least a modified non-natural amino acid polypeptides among the patient, and it can alleviate the symptom of one or more diseases for the treatment of, symptom or illness to a certain extent.In these prophylactic application, described amount is decided by patient's healthy state, body weight and similar factor thereof.Believe that fully the those skilled in the art can determine these prevention significant quantities by normal experiment (increasing clinical trial including (but not limited to) dosage).
As used herein, term " through protection " refers to exist and prevent " protecting group " or the part that the chemical reactivity functional group reacts under some reaction conditions.Protecting group can change according to the type of the chemically reactive group of being protected.Only for instance, if (i) chemically reactive group is amine or hydrazides, protecting group can be selected from tertbutyloxycarbonyl (t-Boc) and 9-fluorenyl methoxy carbonyl (Fmoc) so; If (ii) chemically reactive group is a mercaptan, protecting group can be adjacent pyridyl disulfide so; And if (iii) chemically reactive group is carboxylic acid (such as butyric acid or propionic acid) or hydroxyl, protecting group can be benzyl or alkyl so, such as methyl, ethyl or the tertiary butyl.
Only for instance, obstruction/protecting group (blocking/protecting group) also can be selected from:
Figure A20058004432800631
In addition, protecting group is including (but not limited to) comprising known other protecting groups in group (such as Nvoc and MeNvoc) to photo-labile and the affiliated field.Other protecting groups are described in Greene and Wuts, Protective Groups inOrganic Synthesis, the 3rd edition, John Wiley ﹠amp; Sons, New York, NY, in 1999, it is all to be incorporated herein by reference.
As used herein, term " radioactive segment " refers to the spontaneous group that sends nuclear radiation (such as alpha-particle, beta-particle or γ particle) of its nuclear; Wherein, alpha-particle is a helion, and beta-particle is an electronics, and the γ particle is a high-energy photon.
As used herein, term " reactive compounds " refers under proper condition the compound to another atom, molecule or responding property of compound.
Term " recombinant host cell " (also being known as " host cell ") refers to the cell that comprises exogenous polynucleotide, wherein is used for exogenous polynucleotide is inserted the method for cell including (but not limited to) known additive method in the field under direct absorption, transduction, f-pairing or the generation recombinant host cell.Only for instance, described exogenous polynucleotide can be nonconformity carrier (including (but not limited to) plasmid), maybe can be integrated in the host genome.
As used herein, term " redox active agent " refers to oxidation or reduces another molecule, and redox active agent becomes and is reduced or the molecule of oxidation by this.The example of redox active agent is including (but not limited to) ferrocene, quinone, Ru2+/3+ complex compound, Co2+/3+ complex compound and Os2+/3+ complex compound.
As used herein, term " reductive agent " refers to the compound or the material that can add electronics to institute's reductive compound.For instance, reductive agent is including (but not limited to) dithiothreitol (DTT) (DTT), 2 mercapto ethanol, dithioerythritol, halfcystine, cysteamine (2-aminoothyl mercaptan), and reduced glutathion.Can use these reductive agents (only for instance) sulfydryl remained reduced state and to reduce intramolecularly or intermolecular disulfide bond.
As used herein " more folding " describe will be improper folding or folded state do not change any process, reaction or the method for nature or suitable folded conformation into.Only for instance, the folding again polypeptide that will contain disulfide linkage by improper folding or not folded state change into about the nature of disulfide linkage or suitable folded conformation.These polypeptide that contain disulfide linkage can be natural amino acid polypeptide or non-natural amino acid polypeptides.
As used herein, term " resin " refers to high molecular insoluble polymer bead.Only for instance, these beads can be used as and are used for solid-phase peptide synthetic carrier, or before purifying the site of attachment molecules.
As used herein, term " carbohydrate " refers to a series of carbohydrate, and it is including (but not limited to) sugar, monose, oligosaccharides and polysaccharide.
As used herein, term " security " or " security overview " refer to for the number of times of throwing with medicine and may throw and relevant side effect with medicine.For instance, with throw with repeatedly and only gentle real estate becomes side effect or the medicine that has no side effect is called and has outstanding security overview.The limiting examples of the method for assessment security overview provides in example 92.This method can be used for assessing the security overview of any polypeptide.
As used herein, phrase " selective cross " or " specific hybrid " refer to when specific nucleotide sequence is present in the complex mixture (including (but not limited to) total cell or storehouse DNA or RNA), the combination of molecule and described sequence under stringent hybridization condition, become pair or hybridize.
As used herein, term " spin labeling " refers to and can detect and can be connected to containing on another molecule by ESR spectrum and represent the atom of unpaired electron spin or the molecule of atomic group (promptly stablizing the paramagnetic group).These spin labeling molecules are including (but not limited to) nitroxyl and nitroxide, and can be single spin labeling or dual spin labeling.
As used herein, to refer to the mol ratio of the compound that participates in chemical reaction be about 0.9 to about 1.1 to term " stoichiometry ".
As used herein, term " class stoichiometry " refers to after reaction conditions changes or becomes stoichiometry in the presence of the additive or approaching stoichiometric chemical reaction.The variation of these reaction conditionss raises including (but not limited to) temperature or the pH value changes.These additives contain (but being not limited to) promotor.
Phrase " stringent hybridization condition " refers under the condition of low ion concns and high-temperature, the hybridization of the sequence of DNA, RNA, PNA or other nucleic acid mimics or its combination.For instance, under stringent condition probe can with its target subsequences hybridization in the complex mixture (including (but not limited to) total cell or storehouse DNA or RNA) of nucleic acid, and not with complex mixture in other sequence hybridizations.Stringent condition be sequence dependent and can be different under different situations.For instance, longer sequence specific hybrid under comparatively high temps.Stringent hybridization condition including (but not limited to): (i) determining that the heat fusion joint (Tm) than specificity sequence hangs down about 5 ℃-10 ℃ under ionic concn and the pH value; Be that about 0.01M is to about 1.0M to 8.3 times salt concn of about pH (ii) at about pH 7.0, and for short probe (including (but not limited to) 10 to 50 Nucleotide), temperature is at least about 30 ℃, and is at least about 60 ℃ for long probe (including (but not limited to) more than 50 Nucleotide) temperature; (iii) add destabilizing agent, it is including (but not limited to) methane amide; (iv) 50% methane amide, 5X SSC and 1% SDS cultivate down at 42 ℃, or 5X SSC, 1% SDS cultivates down at 65 ℃, wherein under 65 ℃ in 0.2X SSC and 0.1% SDS between the washing about 5 minutes to about 120 minutes.Only for instance, the detection of selectivity or specific hybrid is at least the twice of background including (but not limited to) positive signal.Detailed guide about nucleic acid hybridization sees Tijssen, Laboratory Techniques in Biochemistry and MolecularBiology-Hybridization with Nucleic Probes is in " Overview of principles of hybridization and thestrategy of nucleic acid assays " (1993).
As used herein, term " person under inspection " refers to the animal as treatment, observation or experimental subjects.Only for instance, the person under inspection can be (but being not limited to) Mammals, and it is including (but not limited to) the mankind.
As used herein, term " purified in fact " refer to can be in fact or be substantially free of before purifying, follow usually the component paid close attention to or with the component of being paid close attention to of other components of the component interaction of being paid close attention to.Only for instance, when the preparation of the component of being paid close attention to contain less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2% or during less than the pollution components of about 1% (with dry weight basis), the component of being paid close attention to can be " purified in fact ".Therefore, the component of being paid close attention to of " purified in fact " can have about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or higher purity level.Only for instance, under the situation of natural amino acid polypeptide that reorganization produces or non-natural amino acid polypeptides, natural amino acid polypeptide or non-natural amino acid polypeptides can be from n cell or host cell purifying.For instance, when preparation contain less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2% or during less than the pollution substance of about 1% (with dry weight basis), the preparation of natural amino acid polypeptide or non-natural amino acid polypeptides can be " purified in fact ".For instance, when natural amino acid polypeptide or non-natural amino acid polypeptides are produced by the host cell reorganization, natural amino acid polypeptide or non-natural amino acid polypeptides can dry cell weights about 30%, about 25%, about 20%, about 15%, about 10%, about 5%, about 4%, about 3%, about 2% about 1% or amount still less exist.For instance, when natural amino acid polypeptide or non-natural amino acid polypeptides were produced by the host cell reorganization, about 5g/L, about 4g/L, about 3g/L, about 2g/L, about 1g/L, about 750mg/L, about 500mg/L, about 250mg/L, about 100mg/L, about 50mg/L, about 10mg/L or about 1mg/L or amount still less that natural amino acid polypeptide or non-natural amino acid polypeptides can dry cell weights were present in the substratum.For instance, the natural amino acid polypeptide of " purified in fact " or non-natural amino acid polypeptides can have about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or the higher purity level of measuring as by proper method (it is including (but not limited to) SDS/PAGE analysis, RP-HPLC, SEC and capillary electrophoresis).
Term " substituting group " (also being known as " non-interfering substituent ") refers to the group that can be used for another group on the displacer molecule.These groups are including (but not limited to) halogen, C 1-C 10Alkyl, C 2-C 10Thiazolinyl, C 2-C 10Alkynyl, C 1-C 10Alkoxyl group, C 5-C 12Aralkyl, C 3-C 12Cycloalkyl, C 4-C 12Cycloalkenyl group, phenyl, be substituted phenyl, tolyl, xylyl, xenyl, C 2-C 12Alkoxyalkyl, C 5-C 12Alkoxy aryl, C 5-C 12Aryloxy alkyl, C 7-C 12Oxygen Ji Fangji, C 1-C 6Alkyl sulphinyl, C 1-C 10Alkyl sulphonyl ,-(CH 2) m-O-(C 1-C 10Alkyl) (wherein m is 1 to 8), aryl, be substituted aryl, be substituted alkoxyl group, fluoroalkyl, heterocyclic radical, be substituted heterocyclic radical, 4-nitro alkyl ,-NO 2,-CN ,-NRC (O)-(C 1-C 10Alkyl) ,-C (O)-(C 1-C 10Alkyl), C 2-C 10Alkylthio alkyl ,-C (O) O-(C 1-C 10Alkyl) ,-OH ,-SO 2,=S ,-COOH ,-NR 2, carbonyl ,-C (O)-(C 1-C 10Alkyl)-CF 3,-C (O)-CF 3,-C (O) NR 2,-(C 1-C 10Aryl)-S-(C 6-C 10Aryl) ,-C (O)-(C 6-C 10Aryl) ,-(CH 2) m-O-(CH 2) m-O-(C 1-C 10Alkyl) (wherein m respectively does for oneself 1 to 8) ,-C (O) NR 2,-C (S) NR 2,-SO 2NR 2,-NRC (O) NR 2,-NRC (S) NR 2, its salt, with and similar group.Each R group in the previous list is including (but not limited to) H, alkyl or be substituted alkyl, aryl or be substituted aryl, or alkaryl.When substituting group was specified by its conventional chemical formula of writing from left to right, it is contained equally can be by writing the chemically identical substituting group that structure produces from right to left, for example, and-CH 2O-equals-OCH 2-.
Only for instance, the substituting group of alkyl and assorted alkyl (comprising those groups that are known as alkylidene group, thiazolinyl, inferior assorted alkyl, assorted thiazolinyl, alkynyl, cycloalkyl, Heterocyclylalkyl, cycloalkenyl group and heterocycloalkenyl) including (but not limited to) :-OR ,=O ,=NR ,=N-OR ,-NR 2,-SR ,-halogen ,-SiR 3,-OC (O) R ,-C (O) R ,-CO 2R ,-CONR 2,-OC (O) NR 2,-NRC (O) R ,-NRC (O) NR 2,-NR (O) 2R ,-NR-C (NR 2)=NR ,-S (O) R ,-S (O) 2R ,-S (O) 2NR 2,-NRSO 2R ,-CN and-NO 2Each R group in the previous list is including (but not limited to) hydrogen, the assorted alkyl that is substituted or is unsubstituted, the aryl (including (but not limited to) the aryl that replaces through 1-3 halogen) that is substituted or is unsubstituted, alkyl, alkoxyl group or thioalkoxy group or the aralkyl that is substituted or is unsubstituted.When two R groups were connected on the same nitrogen-atoms, it can make up to form 5 Yuans rings, 6 Yuans rings or 7 Yuans rings with nitrogen-atoms.For instance ,-NR 2Mean including (but not limited to) 1-pyrrolidyl and 4-morpholinyl.
For instance, the substituting group of aryl and heteroaryl including (but not limited to)-OR ,=O ,=NR ,=N-OR ,-NR 2,-SR ,-halogen ,-SiR 3,-OC (O) R ,-C (O) R ,-CO 2R ,-CONR 2,-OC (O) NR 2,-NRC (O) R ,-NRC (O) NR 2,-NR (O) 2R ,-NR-C (NR 2)=NR ,-S (O) R ,-S (O) 2R ,-S (O) 2NR 2,-NRSO 2R ,-CN ,-NO 2,-R ,-N 3,-CH (Ph) 2, fluorine (C 1-C 4) alkoxyl group and fluorine (C 1-C 4) alkyl, number is in the scope of the sum of zero open valence state on the aromatic ring system; And wherein each the R group in the previous list is including (but not limited to) hydrogen, alkyl, assorted alkyl, aryl and heteroaryl.
As used herein, term " treatment significant quantity " refers to the amount of the composition that contains at least a non-natural amino acid polypeptides and/or at least a modified non-natural amino acid polypeptides of throwing with the patient who has suffered from disease, symptom or illness, being enough to cure or stoping or alleviate to a certain extent to small part the symptom of at least a disease for the treatment of, illness or symptom.The effect of these compositions is decided on following condition, and it is including (but not limited to) the seriousness of disease, illness or symptom and process, previous treatment, patient's state of health with to the reaction of medicine, and attending doctor's judgement.Only for instance, the treatment significant quantity can be determined by normal experiment (increasing clinical trial including (but not limited to) dosage).
As used herein, term " thioalkoxy group " refers to the sulfur-bearing alkyl by Sauerstoffatom and molecular binding.
Term " heat fusion joint " or Tm are 50% and temperature (under definite ionic concn, pH value and the nucleic acid concentration) hybridization of target complementary probe and target sequence under equilibrium state.
As used herein, term " toxicity part " refers to and can damage or dead compound.
As used herein, term " treatment " comprises alleviation, relaxes or improves disease or symptom symptom, prevent other symptoms, improve or the potential metabolic disease of prevention symptom because of, suppress disease or symptom, for example, stop the development of disease or symptom, palliate a disease or symptom, make disease or symptom return, alleviate the symptom that causes by disease or symptom, or stop the symptom of disease or symptom.Term " treatment " is including (but not limited to) preventative and/or therapeutic treatment.
As used herein, term " water-soluble polymers " refers to any polymkeric substance that dissolves in the water-based solvent.These water-soluble polymerss are including (but not limited to) polyoxyethylene glycol, polyoxyethylene glycol propionic aldehyde, its single C 1-C 10Alkoxyl group or aryloxy derivative (are described in United States Patent (USP) the 5th, 252, in No. 714, it is to be incorporated herein by reference), mono methoxy-polyoxyethylene glycol, polyvinylpyrrolidone, polyvinyl alcohol, polyamino acid, the divinyl ether maleic anhydride, N-(2-hydroxypropyl)-Methacrylamide, dextran, glucan derivative (comprising T 500), polypropylene glycol, polyoxytrimethylene/ethylene oxide copolymer, the polyoxyethylene polyvalent alcohol, heparin, heparin fragment, polysaccharide, oligosaccharides, glycan, Mierocrystalline cellulose and derivatived cellulose (including (but not limited to) methylcellulose gum and carboxymethyl cellulose), serum albumin, starch and starch derivative, polypeptide, polyalkylene glycol with and derivative, the multipolymer of polyalkylene glycol and its derivative, the polyvinyl ethyl ether, and alpha-beta-poly-[(2-hydroxyethyl)-DL-l-asparagine, with and analogue or its mixture.Only for instance, the coupling of these water-soluble polymerss and natural amino acid polypeptide or non-natural polypeptide can produce change, it is including (but not limited to) with respect to not modified form, water-soluble increase, serum half-life increases or is regulated, the treatment transformation period increases or is regulated, biological usability increases, biological activity is regulated, prolong cycling time, immunogenicity is regulated, physics association characteristic is regulated, it is including (but not limited to) assembling and polymer formation, receptors bind changes, the combination that combines the collocation thing with one or more changes, and receptor dimerizationization or multimerization effect change.In addition, these water-soluble polymerss can have or can not have the biological activity of himself.
Unless otherwise instructed, mass spectrum, NMR, HPLC, protein chemistry, biological chemistry, recombinant DNA technology and the pharmacological ordinary method in the field otherwise under using.
The compound of Ti Chuing is (including (but not limited to) alpha-non-natural amino acid, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides herein, and the reagent that is used to prepare aforesaid compound) comprise through isotope-labeled compound, it is with identical with those compounds of narrating in the various formulas that propose herein and the structure, but one or more atoms replace through having the different atomic mass of the atomic mass found usually with occurring in nature or total mass number or the atom of total mass number.Can incorporate the isotropic substance that isotopic example in the compound of the present invention comprises hydrogen, carbon, nitrogen, oxygen, fluorine and chlorine into, respectively such as 2H, 3H, 13C, 14C, 15N, 18O, 17O, 35S, 15F, 36Cl.Herein some of Miao Shuing through isotope-labeled compound (for example such as 3H and 14The radio isotope of C is incorporated those compounds wherein into) be applicable to the calibrating of medicine and/or substrate tissue distribution.In addition, use (promptly such as deuterium 2H) the treatment advantage that can provide some to be produced by higher metabolic stability is provided isotropic substance, and for example in vivo the transformation period increases or the reduction of dosage demand.
Some compounds herein (including (but not limited to) alpha-non-natural amino acid, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides, and the reagent that is used to prepare aforesaid compound) have unsymmetrical carbon and can therefore exist with enantiomer or diastereomeric form.Based on its physical chemistry difference, can with the diastereo-isomerism mixture separation its indivedual diastereomers by currently known methods (for example, chromatogram and/or fractional crystallization).Can by by with suitable optically active compound (for example, alcohol) reaction changes the enantiomerism mixture into the diastereo-isomerism mixture, separate diastereomer and with indivedual diastereomers change (for example, hydrolysis) for corresponding pure enantiomer with stage enantiomer separation.All these isomer that comprise diastereomer, enantiomer and composition thereof all are considered the part of the composition of describing herein.
In extra or other embodiment, the compound of Miao Shuing (including (but not limited to) alpha-non-natural amino acid, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides, and the reagent that is used to prepare aforesaid compound) is to use with the form of prodrug herein.In extra or other embodiment, the compound of Miao Shuing is (including (but not limited to) alpha-non-natural amino acid, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides herein, and the reagent that is used to prepare aforesaid compound) throwing the organism metabolism afterwards that produces metabolite with needs, described metabolite is used to produce required effect subsequently, and it comprises required treatment effect.In other or extra embodiment be the active metabolite of alpha-non-natural amino acid and " modified or not modified " non-natural amino acid polypeptides.
Method of Miao Shuing and prescription comprise N-oxide compound, crystallized form (also being known as polymorphic form) or the pharmaceutically acceptable salt that uses alpha-non-natural amino acid, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides herein.In certain embodiments, alpha-non-natural amino acid, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides can tautomeric forms exist.All tautomers all are contained in herein in the category of the alpha-non-natural amino acid that proposes, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides.In addition, the alpha-non-natural amino acid of describing herein, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides can non-solvent compound form and with pharmaceutically acceptable solvent (such as water, ethanol with and analogue) solvate form thereof that forms exists.The solvate form thereof of the alpha-non-natural amino acid of Ti Chuing, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides also is considered as being exposed in herein herein.
Some compounds herein (including (but not limited to) alpha-non-natural amino acid, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides and the reagent that is used to prepare aforesaid compound) can some tautomeric forms exist.All these tautomeric forms all are considered the part of the composition of describing herein.Equally, for example all enols-ketone form of any compound herein (including (but not limited to) alpha-non-natural amino acid, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides and the reagent that is used to prepare aforesaid compound) all is considered the part of the composition of describing herein.
Some compounds herein (including (but not limited to) alpha-non-natural amino acid, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides and any one the reagent that is used to prepare aforesaid compound) have acidity and can with pharmaceutically acceptable salt forming cation.Some compounds herein (including (but not limited to) alpha-non-natural amino acid, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides and the reagent that is used to prepare aforesaid compound) can have alkalescence and therefore can form salt with pharmaceutically acceptable negatively charged ion.Comprise in the category of the composition that all these salt of disalt describe in this article and it can prepare by ordinary method.For instance, salt can prepare by in water-based, non-aqueous or part aqueous medium acid entity is contacted with alkaline entity.By using at least a salt that reclaims in the following technology: filter; With the non-solvent precipitation, then filter; Evaporating solvent; Or (under the situation of the aqueous solution) freeze-drying.
The acid proton that exists in the parent non-natural amino acid polypeptides is replaced through metal ion (for example alkalimetal ion, alkaline-earth metal ions or aluminum ion), or during with the organic bases coordination, can form the pharmaceutically acceptable salt of the non-natural amino acid polypeptides that discloses herein.In addition, the salt form of disclosed non-natural amino acid polypeptides can use the salt of initial substance or intermediate to prepare.The free alkali form that the non-natural amino acid polypeptides of Miao Shuing can be by making herein the non-natural amino acid polypeptides of describing and pharmaceutically acceptable mineral acid or organic acid reaction are prepared as pharmaceutically acceptable acid salt (it is the type of pharmaceutically acceptable salt) herein.Perhaps, the free acid form that the non-natural amino acid polypeptides of describing herein can be by making the non-natural amino acid polypeptides of describing herein and pharmaceutically acceptable mineral alkali or organic bases prepared in reaction are pharmaceutically acceptable base addition salt (it is the type of pharmaceutically acceptable salt).
The type of pharmaceutically acceptable salt is including (but not limited to) acid salt that (1) and mineral acid or organic acid form, all example hydrochloric acids of wherein said mineral acid, Hydrogen bromide, sulfuric acid, nitric acid, phosphoric acid with and similar acid; Described organic acid is such as acetate, propionic acid, caproic acid, the pentamethylene propionic acid, oxyacetic acid, pyruvic acid, lactic acid, propanedioic acid, succsinic acid, oxysuccinic acid, toxilic acid, fumaric acid, tartrate, citric acid, phenylformic acid, 3-(4-hydroxy benzoyl) phenylformic acid, styracin, amygdalic acid, methylsulfonic acid, ethyl sulfonic acid, 1, the 2-ethionic acid, the 2-ethylenehydrinsulfonic acid, Phenylsulfonic acid, the 2-naphthene sulfonic acid, 4-methyl bicycle-[2.2.2] oct-2-ene-1-carboxylic acid, glucoheptonic acid, 4,4 '-methylene-bis-(3-hydroxyl-2-alkene-1-carboxylic acid), the 3-phenylpropionic acid, trimethylacetic acid, tert.-butylacetic acid, lauryl sulfate, gluconic acid, L-glutamic acid, hydroxynaphthoic acid, Whitfield's ointment, stearic acid, sticking furancarboxylic acid with and similar acid; (2) acid proton that exists in parent compound is replaced through metal ion (for example alkalimetal ion, alkaline-earth metal ions or aluminum ion); Or the salt that forms during with the organic bases coordination.Acceptable organic bases comprise thanomin, diethanolamine, trolamine, trometamol, N-methylglucosamine with and analogue.Acceptable mineral alkali comprise aluminium hydroxide, calcium hydroxide, potassium hydroxide, yellow soda ash, sodium hydroxide with and analogue.
The corresponding counter ion of the salt that non-natural amino acid polypeptides is pharmaceutically acceptable can use several different methods to analyze and differentiate that these methods are including (but not limited to) ion-exchange chromatography, chromatography of ions, capillary electrophoresis, inductively coupled plasma, atomic absorption spectrum, mass spectrum or its any combination.In addition, but technology and the method described among the therapeutic activity use-case 87-91 of the pharmaceutically acceptable salt of these non-natural amino acid polypeptides are tested.
Should be appreciated that mentioning of salt comprises its solvent addition form or crystalline form, especially solvate or polymorphic form.Solvate contains the solvent of the amount of stoichiometry or nonstoichiometry, and usually in crystallisation process with pharmaceutically acceptable solvent (such as water, ethanol with and analogue) form.When solvent forms hydrate during for water, or when solvent formation alcoholate when be pure.Polymorphic form comprises the different crystal assembled arrangement of the compound of identical element composition.Polymorphic form has different x-ray diffraction pattern, infrared spectra, fusing point, density, hardness, crystal shape, optical property and electrical properties, stability and solvability usually.Various factors such as recrystallize solvent, crystallization rate and storing temp can make single crystalline form preponderate.
The salt polymorphic form that non-natural amino acid polypeptides is pharmaceutically acceptable and/or the screening of solvate and sign can use multiple technologies to realize, described technology is including (but not limited to) heat analysis, X-ray diffraction, spectroscopy, steam absorption and microscopy.Heat analysis method is absorbed in thermochemistry degraded or thermal physical process (changing including (but not limited to) polymorphic form), and uses relation, gravimetry between these methods analyst polymorphic forms to lose to find out second-order transition temperature or to be used for the vehicle Study on Compatibility.These methods are including (but not limited to) dsc (DSC), modulation dsc (MDSC), thermogravimetric analysis (TGA), and thermogravimetric and infrared analysis (TG/IR).The X-ray diffraction method is including (but not limited to) monocrystalline and powdery diffractometry meter and synchrotron source.The various spectroscopic techniquess that use are including (but not limited to) Raman, FTIR, UVIS and NMR (liquid and solid-state).Various microscopy technology including (but not limited to) polarization microscope, follow energy dispersion type X-ray analysis (EDX) scanning electronic microscope (SEM), follow environmental scanning electron microscope (in gas or steam atmosphere), the IR microscope of EDX and Raman microscope.
Description of drawings
By can obtain better understanding with reference to the following detailed description that proposes illustrative embodiment, utilize the principle of our method, composition, equipment and device among the described embodiment, and subsidiary graphicly be the feature and advantage of method and composition of the present invention:
The schematic representation of the relation of some aspect of the method that Fig. 1 oblatio is described herein, composition, strategy and technology.
The illustrative of the type of the alpha-non-natural amino acid that Fig. 2 oblatio is described herein, limiting examples.These alpha-non-natural amino acids can be used for or incorporate into be used for preparing, method, composition, technology and the strategy of alpha-non-natural amino acid that purifying, sign and use are described herein, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides any one in.
The illustrative of the type of the alpha-non-natural amino acid that Fig. 3 oblatio is described herein, limiting examples.These alpha-non-natural amino acids can be used for or incorporate into be used for preparing, method, composition, technology and the strategy of alpha-non-natural amino acid that purifying, sign and use are described herein, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides any one in.
Fig. 4 oblatio is used to prepare illustrative, the limiting examples of the synthetic method of the alpha-non-natural amino acid of describing herein.These alpha-non-natural amino acids can be used for or incorporate into be used for preparing, method, composition, technology and the strategy of alpha-non-natural amino acid that purifying, sign and use are described herein, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides any one in.
Fig. 5 oblatio is used to prepare illustrative, the limiting examples of the synthetic method of the alpha-non-natural amino acid of describing herein.These alpha-non-natural amino acids can be used for or incorporate into be used for preparing, method, composition, technology and the strategy of alpha-non-natural amino acid that purifying, sign and use are described herein, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides any one in.
Fig. 6 oblatio is used to prepare illustrative, the limiting examples of the synthetic method of the alpha-non-natural amino acid of describing herein.These alpha-non-natural amino acids can be used for or incorporate into be used for preparing, method, composition, technology and the strategy of alpha-non-natural amino acid that purifying, sign and use are described herein, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides any one in.
Fig. 7 oblatio contains the non-natural amino acid polypeptides of carbonyl to form the modified illustrative that contains the oxime non-natural amino acid polypeptides, limiting examples with the reagent posttranslational modification that contains azanol.These non-natural amino acid polypeptides can be used for or incorporate into be used for preparing, method, composition, technology and the strategy of alpha-non-natural amino acid that purifying, sign and use are described herein, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides any one in.
Fig. 8 oblatio can be used for strengthening the reaction of non-natural amino acid polypeptides that contains carbonyl and the reagent that contains azanol to form illustrative, the limiting examples of the modified additive that contains the oxime non-natural amino acid polypeptides.
Fig. 9 oblatio contains the non-natural amino acid polypeptides of oxime to form the modified illustrative that contains the oxime non-natural amino acid polypeptides, limiting examples with the reagent posttranslational modification that contains carbonyl.These non-natural amino acid polypeptides can be used for or incorporate into be used for preparing, method, composition, technology and the strategy of alpha-non-natural amino acid that purifying, sign and use are described herein, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides any one in.
Figure 10 oblatio contains the non-natural amino acid polypeptides of azanol to form the modified illustrative that contains the oxime non-natural amino acid polypeptides, limiting examples with the reagent posttranslational modification that contains carbonyl.These non-natural amino acid polypeptides can be used for or incorporate into be used for preparing, method, composition, technology and the strategy of alpha-non-natural amino acid that purifying, sign and use are described herein, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides any one in.
Figure 11 oblatio can be used for modifying non-natural amino acid polypeptides contains PEG with formation the illustrative that contains PEG reagent, limiting examples through the non-natural amino acid polypeptides of oxime binding.
Figure 12 oblatio can be used for modifying non-natural amino acid polypeptides contains PEG with formation the synthetic illustrative, the limiting examples that contain PEG reagent through the non-natural amino acid polypeptides of oxime binding.
Figure 13 oblatio can be used for modifying non-natural amino acid polypeptides contains PEG with formation the synthetic illustrative, the limiting examples that contain amide group PEG reagent through the non-natural amino acid polypeptides of oxime binding.
Figure 14 oblatio can be used for modifying non-natural amino acid polypeptides contains PEG with formation the synthetic illustrative, the limiting examples that contain carbamate groups PEG reagent through the non-natural amino acid polypeptides of oxime binding.
Figure 15 oblatio can be used for modifying non-natural amino acid polypeptides contains PEG with formation the synthetic illustrative, the limiting examples that contain carbamate groups PEG reagent through the non-natural amino acid polypeptides of oxime binding.
Figure 16 oblatio can be used for modifying non-natural amino acid polypeptides contains PEG with formation the synthetic illustrative, the limiting examples that contain simple PEG reagent through the non-natural amino acid polypeptides of oxime binding.
Figure 17 oblatio can be used for modifying non-natural amino acid polypeptides and contains illustrative, the limiting examples through the reagent that contains branch PEG of the non-natural amino acid polypeptides of oxime binding of PEG with formation, and a kind of this reagent is used to modify the purposes based on the non-natural amino acid polypeptides of carbonyl.
Figure 18 oblatio can be used for modifying synthetic illustrative, the limiting examples with the difunctionality connexon group of binding non-natural amino acid polypeptides.
Figure 19 oblatio can be used for modifying illustrative, the limiting examples with the multifunctional connexon group of binding non-natural amino acid polypeptides.
Figure 20 oblatio difunctionality connexon group be used to modify non-natural amino acid polypeptides and with the non-natural amino acid polypeptides binding to the illustrative of the purposes of PEG group, non-limiting representative graph.
Figure 21 oblatio difunctionality connexon group is used to modify non-natural amino acid polypeptides and with illustrative, the limiting examples of non-natural amino acid polypeptides binding to the purposes of PEG group.
Figure 22 oblatio difunctionality connexon group with two non-natural amino acid polypeptides bindings together with illustrative, the non-limiting representative graph of the purposes that forms homodimer.
Figure 23 oblatio difunctionality connexon group with two different non-natural amino acid polypeptides bindings together with illustrative, the non-limiting representative graph of the purposes that forms heterodimer.
Figure 24 oblatio contains the synthetic illustrative of the alpha-non-natural amino acid of carbonyl, non-limiting representative graph.These alpha-non-natural amino acids can be used for or incorporate into be used for preparing, method, composition, technology and the strategy of alpha-non-natural amino acid that purifying, sign and use are described herein, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides any one in.
Figure 25 oblatio contains the synthetic illustrative of the alpha-non-natural amino acid of dicarbapentaborane, non-limiting representative graph.These alpha-non-natural amino acids can be used for or incorporate into be used for preparing, method, composition, technology and the strategy of alpha-non-natural amino acid that purifying, sign and use are described herein, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides any one in.
Figure 26 oblatio contains the synthetic illustrative of the alpha-non-natural amino acid of dicarbapentaborane, non-limiting representative graph.
Figure 27 oblatio contains the synthetic illustrative of the alpha-non-natural amino acid of dicarbapentaborane, non-limiting representative graph.These alpha-non-natural amino acids can be used for or incorporate into be used for preparing, method, composition, technology and the strategy of alpha-non-natural amino acid that purifying, sign and use are described herein, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides any one in.
Figure 28 oblatio contains the synthetic illustrative of the alpha-non-natural amino acid of dicarbapentaborane, non-limiting representative graph.These alpha-non-natural amino acids can be used for or incorporate into be used for preparing, method, composition, technology and the strategy of alpha-non-natural amino acid that purifying, sign and use are described herein, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides any one in.
Figure 29 oblatio contains the synthetic illustrative of the alpha-non-natural amino acid of dicarbapentaborane, non-limiting representative graph.These alpha-non-natural amino acids can be used for or incorporate into be used for preparing, method, composition, technology and the strategy of alpha-non-natural amino acid that purifying, sign and use are described herein, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides any one in.
Figure 30 oblatio contains the synthetic illustrative of the alpha-non-natural amino acid of dicarbapentaborane, non-limiting representative graph.These alpha-non-natural amino acids can be used for or incorporate into be used for preparing, method, composition, technology and the strategy of alpha-non-natural amino acid that purifying, sign and use are described herein, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides any one in.
Figure 31 oblatio contains the synthetic illustrative of the alpha-non-natural amino acid of dicarbapentaborane, non-limiting representative graph.These alpha-non-natural amino acids can be used for or incorporate into be used for preparing, method, composition, technology and the strategy of alpha-non-natural amino acid that purifying, sign and use are described herein, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides any one in.
Figure 32 oblatio contains the synthetic illustrative of the alpha-non-natural amino acid of dicarbapentaborane, non-limiting representative graph.These alpha-non-natural amino acids can be used for or incorporate into be used for preparing, method, composition, technology and the strategy of alpha-non-natural amino acid that purifying, sign and use are described herein, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides any one in.
Figure 33 oblatio contains the synthetic illustrative of the alpha-non-natural amino acid of dicarbapentaborane, non-limiting representative graph.These alpha-non-natural amino acids can be used for or incorporate into be used for preparing, method, composition, technology and the strategy of alpha-non-natural amino acid that purifying, sign and use are described herein, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides any one in.
Figure 34 oblatio contains the synthetic illustrative of the alpha-non-natural amino acid of dicarbapentaborane, non-limiting representative graph.These alpha-non-natural amino acids can be used for or incorporate into be used for preparing, method, composition, technology and the strategy of alpha-non-natural amino acid that purifying, sign and use are described herein, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides any one in.
Figure 35 oblatio contains the alpha-non-natural amino acid of carbonyl and contains the illustrative of the alpha-non-natural amino acid of dicarbapentaborane, non-limiting representative graph.These alpha-non-natural amino acids can be used for or incorporate into be used for preparing, method, composition, technology and the strategy of alpha-non-natural amino acid that purifying, sign and use are described herein, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides any one in.
The synthetic illustrative of Figure 36 oblatio alpha-non-natural amino acid, non-limiting representative graph.These alpha-non-natural amino acids can be used for or incorporate into be used for preparing, method, composition, technology and the strategy of alpha-non-natural amino acid that purifying, sign and use are described herein, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides any one in.
Figure 37 oblatio contains the alpha-non-natural amino acid of carbonyl and contains the synthetic illustrative of the alpha-non-natural amino acid of dicarbapentaborane, non-limiting representative graph.These alpha-non-natural amino acids can be used for or incorporate into be used for preparing, method, composition, technology and the strategy of alpha-non-natural amino acid that purifying, sign and use are described herein, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides any one in.
Figure 38 oblatio contains the alpha-non-natural amino acid of carbonyl and contains the synthetic illustrative of the alpha-non-natural amino acid of dicarbapentaborane, non-limiting representative graph.These alpha-non-natural amino acids can be used for or incorporate into be used for preparing, method, composition, technology and the strategy of alpha-non-natural amino acid that purifying, sign and use are described herein, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides any one in.
Figure 39 oblatio contains the alpha-non-natural amino acid of carbonyl and contains the synthetic illustrative of the alpha-non-natural amino acid of dicarbapentaborane, non-limiting representative graph.These alpha-non-natural amino acids can be used for or incorporate into be used for preparing, method, composition, technology and the strategy of alpha-non-natural amino acid that purifying, sign and use are described herein, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides any one in.
Figure 40 oblatio contains the illustrative of the alpha-non-natural amino acid of dicarbapentaborane, non-limiting representative graph.These alpha-non-natural amino acids can be used for or incorporate into be used for preparing, method, composition, technology and the strategy of alpha-non-natural amino acid that purifying, sign and use are described herein, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides any one in.
Figure 41 oblatio contains the illustrative of the alpha-non-natural amino acid of dicarbapentaborane, non-limiting representative graph.These alpha-non-natural amino acids can be used for or incorporate into be used for preparing, method, composition, technology and the strategy of alpha-non-natural amino acid that purifying, sign and use are described herein, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides any one in.
Figure 42 oblatio contains the illustrative of the alpha-non-natural amino acid of dicarbapentaborane, non-limiting representative graph.These alpha-non-natural amino acids can be used for or incorporate into be used for preparing, method, composition, technology and the strategy of alpha-non-natural amino acid that purifying, sign and use are described herein, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides any one in.
Figure 43 oblatio (a) contains 1, the alpha-non-natural amino acid of 3-keto-aldehyde, and the illustrative, the non-limiting representative graph that (b) contain the alpha-non-natural amino acid of 1-3-ketone carboxyl (sulphur) ester through protection or without what protect.These alpha-non-natural amino acids can be used for or incorporate into be used for preparing, method, composition, technology and the strategy of alpha-non-natural amino acid that purifying, sign and use are described herein, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides any one in.
Figure 44 oblatio contains the illustrative of the alpha-non-natural amino acid of hydrazides, non-limiting representative graph.These alpha-non-natural amino acids can be used for or incorporate into be used for preparing, method, composition, technology and the strategy of alpha-non-natural amino acid that purifying, sign and use are described herein, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides any one in.
Figure 45 oblatio contains the illustrative of the alpha-non-natural amino acid of hydrazides, non-limiting representative graph.These alpha-non-natural amino acids can be used for or incorporate into be used for preparing, method, composition, technology and the strategy of alpha-non-natural amino acid that purifying, sign and use are described herein, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides any one in.
Figure 46 A and 46B oblatio contain the illustrative of the alpha-non-natural amino acid of oxime, non-limiting representative graph, and Figure 46 C oblatio contains the illustrative of the alpha-non-natural amino acid of hydrazine, non-limiting representative graph.These alpha-non-natural amino acids can be used for or incorporate into be used for preparing, method, composition, technology and the strategy of alpha-non-natural amino acid that purifying, sign and use are described herein, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides any one in.
One step of Figure 47 oblatio is engaged to non-natural amino acid polypeptides and two steps were engaged to the illustrative of non-natural amino acid polypeptides, non-limiting representative graph.For instance, these joints comprise the PEGization of non-natural amino acid polypeptides.
Synthetic illustrative, the non-limiting representative graph of Figure 48 oblatio mPEG-hydroxylamine compound.These alpha-non-natural amino acids can be used for or incorporate into be used for preparing, method, composition, technology and the strategy of alpha-non-natural amino acid that purifying, sign and use are described herein, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides any one in.
Synthetic illustrative, the non-limiting representative graph of Figure 49 oblatio mPEG-hydroxylamine compound.These alpha-non-natural amino acids can be used for or incorporate into be used for preparing, method, composition, technology and the strategy of alpha-non-natural amino acid that purifying, sign and use are described herein, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides any one in.
Synthetic illustrative, the non-limiting representative graph of Figure 50 oblatio mPEG-hydroxylamine compound.These alpha-non-natural amino acids can be used for or incorporate into be used for preparing, method, composition, technology and the strategy of alpha-non-natural amino acid that purifying, sign and use are described herein, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides any one in.
Synthetic illustrative, the non-limiting representative graph of Figure 51 oblatio mPEG-hydroxylamine compound.These alpha-non-natural amino acids can be used for or incorporate into be used for preparing, method, composition, technology and the strategy of alpha-non-natural amino acid that purifying, sign and use are described herein, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides any one in.
Synthetic illustrative, the non-limiting representative graph of Figure 52 oblatio mPEG-hydroxylamine compound.These alpha-non-natural amino acids can be used for or incorporate into be used for preparing, method, composition, technology and the strategy of alpha-non-natural amino acid that purifying, sign and use are described herein, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides any one in.
Synthetic illustrative, the non-limiting representative graph of Figure 53 oblatio mPEG-hydroxylamine compound.These alpha-non-natural amino acids can be used for or incorporate into be used for preparing, method, composition, technology and the strategy of alpha-non-natural amino acid that purifying, sign and use are described herein, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides any one in.
Synthetic illustrative, the non-limiting representative graph of Figure 54 oblatio mPEG-hydroxylamine compound.These alpha-non-natural amino acids can be used for or incorporate into be used for preparing, method, composition, technology and the strategy of alpha-non-natural amino acid that purifying, sign and use are described herein, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides any one in.
Synthetic illustrative, the non-limiting representative graph of Figure 55 oblatio mPEG-hydroxylamine compound.These alpha-non-natural amino acids can be used for or incorporate into be used for preparing, method, composition, technology and the strategy of alpha-non-natural amino acid that purifying, sign and use are described herein, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides any one in.
Synthetic illustrative, the non-limiting representative graph of Figure 56 oblatio mPEG-hydroxylamine compound.These alpha-non-natural amino acids can be used for or incorporate into be used for preparing, method, composition, technology and the strategy of alpha-non-natural amino acid that purifying, sign and use are described herein, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides any one in.
Synthetic illustrative, the non-limiting representative graph of Figure 57 oblatio mPEG-hydroxylamine compound.These alpha-non-natural amino acids can be used for or incorporate into be used for preparing, method, composition, technology and the strategy of alpha-non-natural amino acid that purifying, sign and use are described herein, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides any one in.
Synthetic illustrative, the non-limiting representative graph of Figure 58 oblatio mPEG-hydroxylamine compound.These alpha-non-natural amino acids can be used for or incorporate into be used for preparing, method, composition, technology and the strategy of alpha-non-natural amino acid that purifying, sign and use are described herein, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides any one in.
Synthetic illustrative, the non-limiting representative graph of Figure 59 oblatio mPEG-hydroxylamine compound.These alpha-non-natural amino acids can be used for or incorporate into be used for preparing, method, composition, technology and the strategy of alpha-non-natural amino acid that purifying, sign and use are described herein, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides any one in.
The synthetic illustrative of Figure 60 oblatio hydroxylamine compound, non-limiting representative graph.These alpha-non-natural amino acids can be used for or incorporate into be used for preparing, method, composition, technology and the strategy of alpha-non-natural amino acid that purifying, sign and use are described herein, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides any one in.
Synthetic illustrative, the non-limiting representative graph of Figure 61 oblatio mPEG-hydroxylamine compound.These alpha-non-natural amino acids can be used for or incorporate into be used for preparing, method, composition, technology and the strategy of alpha-non-natural amino acid that purifying, sign and use are described herein, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides any one in.
Synthetic illustrative, the non-limiting representative graph of Figure 62 A oblatio hydroxylamine compound; Synthetic illustrative, the non-limiting representative graph of Figure 62 B oblatio mPEG compound.These alpha-non-natural amino acids can be used for or incorporate into be used for preparing, method, composition, technology and the strategy of alpha-non-natural amino acid that purifying, sign and use are described herein, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides any one in.
Figure 63 oblatio (A) is to contain that (comprise and contain dicarbapentaborane) non-natural amino acid polypeptides of carbonyl is modified non-natural amino acid polypeptides by chemical transformation and (B) is to contain illustrative, the limiting examples that the non-natural amino acid polypeptides of azanol is modified non-natural amino acid polypeptides by chemical transformation.These non-natural amino acid polypeptides can be used for or incorporate into be used for preparing, method, composition, technology and the strategy of alpha-non-natural amino acid that purifying, sign and use are described herein, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides any one in.
Synthetic illustrative, the non-limiting representative graph of Figure 64 oblatio PEG-hydroxylamine compound.These alpha-non-natural amino acids can be used for or incorporate into be used for preparing, method, composition, technology and the strategy of alpha-non-natural amino acid that purifying, sign and use are described herein, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides any one in.
Synthetic illustrative, the non-limiting representative graph of Figure 65 oblatio PEG-hydroxylamine compound.These alpha-non-natural amino acids can be used for or incorporate into be used for preparing, method, composition, technology and the strategy of alpha-non-natural amino acid that purifying, sign and use are described herein, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides any one in.
Embodiment
I. foreword
In recent years, reported the brand new technical in the protein science, its hope overcomes the relevant restriction of numerous and proteinic site-specific sex modification.Specifically, new component has been added to prokaryotic organism intestinal bacteria (E.coli) (for example, people such as L.Wang, (2001), Science292:498-500) and eukaryote yeast saccharomyces cerevisiae (Sacchromycescerevisiae) (S.cerevisiae) (for example, people such as J.Chin, Science301:964-7 (2003)) in the protein biosynthesizing mechanism, it makes it possible in vivo alpha-non-natural amino acid be incorporated in the protein.Use this method, the many amino acids (but comprising photoaffinity mark and photoisomerization amino acid, keto amino acid and glycosylation amino acid) with novel chemistry, physics or biological property effectively and hi-fi ground incorporate in the protein in intestinal bacteria and the yeast with response amber codon TAG.Referring to people such as (for example) J.W.Chin, (2002), Journal of the American Chemical Society124:9026-9027 (all incorporating into by reference); J.W.Chin and P.G.Schultz, (2002), ChemBioChem3 (11): 1135-1137 (all incorporating into by reference); J.W.Chin waits the people, and (2002), PNAS United States of America99 (17): 11020-11024 (all incorporating into by reference); And, L.Wang and P.G.Schultz, (2002), Chem.Comm.,1-11 (all incorporating into by reference).These researchs have confirmed might selectivity and introduce non-existent chemical functional group in the protein routinely, it is chemically inert to all functional groups that exist in 20 kinds of common heredity coded amino acids, and it can be used for effectively and optionally reacting to form stable covalent linkage.
II. general introduction
Fig. 1 oblatio is to the general introduction of composition, method and the technology of description herein.In a way, the means (method, composition, technology) be used to produce and use the polypeptide of the alpha-non-natural amino acid that comprises at least one alpha-non-natural amino acid or modify through carbonyl, dicarbapentaborane, oximido or azanol base are described herein.These alpha-non-natural amino acids can contain other functional groups, and it is including (but not limited to) mark; Dyestuff; Polymkeric substance; Water-soluble polymers; The derivative of polyoxyethylene glycol; Photocrosslinking agent; Cytotoxic compound; Medicine; Affinity marker; The photoaffinity mark; Reactive compounds; Resin; Second protein or polypeptide or polypeptide analog; Antibody or antibody fragment; Metal chelator; Cofactor; Lipid acid; Carbohydrate; Polynucleotide; DNA; RNA; Antisense polynucleotide; Carbohydrate, water-soluble branch-shape polymer, cyclodextrin, biomaterial; Nanoparticle; Spin labeling; Fluorophore; Metallic part; Radioactive segment; Novel functional group; With the covalently or non-covalently interactional group of other molecules; The light cage covers part; But the part that actinic radiation excites; Ligand; But photoisomerization part; Vitamin H; The vitamin H analogue; Be combined with the part of heavy atom; But the group of chemical cracking; But the group of photodestruciton; The side chain that prolongs; The sugar of carbon bond connection; Redox active agent; Amino thioic acid sulfoacid; The toxicity part; Through isotope-labeled part; The biophysics probe; The phosphorescence group; Chemiluminescent groups; The electron dense group; The magnetic group; Insert group; Chromophoric group; Energy transfer agent; Biologically active agent (in the case, biologically active agent can comprise medicament with therapeutic activity and non-natural amino acid polypeptides or modified alpha-non-natural amino acid can serve as with attached therapeutical agent common altogether therapeutical agent or as means with therapeutic agent delivery required site to the organism); Detectable label; Small molecules; Inhibition Yeast Nucleic Acid; The radioactive nuleus thuja acid; Neutron capture agent; The derivative of vitamin H; Quantum dot; Nanometer is transmitted plain; Radioactivity is transmitted plain; Abzyme, activation composite activating agent, virus, adjuvant, aglycone, anaphylactogen, angiostatin, hormone antagonist, antioxidant, fit, guide RNA, Saponin/TSM, shuttle vectors, macromole, plan epitope, acceptor, reverse micelle with and any combination.It should be noted that various aforementioned functional groups mean the member that the member who hints a kind of functional group can not be categorized as another kind of functional group.Certainly, it can be according to particular case and overlapping.Only for instance, the derivative of water-soluble polymers and polyoxyethylene glycol is overlapping on scope, however overlapping and not exclusively and therefore two kinds of functional groups all list in above.
As shown in fig. 1, on the one hand for using method, composition and the technology described to select and design the method for desiring modified polypeptides herein.From the beginning novel polypeptide can design, and it comprises (only for instance) as the part of high-throughput screening method (can design in the case,, synthesize, characterize and/or test numerous polypeptide) or according to investigator's interest design.Novel polypeptide also can design according to the structure known or polypeptide that part characterizes.Only for instance, growth hormone gene superfamily (Growth Hormone Gene Superfamily) (seeing below) has become the theme of scientific community's broad research; Novel polypeptide can design according to the member's of this gene superfamily structure.The principle of selecting which amino acid to replace and/or modifying is described in herein separately.Use the selection of which kind of modification also to be described in herein, and can be used for satisfying experimenter or end user's demand.These demands can be including (but not limited to) the therapeutic efficiency of handling polypeptide; The security overview of improvement polypeptide; Regulate pharmacokinetics, pharmacology and/or the drug effect of polypeptide; Such as, (only for instance) increases water-soluble, biological usability; Increase serum half-life; Increase the treatment transformation period; Regulate immunogenicity; Regulate biological activity; Or prolong cycling time.In addition, these modifications comprise (only for instance) provides other functional groups to polypeptide; In polypeptide, incorporate label, mark or detectable signal into; The separating property of facilitation polypeptide, and any combination of aforementioned modification.
Also describe modified herein or can be modified to contain the alpha-non-natural amino acid of oximido, carbonyl, dicarbapentaborane or azanol base.This aspect comprises and is used to prepare, the method for purifying, sign and these alpha-non-natural amino acids of use.Describe herein on the other hand at least one described alpha-non-natural amino acid being incorporated into method, strategy and the technology in the polypeptide.The method that this aspect comprises equally and is used to prepare, purifying, sign and use contain these polypeptide of at least one described alpha-non-natural amino acid.This aspect comprises equally and is used for preparation, purifying, sign and use and can be used for preparation (at least in part) and contain the composition and the method for the oligonucleotide (comprising DNA and RNA) of the polypeptide of at least one alpha-non-natural amino acid.This aspect comprises equally and is used for preparation, purifying, sign and use and can expresses and can be used for making (at least in part) and contain the composition and the method for cell of these oligonucleotide of the polypeptide of at least one alpha-non-natural amino acid.
Therefore, provide and describe the polypeptide of the alpha-non-natural amino acid that comprises at least one alpha-non-natural amino acid or modify through carbonyl, dicarbapentaborane, oximido or azanol base herein.In certain embodiments, has at least one alpha-non-natural amino acid or the polypeptide of the alpha-non-natural amino acid modified through carbonyl, dicarbapentaborane, oximido or azanol base is included in some locational at least posttranslational modifications on the polypeptide.In certain embodiments; translation or posttranslational modification are by cell mechanism (for example altogether; glycosylation, acetylize, acidylate, lipid-modified, palmitoylation, palmitate addition, phosphorylation, glycolipid key are modified; with and similar mechanism) take place; in many cases; these betide the natural amino acid sites place that exists on the polypeptide based on the common translation of cell mechanism or posttranslational modification; yet; in certain embodiments, occur on the alpha-non-natural amino acid site on the polypeptide based on the common translation of cell mechanism or posttranslational modification.
In other embodiments, posttranslational modification does not utilize cell mechanism, but passes through to utilize the chemical process of describing herein, or is applicable to that the additive method of specific reactivity group will comprise that the molecule of second reactive group is (including (but not limited to) mark; Dyestuff; Polymkeric substance; Water-soluble polymers; The derivative of polyoxyethylene glycol; Photocrosslinking agent; Cytotoxic compound; Medicine; Affinity marker; The photoaffinity mark; Reactive compounds; Resin; Second protein or polypeptide or polypeptide analog; Antibody or antibody fragment; Metal chelator; Cofactor; Lipid acid; Carbohydrate; Polynucleotide; DNA; RNA; Antisense polynucleotide; Carbohydrate, water-soluble branch-shape polymer, cyclodextrin, biomaterial; Nanoparticle; Spin labeling; Fluorophore, metallic part; Radioactive segment; Novel functional group; With the covalently or non-covalently interactional group of other molecules; The light cage covers part; But the part that actinic radiation excites; Ligand; But photoisomerization part; Vitamin H; The vitamin H analogue; Be combined with the part of heavy atom; But the group of chemical cracking; But the group of photodestruciton; The side chain that prolongs; The sugar of carbon bond connection; Redox active agent; Amino thioic acid sulfoacid; The toxicity part; Through isotope-labeled part; The biophysics probe; The phosphorescence group; Chemiluminescent groups; The electron dense group; The magnetic group; Insert group; Chromophoric group; Energy transfer agent; Biologically active agent; Detectable label; Small molecules; Inhibition Yeast Nucleic Acid, radioactive nuleus thuja acid; Neutron capture agent; The derivative of vitamin H; Quantum dot; Nanometer is transmitted plain; Radioactivity is transmitted plain; Abzyme, the activation composite activating agent, virus, adjuvant, aglycone, anaphylactogen, angiostatin, hormone antagonist, antioxidant, fit, guide RNA, Saponin/TSM, shuttle vectors, macromole, intend epitope, acceptor, reverse micelle with and any combination) be connected at least a first reactive group that comprises (including (but not limited to) containing ketone, aldehyde, acetal, hemiacetal, oxime or azanol functional group's alpha-non-natural amino acid) alpha-non-natural amino acid provides the functional group.In certain embodiments, translation or posttranslational modification are in vivo to carry out in eukaryote or non-eukaryote altogether.In certain embodiments, posttranslational modification is not utilize cell mechanism to carry out in vitro.This aspect comprises equally and is used to prepare, purifying, sign and use contain at least one these through methods of these polypeptide of the alpha-non-natural amino acids of translation or posttranslational modification altogether.
Also comprise in the category of the method for Miao Shuing, composition, strategy and technology herein can with as alpha-non-natural amino acid (contain carbonyl or dicarbapentaborane, oximido, azanol base, or it being sheltered or the protection form) reaction of the part of polypeptide to produce the reagent of any aforementioned posttranslational modification.In general, gained should contain at least one oximido through the alpha-non-natural amino acid of posttranslational modification; Gained is modified contains the oxime alpha-non-natural amino acid and can stand subsequently modification reaction.The described compositions and methods that this aspect also comprises and is used to prepare, purifying, sign and use can be carried out any described posttranslational modification of described alpha-non-natural amino acid.
In certain embodiments, polypeptide comprises at least a common translation or the posttranslational modification of being carried out by a kind of host cell in vivo, and wherein posttranslational modification be can't help another kind of host cell type usually and carried out.In certain embodiments, polypeptide comprises at least a common translation or the posttranslational modification of being carried out by eukaryote in vivo, and wherein altogether translation or posttranslational modification be can't help non-eukaryote usually and carried out.These altogether the example of translation or posttranslational modification modify including (but not limited to) glycosylation, acetylize, acidylate, lipid-modified, palmitoylation, palmitate addition, phosphorylation, glycolipid key, with and similar modification.In one embodiment, altogether translation or posttranslational modification comprise by GlcNAc-l-asparagine binding with oligosaccharides be connected to l-asparagine (including (but not limited to), wherein oligosaccharides comprises (GlcNAc-Man) 2-Man-GlcNAc-GlcNAc, with and analogue).In another embodiment, altogether translation or posttranslational modification comprise by GalNAc-Serine, GalNAc-Threonine, GlcNAc-Serine or GlcNAc-Threonine binding oligosaccharides (including (but not limited to) Gal-GalNAc, Gal-GlcNAc etc.) are connected to Serine or Threonine.In certain embodiments, protein or polypeptide can comprise secretion or positioning sequence, epitope label, FLAG label, polyhistidyl label, GST fusion and/or its analogue.This aspect comprises equally and is used to prepare, purifying, sign and use contain at least a this method of these polypeptide of translation or posttranslational modification altogether.In other embodiments, the glycosylation non-natural amino acid polypeptides is with non-glycosylated form preparation.The non-glycosylated form of this of glycosylation alpha-non-natural amino acid can be by the combination results of following method or any of these method, described method comprise from through separate or purified in fact or not purified glycosylation non-natural amino acid polypeptides chemistry or enzymatic remove the oligosaccharides group; (described host comprises through through engineering approaches or suddenlys change with the prokaryotic organism or the eukaryote of this polypeptide of not glycosylation) generation alpha-non-natural amino acid in the host of this non-natural amino acid polypeptides of not glycosylation is to introduce glycosylation inhibitor in the cell culture medium that is produced by common eukaryote of understanding this polypeptide of glycosylation to wherein said non-natural amino acid polypeptides.These non-glycosylated forms of common glycosylation non-natural amino acid polypeptides (usually glycosylation means when understanding glycosylated polypeptide when existing polypeptide to prepare natural under glycosylated condition) are also described herein.Certainly, usually these non-glycosylated forms of glycosylation non-natural amino acid polypeptides (or any polypeptide of describing in fact herein) can be not purified form, purified in fact form or separated form.
In certain embodiments, non-natural amino acid polypeptides comprises at least a posttranslational modification of carrying out in the presence of promotor, and wherein posttranslational modification is stoichiometry, class stoichiometry or approaching stoichiometric.In other embodiments, polypeptide is contacted with the reagent of formula (XIX).In other embodiments, promotor being is selected from the group that is made up of following each thing:
Figure A20058004432800811
The polypeptide that contains alpha-non-natural amino acid can contain at least one, contain carbonyl or dicarbapentaborane, oximido, azanol base or its alpha-non-natural amino acid through the protection form at least at least at least at least at least at least at least at least more than two, three, four, five, six, seven, eight, nine or ten or ten.Alpha-non-natural amino acid can be identical or different, for example, in the protein that comprises different alpha-non-natural amino acids more than 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20 or 20, can there be 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20 or 20 above different loci.In certain embodiments, at least one in the specific amino acids that exists with proteinic natural existence form (but being less than all) replaces through alpha-non-natural amino acid.
The method and composition that provides herein and describe comprises and comprises that at least one contains carbonyl or dicarbapentaborane, oximido, azanol base or it is through protecting or shelter the polypeptide of the alpha-non-natural amino acid of form.With at least one alpha-non-natural amino acid introduce in polypeptide tolerable use comprise the particular chemical reaction (including (but not limited to) with one or more alpha-non-natural amino acid reaction, and with the 20 seed amino acids reaction of common existence) engage chemistry.In case incorporate into, then non-natural exist amino acid side chain also can utilize herein to describe or be applicable to that the particular functional base or the substituent chemical process that exist in the natural coded amino acid modify.
Herein the alpha-non-natural amino acid method and composition of Miao Shuing provide have multiple functional group, substituting group or the material of part and the joiner of other materials, described other materials are including (but not limited to) mark; Dyestuff; Polymkeric substance; Water-soluble polymers; The derivative of polyoxyethylene glycol; Photocrosslinking agent; Cytotoxic compound; Medicine; Affinity marker; The photoaffinity mark; Reactive compounds; Resin; Second protein or polypeptide or polypeptide analog; Antibody or antibody fragment; Metal chelator; Cofactor; Lipid acid; Carbohydrate; Polynucleotide; DNA; RNA; Antisense polynucleotide; Carbohydrate, water-soluble branch-shape polymer, cyclodextrin, biomaterial; Nanoparticle; Spin labeling; Fluorophore; Metallic part; Radioactive segment; Novel functional group; With the covalently or non-covalently interactional group of other molecules; The light cage covers part; But the part that actinic radiation excites; Ligand; But photoisomerization part; Vitamin H; The vitamin H analogue; Be combined with the part of heavy atom; But the group of chemical cracking; But the group of photodestruciton; The side chain that prolongs; The sugar of carbon bond connection; Redox active agent; Amino thioic acid sulfoacid; The toxicity part; Through isotope-labeled part; The biophysics probe; The phosphorescence group; Chemiluminescent groups; The electron dense group; The magnetic group; Insert group; Chromophoric group; Energy transfer agent; Biologically active agent; Detectable label; Small molecules; Inhibition Yeast Nucleic Acid; The radioactive nuleus thuja acid; Neutron capture agent; The derivative of vitamin H; Quantum dot; Nanometer is transmitted plain; Radioactivity is transmitted plain; Abzyme, activation composite activating agent, virus, adjuvant, aglycone, anaphylactogen, angiostatin, hormone antagonist, antioxidant, fit, guide RNA, Saponin/TSM, shuttle vectors, macromole, plan epitope, acceptor, reverse micelle with and any combination.
In certain embodiments, alpha-non-natural amino acid, non-natural amino acid polypeptides, connexon and the reagent of the compound of describing herein that comprises formula (I)-(XXXIII) under appropriate acidic conditions (including (but not limited to) pH 2 to 8) are stable in the aqueous solution.In other embodiments, these compounds are stablized at least one month under appropriate acidic conditions.In other embodiments, these compounds stable at least 2 weeks under appropriate acidic conditions.In other embodiments, these compounds were stablized 5 days under appropriate acidic conditions at least.
The composition of Miao Shuing, method, technology and strategy on the other hand for being used for research or using any one the method for aforementioned " modified or not modified " non-natural amino acid polypeptides herein.Comprise treatment that (only for instance) can benefit, diagnosis in this aspect from comprise " modified or not modified " non-natural amino acid polypeptides or proteinic polypeptide, based on calibrating, industry, makeup, plant biology, environment, energy generation, consumer products and/or military purposes.
III. the location of alpha-non-natural amino acid in the polypeptide
The method and composition of Miao Shuing comprises one or more alpha-non-natural amino acid is incorporated in the polypeptide herein.One or more alpha-non-natural amino acid can be incorporated in one or more specific location of not destroying polypeptide active.This measure can replace by carrying out " conservative property " (including (but not limited to) replacing hydrophobic amino acid with non-natural or natural hydrophobic amino acid, with non-natural or the huge amino acid of natural huge aminoacid replacement, with non-natural or natural hydrophilic aminoacid replacement hydrophilic amino acid) and/or alpha-non-natural amino acid inserted do not need to realize in the active position.
Can use multiple biological chemistry and structural approach to be chosen in the interior required site that replaces through alpha-non-natural amino acid of polypeptide.Any position of polypeptide chain is suitable for selecting incorporating alpha-non-natural amino acid into, and for any or do not have specific required purpose, can be according to appropriate design or by being selected at random.The selection in required site can have any required character or activity ((including (but not limited to) agonist based on generation, super agonist, the part agonist, reverse agonist, antagonist, the receptors bind conditioning agent, receptor activity modulators, with combine collocation thing bonded conditioning agent, in conjunction with collocation thing active regulator, in conjunction with collocation thing conformation conditioning agent, dimer or polymer form), compare the non-natural amino acid polypeptides (it can or keep not modified through further modification) that non-activity or character change with natural molecule, or any physics or the chemical property of manipulation polypeptide, such as solvability, assemble or stability.For instance, can use including (but not limited to) the method for scanning of point mutation analysis, L-Ala or homologue scanning method and differentiate position in the bioactive polypeptide that needs polypeptide.Except that differentiating the good candidate of required activity of looking for for polypeptide for to the possibility of the residue those residues of biological activity key basis for replacing with alpha-non-natural amino acid through scan the method that forms of suddenling change including (but not limited to) L-Ala or homologue.Perhaps, also may be through discriminating once more according to the good candidate of the required activity of looking for for polypeptide for replacing with alpha-non-natural amino acid for site to the biological activity key.Another replacement method can be with alpha-non-natural amino acid and simply carries out the continuous replacement in each position on the polypeptide chain and observe active influence to polypeptide.Selection is applicable to method, technology and the composition of describing herein with any means, technology or the method for the position of alpha-non-natural amino acid replacement in any polypeptide.
Also can study the natural structure of mutant and the activity of existing of the polypeptide that contains disappearance and may allow the protein zone that replaces with alpha-non-natural amino acid to measure.May allow the residue that replaces with alpha-non-natural amino acid in case remove, just can use including (but not limited to) what the method for related polypeptide and any association ligand or protein-bonded three-dimensional structure was researched and proposed and be substituted in influence on each rest position.Protein Data Bank (Protein Data Bank) (PDB, Www.rcsb.org) can obtain the X ray crystallization and the NMR structure of many polypeptide in (integrated data store that contains the macromolecular three-dimensional structure data of protein and nucleic acid), it can be used for the amino acid position of differentiating that available alpha-non-natural amino acid replaces.In addition, if three-dimensional structure data can not obtain, can make secondary structure and tertiary structure that model is studied polypeptide so.Therefore, can be easy to obtain the amino acid position itself that available alpha-non-natural amino acid replaces.
The exemplary site of incorporating alpha-non-natural amino acid into is including (but not limited to) those sites of getting rid of potential receptors bind zone, or with conjugated protein or ligand bonded zone wholly or in part solvent expose, have minimum or do not have interaction of hydrogen bond with contiguous residue, but minimum level ground is exposed to contiguous reactive residue, and/or can be in the zone as associated mutually by specific polypeptide and its acceptor, ligand or highly flexible that protein-bonded three-dimensional crystalline structure is predicted as.
Multiple alpha-non-natural amino acid can replace or incorporate in the given position in the polypeptide.For instance, can be according to the specific alpha-non-natural amino acid of the research of polypeptide and its association ligand, acceptor and/or protein-bonded three-dimensional crystalline structure being selected be used to incorporate into, preferred conservative property replaces.
In one embodiment, the method for Miao Shuing comprises and incorporate alpha-non-natural amino acid in polypeptide herein, and wherein alpha-non-natural amino acid comprises first reactive group; And make polypeptide and comprise that the molecule of second reactive group is (including (but not limited to) mark; Dyestuff; Polymkeric substance; Water-soluble polymers; The derivative of polyoxyethylene glycol; Photocrosslinking agent; Cytotoxic compound; Medicine; Affinity marker; The photoaffinity mark; Reactive compounds; Resin; Second protein or polypeptide or polypeptide analog; Antibody or antibody fragment; Metal chelator; Cofactor; Lipid acid; Carbohydrate; Polynucleotide; DNA; RNA; Antisense polynucleotide; Carbohydrate, water-soluble branch-shape polymer, cyclodextrin, biomaterial; Nanoparticle; Spin labeling; Fluorophore, metallic part; Radioactive segment; Novel functional group; With the covalently or non-covalently interactional group of other molecules; The light cage covers part; But the part that actinic radiation excites; Ligand; But photoisomerization part; Vitamin H; The vitamin H analogue; Be combined with the part of heavy atom; But the group of chemical cracking; But the group of photodestruciton; The side chain that prolongs; The sugar of carbon bond connection; Redox active agent; Amino thioic acid sulfoacid; The toxicity part; Through isotope-labeled part; The biophysics probe; The phosphorescence group; Chemiluminescent groups; The electron dense group; The magnetic group; Insert group; Chromophoric group; Energy transfer agent; Biologically active agent; Detectable label; Small molecules; Inhibition Yeast Nucleic Acid, radioactive nuleus thuja acid; Neutron capture agent; The derivative of vitamin H; Quantum dot; Nanometer is transmitted plain; Radioactivity is transmitted plain; Abzyme, activation composite activating agent, virus, adjuvant, aglycone, anaphylactogen, angiostatin, hormone antagonist, antioxidant, fit, guide RNA, Saponin/TSM, shuttle vectors, macromole, plan epitope, acceptor, reverse micelle with and any combination) contact.In certain embodiments, first reactive group is that carbonyl or the dicarbapentaborane part and second reactive group are the azanol part, forms the oxime key by this.In certain embodiments, first reactive group is that the azanol part and second reactive group are carbonyl or dicarbapentaborane part, forms the oxime key by this.In certain embodiments, first reactive group is that carbonyl or the dicarbapentaborane part and second reactive group are the oxime part, and the oxime permutoid reaction takes place by this.In certain embodiments, first reactive group is that the oxime part and second reactive group are carbonyl or dicarbapentaborane part, and the oxime permutoid reaction takes place by this.
In some cases, alpha-non-natural amino acid replace or incorporate into with polypeptide in other interpolations, replacement or disappearance combination to influence other chemistry, physics, pharmacology and/or biological nature.In some cases, other add, replace or lack the stability (including (but not limited to) the resistance to proteolytic degradation) that can increase polypeptide or increase polypeptide to its suitable acceptor, ligand and/or protein-bonded affinity.In some cases, other add, replace or disappearance can increase polypeptide solubleness (including (but not limited to), when with intestinal bacteria or other host cell expressions).In certain embodiments, selection is used for the site with natural coding or alpha-non-natural amino acid replacement, in addition, select another site to be used to incorporate into alpha-non-natural amino acid, express increase polypeptide deliquescent purpose in back in intestinal bacteria or other recombinant host cells to be implemented in.In certain embodiments, polypeptide comprise adjusting to affinity, adjusting (including (but not limited to) increasing or the reducing) receptor dimerizationization of association ligand, conjugated protein and/or acceptor, stablize receptor dimer, regulate circulating half-life, adjustment release or biological usability, be beneficial to purifying, or improve or change another interpolation, replacement or the disappearance of specific dosing way.Similarly, non-natural amino acid polypeptides can comprise detection (including (but not limited to) GFP), purifying, transmission penetrate tissue or the cytolemma, prodrug release or the activation that improve polypeptide, size reduces or the chemistry of other characteristics or enzymatic lysis sequence, protease cracking sequence, reactive group, antibody binding domain (including (but not limited to) FLAG or poly-His) or other sequences based on affinity (including (but not limited to) FLAG, poly-His, GST etc.) or binding molecule (including (but not limited to) vitamin H).
IV. as the tethelin supergene family of example
The method of Miao Shuing, composition, strategy and technology are not limited to specific polypeptide or protein type, kind or family herein.Really, any in fact polypeptide can or be modified to comprise " modified or not modified " alpha-non-natural amino acid that at least one is described herein through design.Only for instance, polypeptide can with the treatment protein homology that is selected from the group that forms by following each thing: α-1 antitrypsin, angiostatin, AHF, antibody, antibody fragment, lipophorin, apoprotein, atrionatriuretic factor, atrial natriuretic polypeptins, atrial natriuretic peptide, the C-X-C chemokine, T39765, NAP-2, ENA-78, gro-a, gro-b, gro-c, IP-10, GCP-2, NAP-4, SDF-1, PF4, MIG, calcitonin, the c-kit ligand, cytokine, the CC chemokine, MCP-1, MCP-2, monocyte chemoattractant protein-3, monocyte inflammatory protein-1 α, monocyte inflammatory protein-i β, RANTES, 1309, R83915, R91733, HCC1, T58847, D31065, T64262, CD40, the CD40 ligand, the c-kit ligand, collagen protein, G CFS (CSF), complement factor 5a, complement inhibitor, complement receptor 1, cytokine, epithelium neutrophilic granulocyte activation peptide-78, MIP-16, MCP-1, Urogastron (EGF), epithelium neutrophilic granulocyte activation peptide, erythropoietin (EPO), exfoliative toxin,or exfoliatin, factors IX, factor VII, Factor IX, factor X, fibroblast growth factor (FGF), Fibrinogen, fibronectin, four-helix bundle albumen, G-CSF, glp-1, GM-CSF, glucocerebrosidase, gonad-stimulating hormone, somatomedin, growth factor receptors, grf, Shh, hemochrome, pHGF (hGF), r-hirudin, human growth hormone (hGH), the human serum albumin, ICAM-1, the ICAM-1 acceptor, LFA-1, the LFA-1 acceptor, Regular Insulin, insulin-like growth factor (IGF), IGF-I, IGF-II, Interferon, rabbit (IFN), IFN-α, IFN-β, IFN-γ, interleukin-(IL), IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, keratinocyte growth factor (KGF), lactoferrin, leukaemia inhibitory factor, luciferase, neural plain, neutrophil inhibitory factor (nif) (NIF), oncostatin M, bone morphogenic protein, oncoprotein, paracitonin, Rat parathyroid hormone 1-34, PD-ECSF, PDGF, peptide hormone, PTN, albumin A, Protein G, pth, pyrogenic exotoxin A, pyrogenic exotoxin B, pyrogenic exotoxin C, pyy, Relaxin, feritin, SCF, the atom synthetic proteins, soluble complement acceptor I, solubility I-CAM 1, soluble interleukin receptor, soluble TNF acceptor, somatomedin, Somat, tethelin (somatotropin), streptokinase, superantigen, staphyloentero-toxin, SEA, SEB, SEC1, SEC2, SEC3, SED, SEE, steroid hormone receptor, superoxide-dismutase, the toxic shock syndrome toxin, thymosin, the histiotype plasminogen activation factor, tumor growth factor (TGF), tumour necrosis factor, tumor necrosis factor alpha, tumor necrosis factor, Tumor Necrosis Factor Receptors (TNFR), VLA-4 albumen, VCAM-1 albumen, vascular endothelial growth factor (VEGF), urokinase, mos, ras, raf, met, p53, tat, fos, myc, jun, myb, rel, estrogen receptor, PgR, the testosterone acceptor, aldosterone receptor, ldl receptor and Kendall compound.
Therefore, the following description of tethelin (GH) supergene family is for the illustrative purpose and only provides as an example, and is not that conduct is to the restriction of the category of method, composition, strategy and the technology of description herein.In addition, in the application's case mentioning of GH polypeptide is intended to use the example of general terms as any member in the GH supergene family.Therefore, should be appreciated that about GH polypeptide or the proteinic modification of describing and chemical any member that can be applied to equally in the GH supergene family, it comprises specific those members that list herein herein.
Following protein comprises by those protein of the genes encoding of tethelin (GH) supergene family (Bazan, F., Immunology Today 11:350-354 (1990); Bazan, J.F.Science 257:410-411 (1992); Mott, H.R. and Campbell, I.D., Current Opinion in Structural Biology 5:114-121 (1995); Silvennoinen, O. and Ihle, J.N., Signalling by the Hematopoietic Cytokine Receptors (1996)): tethelin, prolactin antagonist, human placental lactogen, erythropoietin (EPO), thrombopoietin (TPO), interleukin II (IL-2), IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-11, IL-12 (p35 unit), IL-13, IL-15, oncostatin M, ciliary neurotrophic factor, leukaemia inhibitory factor, interferon-alpha, interferon-, the ε Interferon, rabbit, IFN-, omega interferon, the τ Interferon, rabbit, granulocyte-G CFS (G-CSF), granulocyte-macrophage colony stimutaing factor (GM-CSF), macrophage colony stimulating factor (M-CSF) and myocardial nutrition element-1 (CT-1) (" GH supergene family ").Other members that estimate this gene family differentiate by gene clone and order-checking in the future.The member of GH supergene family has similar secondary structure and tertiary structure, although they have limited amino acid or consensus dna sequence usually.The common constitutional features makes the newcomer of described gene family be easy to differentiate and uses the alpha-non-natural amino acid method and composition of describing similarly herein.
Comprise G-CSF (people such as Zink, FEBS Lett.314:435 (1992); People such as Zink, Biochemistry 33:8453 (1994); People such as Hill, Proc.Natl.Acad.Sci.USA 90:5167 (1993)), GM-CSF (Diederichs, people such as K., Science 154:1779-1782 (1991); People such as Walter, J.Mol.Biol.224:1075-1085 (1992)), IL-2 (Bazan, J.F. and McKay, D.B., Science 257:410-413 (1992)); IL-4 (people such as Redfield, Biochemistry 30:11029-11035 (1991); People such as Powers, Science 256:1673-1677 (1992)) and IL-5 (people such as Milburn, the structure of numerous cytokines Nature 363:172-176 (1993)) is studied the surprising conservative property of mensuration and displaying and GH structure by X-ray diffraction and NMR, but lacks significant primary sequence homology.Think that according to model and other researchs IFN is member (people such as Lee, the J.Interferon Cytokine Res.15:341 (1995) of this family; People such as Murgolo, Proteins 17:62 (1993); People such as Radhakrishnan, Structure 4:1453 (1996); People such as Klaus, J.Mol.Biol.274:661 (1997)).Comprise ciliary neurotrophic factor (CNTF), leukaemia inhibitory factor (LIF), thrombopoietin (TPO), oncostatin M, macrophage colony stimulating factor (M-CSF), IL-3, IL-6, IL-7, IL-9, IL-12, IL-13, IL-15 and granulocyte-G CFS (G-CSF) and such as a large amount of other cytokines of the IFN of α, β, ω, τ, ε and IFN-and somatomedin belong to this family (in Mott and Campbell, Current Opinion in Structural Biology 5:114-121 (1995); Look back among Silvennoinen and Ihle (1996) the Signalling by the Hematopoietic Cytokine Receptors).Nowadays think that all above-mentioned cytokines and somatomedin comprise a big gene family.
Except total similar secondary structure and tertiary structure, total its of the member of this family must make the character of cell surface receptor oligomerization with signal transduction pathway in the active cells.The receptors bind of some GH family members (including (but not limited to) GH and EPO) and single type and make it form homodimer.Including (but not limited to) other family members of IL-2, IL4 and IL-6 and the receptors bind more than a type and make these acceptors form heterodimers or high-grade aggregate (people such as Davis, (1993) Science 260:1805-1808 more; People such as Paonessa, (1995) EMBOJ.14:1942-1951; Mott and Campbell, Current Opinion in Structural Biology 5:114-121 (1995)).Mutagenesis research is showed, as GH, these other cytokines and somatomedin contain a plurality of receptor binding sites (common two), and combine (Mott and Campbell, Current Opinion inStructural Biology 5:114-121 (1995) successively with its homoreceptor; People such as Matthews, (1996) Proc.Natl.Acad.Sci.USA 93:9471-9476).As GH, these other family members' principal recipient binding site mainly is present in four α spirals and the A-B ring.Specific amino acids in the helical bundle of participation receptors bind is different in the family member.With relevant on the interactional most cells surface receptor of the member of the GH supergene family structure and constitute second big multigene family.Referring to No. the 6th, 608,183, (for example) United States Patent (USP), it is all to be incorporated herein by reference.
The common conclusions that is obtained by the mutation research to multiple GH supergene family member is that the ring that connects the α spiral trends towards not participating in receptors bind usually.Specifically, for the receptors bind among great majority (if not all) family member, short B-C ring seems and is inessential.Reason for this reason, in the member of GH supergene family, the B-C ring can replace through alpha-non-natural amino acid as described herein.A-B ring, C-D ring (and the class IL-10 member's of Interferon, rabbit/GH superfamily D-E ring) also can replace through alpha-non-natural amino acid.Also trend towards not participating in receptors bind and also can be the site that is used to introduce alpha-non-natural amino acid near spiral A and away from the amino acid of last spiral.In certain embodiments, alpha-non-natural amino acid is in the preceding any position replacement in the amino acid whose ring structure more than 1,2,3,4,5,6,7 or 7 including (but not limited to) A-B, B-C, C-D or D-E ring.In certain embodiments, alpha-non-natural amino acid replaces in upper amino acid A-B, B-C, C-D or D-E ring back 1,2,3,4,5,6,7 or 7.
Some member of GH family (including (but not limited to) EPO, IL-2, IL-3, IL-4, IL-6, IFN, GM-CSF, TPO, IL-10, IL-12 p35, IL-13, IL-15 and interferon-) is contained the sugar of N-binding and/or O-binding.Glycosylation site in the protein almost all is present in the ring zone and is not to be present in the α helical bundle.Because the ring zone does not participate in the receptors bind usually and because it is to be used for the site that covalently bound glycosyl is rolled into a ball, is used for alpha-non-natural amino acid is replaced the suitable site of introducing protein so it can be.The amino acid that comprises N-binding glycosylation site and O-binding glycosylation site in the protein can be the site that alpha-non-natural amino acid replaces, and exposes because these amino acid are the surface.Therefore, the natural protein tolerable is connected to huge glycosyl group on the protein and glycosylation site in these site and trends towards being positioned at position away from receptor binding site.
Might be other members that find in the future the GH gene family.The newcomer of GH supergene family can be by predicted protein matter sequence auxiliary secondary structure of computer and tertiary structure analysis, and be used for differentiating that by design the selection technology with specific target bonded molecule differentiates.The member of GH supergene family has four or five amphipathic molecule spirals that connected by non-helical shape amino acid (ring zone) usually.Protein can contain the hydrophobic signal sequence to promote from emiocytosis at its N-end.These GH supergene family members that found afterwards are also contained in the method and composition of describing herein.
V. alpha-non-natural amino acid
The alpha-non-natural amino acid that uses in the method and composition of Miao Shuing has at least one in following four kinds of character herein: the functional group on the side chain of (1) at least one alpha-non-natural amino acid has at least a and 20 kinds of common heredity coded amino acid (that is L-Ala,, arginine, l-asparagine, aspartic acid, halfcystine, glutamine, L-glutamic acid, glycine, Histidine, Isoleucine, leucine, Methionin, methionine(Met), phenylalanine, proline(Pro), Serine, Threonine, tryptophane, tyrosine and Xie Ansuan) the chemical reactivity quadrature, or at least with the polypeptide that comprises alpha-non-natural amino acid in the natural orthogonal feature of amino acid whose chemical reactivity and/or active and/or reactive that exists that exists; (2) alpha-non-natural amino acid through introducing is essentially chemically inert to 20 kinds of common heredity coded amino acids; (3) alpha-non-natural amino acid can stably be incorporated in the polypeptide, preferably have with natural to have the suitable stability of amino acid or have stability under typical physiological condition, and further preferably, described incorporating into can be by in vivo system's generation; And (4) alpha-non-natural amino acid comprise the oxime functional group maybe can be by the functional group of changing oximido into reagent react, preferably under the condition of the biological property that does not destroy the polypeptide that comprises alpha-non-natural amino acid (unless the destruction of this biological property is the purpose of modification/transformation certainly), or wherein change and can take place under aqueous conditions under the pH value between about 4 and about 8, or wherein the reactive site on the alpha-non-natural amino acid is the electrophilic site.Amino acid whose illustrative, the limiting examples about described four kinds of character of alpha-non-natural amino acid of satisfying of composition that can be used for describing herein and method is and is shown among Fig. 2, Fig. 3, Figure 35 and Figure 40-43.The alpha-non-natural amino acid of arbitrary number can be introduced in the polypeptide.Alpha-non-natural amino acid also can comprise through protection or the oxime sheltered or can blocking group is gone to protect maybe will shelter group change into after going to shelter oximido through protecting or shelter group.Alpha-non-natural amino acid also can comprise through protection or carbonyl or the dicarbapentaborane sheltered, its can blocking group is gone protection maybe will shelter group change into after going to shelter carbonyl or dicarbapentaborane and and then can be used for azanol or oxime reaction with the formation oximido.
But the alpha-non-natural amino acid in the method and composition that can be used for describing herein is including (but not limited to) the amino acid that comprises the photoactivated cross-linking agent, amino acid through spin labeling, fluorescence amino acid, melts combine amino acid, containing metal amino acid, radioactivity amino acid, amino acid with novel functional group, with the covalently or non-covalently interactional amino acid of other molecules, but the light cage covers and/or photoisomerization amino acid, the amino acid that comprises vitamin H or vitamin H analogue, glycosylation amino acid (such as through sugar-substituted Serine), other are through the amino acid of carbohydrate modification, contain keto amino acid, contain aldehyde amino acid, the amino acid that comprises polyoxyethylene glycol or other polyethers, amino acid through the heavy atom replacement, but but the amino acid of chemical cracking and/or photodestruciton, have compare the side chain of prolongation with natural amino acid amino acid (including (but not limited to) polyethers or long chain hydrocarbon, it is including (but not limited to) greater than about 5 carbon or greater than about 10 carbon), contain amino acid through the sugar of carbon bond connection, redox active amino acids, contain the amino acid of amino thioic acid sulfoacid, and the amino acid that comprises one or more toxicity parts.
In certain embodiments, alpha-non-natural amino acid comprises the carbohydrate part.These amino acid whose examples comprise N-ethanoyl-L-glucose amido-L-Serine, N-ethanoyl-L-semi-lactosi amido-L-Serine, N-ethanoyl-L-glucose amido-L-Threonine, N-ethanoyl-L-glucose amido-altheine and O-seminose amido-L-Serine.These amino acid whose examples also comprise wherein the natural N-of the existence key between the amino acid and sugar or O-key by the common non-existent covalent linkage of occurring in nature (including (but not limited to) alkene, oxime, thioether, acid amides with and analogue) displaced example.These amino acid whose examples also comprise the natural common non-existent carbohydrate in the protein that exists, such as 2-deoxidation-glucose, 2-deoxy-galactose with and analogue.
Provide the operation of multiple advantage and polypeptide by alpha-non-natural amino acid being incorporated into the chemical part of incorporating in these polypeptide in the polypeptide.For instance, unique reactivity of carbonyl or dicarbapentaborane functional group (comprising ketone functional group or aldehyde functional group) allow with numerous contain hydrazine reagent or contain in the azanol reagent any one carry out proteinic selective modification in vivo and in vitro.Heavy atom alpha-non-natural amino acid (for example) is applicable to phasing x-ray structure data.Selectivity and handiness when using alpha-non-natural amino acid locus specificity introducing heavy atom also to be provided at the position of selecting heavy atom.Photoreactivity alpha-non-natural amino acid (including (but not limited to) amino acid) (for example) with benzophenone and aromatic yl azide (including (but not limited to) azidomethyl phenyl nitrogenize thing) side chain make polypeptide in vivo and in vitro photo-crosslinking is effective.The example of photoreactivity alpha-non-natural amino acid is including (but not limited to) to azido--phenylalanine with to benzoyl-phenylalanine.Polypeptide with photoreactivity alpha-non-natural amino acid subsequently can by photoreactive group to excite (Instantaneous Control is provided) to come crosslinked arbitrarily.In limiting examples, the methyl of alpha-non-natural amino acid can replace (including (but not limited to) through methyl substituted) through isotopic labeling, as local structure and dynamic (dynamical) probe (including (but not limited to) by means of nucleus magnetic resonance and vibrational spectrum).
A. alpha-non-natural amino acid: carbonyl, class carbonyl, through sheltering carbonyl and through the structure of protection carbonyl and synthetic
Amino acid with electrophilic reactivity group allows that multiple reaction is to pass through various chemical reactions (including (but not limited to) nucleophilic addition) binding molecule.These electrophilic reactivity groups comprise carbonyl or dicarbapentaborane (comprising ketone group or aldehyde radical), class carbonyl or class dicarbapentaborane group (its have be similar to the reactive and structurally similar with carbonyl or dicarbapentaborane of carbonyl or dicarbapentaborane), the carbonyl through sheltering or the dicarbapentaborane through sheltering (it can be easy to change into carbonyl or dicarbapentaborane), or through the carbonyl of protection or through the dicarbapentaborane of protection (it has and the similar reactivity of carbonyl or dicarbapentaborane to go to the protection back).These amino acid comprise the have formula amino acid of structure of (I):
Figure A20058004432800901
Wherein:
A is optionally, and when exist, be low-grade alkylidene, be substituted low-grade alkylidene, rudimentary cycloalkylidene, be substituted rudimentary cycloalkylidene, lower alkenylene, be substituted lower alkenylene, alkynylene, the assorted alkyl in rudimentary Asia, be substituted inferior mix alkyl, rudimentary inferior Heterocyclylalkyl, be substituted rudimentary inferior Heterocyclylalkyl, arylidene, be substituted arylidene, inferior heteroaryl, be substituted inferior heteroaryl, alkarylene, be substituted alkarylene, inferior aralkyl or be substituted inferior aralkyl;
B is optionally, and is the connexon that is selected from the group that is made up of following each group when exist: low-grade alkylidene, be substituted low-grade alkylidene, lower alkenylene, be substituted lower alkenylene, alkyl is mixed in rudimentary Asia, be substituted rudimentary Asia mix alkyl ,-O-,-O-(alkylidene group or be substituted alkylidene group)-,-S-,-S-(alkylidene group or be substituted alkylidene group)-,-S (O) k-(wherein k is 1,2 or 3) ,-S (O) k(alkylidene group or be substituted alkylidene group)-,-C (O)-,-NS (O) 2-,-OS (O) 2-,-C (O)-(alkylidene group or be substituted alkylidene group)-,-C (S)-,-C (S)-(alkylidene group or be substituted alkylidene group)-,-N (R ')-,-NR '-(alkylidene group or be substituted alkylidene group)-,-C (O) N (R ')-,-CON (R ')-(alkylidene group or be substituted alkylidene group)-,-CSN (R ')-,-CSN (R ')-(alkylidene group or be substituted alkylidene group)-,-N (R ') CO-(alkylidene group or be substituted alkylidene group)-,-N (R ') C (O) O-,-S (O) kN (R ')-,-N (R ') C (O) N (R ')-,-N (R ') C (S) N (R ')-,-N (R ') S (O) kN (R ')-,-N (R ')-N=,-C (R ')=N-,-C (R ')=N-N (R ')-,-C (R ')=N-N=,-C (R ') 2-N=N-and-C (R ') 2-N (R ')-N (R ')-, wherein R ' is H, alkyl independently of one another or is substituted alkyl;
J is
Figure A20058004432800902
Or
Figure A20058004432800903
R is H, alkyl, is substituted alkyl, cycloalkyl or is substituted cycloalkyl;
R " independently of one another for H, alkyl, " during group, two R " forms Heterocyclylalkyl according to circumstances to be substituted alkyl or protecting group, maybe when there being more than one R;
R 1Be H, amino protecting group, resin, amino acid, polypeptide or polynucleotide; And
R 2Be OH, ester protecting group, resin, amino acid, polypeptide or polynucleotide;
R 3With R 4Be H, halogen, low alkyl group or be substituted low alkyl group independently of one another, or R 3With R 4Or two R 3Group forms cycloalkyl or Heterocyclylalkyl according to circumstances;
Or-the A-B-J-R group common form comprise at least one carbonyl (comprising dicarbapentaborane), through protection carbonyl (comprising) or dicyclo or three cyclic rings alkyl or Heterocyclylalkyls through sheltering carbonyl (comprising) through sheltering dicarbapentaborane through the protection dicarbapentaborane;
Or-the J-R group common form comprise at least one carbonyl (comprising dicarbapentaborane), through protection carbonyl (comprising) or through sheltering monocycle or the double-ring cycloalkyl or the Heterocyclylalkyl of carbonyl (comprising) through sheltering dicarbapentaborane through the protection dicarbapentaborane;
Its restricted condition is phenylene and R for working as A 3When respectively doing for oneself H, B exists; And when A is-(CH 2) 4-and R 3When respectively doing for oneself H, B is not-NHC (O) (CH 2CH 2)-; And working as A and B does not exist and R 3When respectively doing for oneself H, R is not a methyl.These alpha-non-natural amino acids can be the form of salt, maybe can incorporate in non-natural amino acid polypeptides, polymkeric substance, polysaccharide or the polynucleotide and according to circumstances through posttranslational modification.
In certain embodiments, formula (I) compound was stablized 1 month in the aqueous solution under appropriate acidic conditions at least.In certain embodiments, formula (I) compound stable at least 2 weeks under appropriate acidic conditions.In certain embodiments, formula (I) compound was stablized 5 days under appropriate acidic conditions at least.In certain embodiments, described acidic conditions is pH 2 to 8.
In some embodiment of formula (I) compound, B is low-grade alkylidene, be substituted low-grade alkylidene ,-O-(alkylidene group or be substituted alkylidene group)-,-C (R ')=N-N (R ')-,-N (R ') CO-,-C (O)-,-C (R ')=N-,-C (O)-(alkylidene group or be substituted alkylidene group)-,-CON (R ')-(alkylidene group or be substituted alkylidene group)-,-S (alkylidene group or be substituted alkylidene group)-,-S (O) (alkylidene group or be substituted alkylidene group)-or-S (O) 2(alkylidene group or be substituted alkylidene group)-.In some embodiment of formula (I) compound, B is-O (CH 2)-,-CH=N-,-CH=N-NH-,-NHCH 2-,-NHCO-,-C (O)-,-C (O)-(CH 2)-,-CONH-(CH 2)-,-SCH 2-,-S (=O) CH 2-or-S (O) 2CH 2-.In some embodiment of formula (I) compound, R is C 1-6Alkyl or cycloalkyl.In some embodiment of formula (I) compound, R is-CH 3,-CH (CH 3) 2Or cyclopropyl.In some embodiment of formula (I) compound, R 1Be H, tertbutyloxycarbonyl (Boc), 9-fluorenyl methoxy carbonyl (Fmoc), N-ethanoyl, tetrafluoro ethanoyl (TFA) or carbobenzoxy-(Cbz) (Cbz).In some embodiment of formula (I) compound, R 1Be resin, amino acid, polypeptide or polynucleotide.In some embodiment of formula (I) compound, R 2Be OH, O-methyl, O-ethyl or the O-tertiary butyl.In some embodiment of formula (I) compound, R 2Be resin, amino acid, polypeptide or polynucleotide.In some embodiment of formula (I) compound, R 2Be polynucleotide.In some embodiment of formula (I) compound, R 2Be Yeast Nucleic Acid (RNA).In some embodiment of formula (I) compound, R 2Be tRNA.In some embodiment of formula (I) compound, the tRNA specific recognition is selected codon.In some embodiment of formula (I) compound, selecting codon is to be selected from by amber codon, ocher codon, opal codon, unique codon, rare codon, non-natural codon, five base codons and the molecular group of four base passwords.In some embodiment of formula (I) compound, R 2For suppressing tRNA.
In some embodiment of formula (I) compound,
Figure A20058004432800921
Be to be selected from the group that forms by following each thing:
(i) A is for being substituted low-grade alkylidene, C 4-arylidene, be substituted arylidene, inferior heteroaryl, be substituted inferior heteroaryl, alkarylene, be substituted alkarylene, inferior aralkyl or be substituted inferior aralkyl;
B is optionally, and is the divalence connexon that is selected from the group that is made up of following each group when existing: low-grade alkylidene, be substituted low-grade alkylidene, lower alkenylene, be substituted lower alkenylene ,-O-,-O-(alkylidene group or be substituted alkylidene group)-,-S-,-S (O)-,-S (O) 2-,-NS (O) 2-,-OS (O) 2-,-C (O)-,-C (O)-(alkylidene group or be substituted alkylidene group)-,-C (S)-,-N (R ')-,-C (O) N (R ')-,-CON (R ')-(alkylidene group or be substituted alkylidene group)-,-CSN (R ')-,-N (R ') CO-(alkylidene group or be substituted alkylidene group)-,-N (R ') C (O) O-,-N (R ') C (S)-,-S (O) N (R ') ,-S (O) 2N (R ') ,-N (R ') C (O) N (R ')-,-N (R ') C (S) N (R ')-,-N (R ') S (O) N (R ')-,-N (R ') S (O) 2N (R ')-,-N (R ')-N=,-C (R ')=N-N (R ')-,-C (R ')=N-N=,-C (R ') 2-N=N-and-C (R ') 2-N (R ')-N (R ')-;
(ii) A is optionally, and when existing for being substituted low-grade alkylidene, C 4-arylidene, be substituted arylidene, inferior heteroaryl, be substituted inferior heteroaryl, alkarylene, be substituted alkarylene, inferior aralkyl or be substituted inferior aralkyl;
B is the divalence connexon that is selected from the group that is made up of following each group: low-grade alkylidene, be substituted low-grade alkylidene, lower alkenylene, be substituted lower alkenylene ,-O-,-O-(alkylidene group or be substituted alkylidene group)-,-S-,-S (O)-,-S (O) 2-,-NS (O) 2-,-OS (O) 2-,-C (O)-,-C (O)-(alkylidene group or be substituted alkylidene group)-,-C (S)-,-N (R ')-,-C (O) N (R ')-,-CON (R ')-(alkylidene group or be substituted alkylidene group)-,-CSN (R ')-,-N (R ') CO-(alkylidene group or be substituted alkylidene group)-,-N (R ') C (O) O-,-N (R ') C (S)-,-S (O) N (R ') ,-S (O) 2N (R ') ,-N (R ') C (O) N (R ')-,-N (R ') C (S) N (R ')-,-N (R ') S (O) N (R ')-,-N (R ') S (O) 2N (R ')-,-N (R ')-N=,-C (R ')=N-N (R ')-,-C (R ')=N-N=,-C (R ') 2-N=N-and-C (R ') 2-N (R ')-N (R ')-;
(iii) A is a low-grade alkylidene;
B is optionally, and is the divalence connexon that is selected from the group that is made up of following each group when existing: low-grade alkylidene, be substituted low-grade alkylidene, lower alkenylene, be substituted lower alkenylene ,-O-,-O-(alkylidene group or be substituted alkylidene group)-,-S-,-S (O)-,-S (O) 2-,-NS (O) 2-,-OS (O) 2-,-C (O)-,-C (O)-(alkylidene group or be substituted alkylidene group)-,-C (S)-,-N (R ')-,-C (O) N (R ')-,-CSN (R ')-,-CON (R ')-(alkylidene group or be substituted alkylidene group)-,-N (R ') C (O) O-,-N (R ') C (S)-,-S (O) N (R ') ,-S (O) 2N (R ') ,-N (R ') C (O) N (R ')-,-N (R ') C (S) N (R ')-,-N (R ') S (O) N (R ')-,-N (R ') S (O) 2N (R ')-,-N (R ')-N=,-C (R ')=N-N (R ')-,-C (R ')=N-N=,-C (R ') 2-N=N-and-C (R ') 2-N (R ')-N (R ')-; And
(iv) A is a phenylene;
B is the divalence connexon that is selected from the group that is made up of following each group: low-grade alkylidene, be substituted low-grade alkylidene, lower alkenylene, be substituted lower alkenylene ,-O-,-O-(alkylidene group or be substituted alkylidene group)-,-S-,-S (O)-,-S (O) 2-,-NS (O) 2-,-OS (O) 2-,-C (O)-,-C (O)-(alkylidene group or be substituted alkylidene group)-,-C (S)-,-N (R ')-,-C (O) N (R ')-,-CON (R ')-(alkylidene group or be substituted alkylidene group)-,-CSN (R ')-,-N (R ') CO-(alkylidene group or be substituted alkylidene group)-,-N (R ') C (O) O-,-N (R ') C (S)-,-S (O) N (R ') ,-S (O) 2N (R ') ,-N (R ') C (O) N (R ')-,-N (R ') C (S) N (R ') ,-N (R ') S (O) N (R ')-,-N (R ') S (O) 2N (R ')-,-N (R ')-N=,-C (R ')=N-N (R ')-,-C (R ')=N-N=,-C (R ') 2-N=N-and-C (R ') 2-N (R ')-N (R ')-;
J is
Figure A20058004432800931
Or
Figure A20058004432800932
R ' is H, alkyl independently of one another or is substituted alkyl;
R 1For optionally, and when existing H, amino protecting group, resin, amino acid, polypeptide or polynucleotide; And
R 2For optionally, and when existing OH, ester protecting group, resin, amino acid, polypeptide or polynucleotide; And
R 3With R 4Be H, halogen, low alkyl group or be substituted low alkyl group independently of one another;
R is H, alkyl, is substituted alkyl, cycloalkyl or is substituted cycloalkyl;
In addition, comprise the have formula amino acid of structure of (II):
Figure A20058004432800933
Wherein:
A is optionally, and when exist, be low-grade alkylidene, be substituted low-grade alkylidene, rudimentary cycloalkylidene, be substituted rudimentary cycloalkylidene, lower alkenylene, be substituted lower alkenylene, alkynylene, the assorted alkyl in rudimentary Asia, be substituted inferior mix alkyl, rudimentary inferior Heterocyclylalkyl, be substituted rudimentary inferior Heterocyclylalkyl, arylidene, be substituted arylidene, inferior heteroaryl, be substituted inferior heteroaryl, alkarylene, be substituted alkarylene, inferior aralkyl or be substituted inferior aralkyl;
B is optionally, and is the connexon that is selected from the group that is made up of following each group when exist: low-grade alkylidene, be substituted low-grade alkylidene, lower alkenylene, be substituted lower alkenylene, alkyl is mixed in rudimentary Asia, be substituted rudimentary Asia mix alkyl ,-O-,-O-(alkylidene group or be substituted alkylidene group)-,-S-,-S-(alkylidene group or be substituted alkylidene group)-,-S (O) k-(wherein k is 1,2 or 3) ,-S (O) k(alkylidene group or be substituted alkylidene group)-,-C (O)-,-NS (O) 2-,-OS (O) 2-,-C (O)-(alkylidene group or be substituted alkylidene group)-,-C (S)-,-C (S)-(alkylidene group or be substituted alkylidene group)-,-N (R ')-,-NR '-(alkylidene group or be substituted alkylidene group)-,-C (O) N (R ')-,-CON (R ')-(alkylidene group or be substituted alkylidene group)-,-CSN (R ')-,-CSN (R ')-(alkylidene group or be substituted alkylidene group)-,-N (R ') CO-(alkylidene group or be substituted alkylidene group)-,-N (R ') C (O) O-,-S (O) kN (R ')-,-N (R ') C (O) N (R ')-,-N (R ') C (S) N (R ')-,-N (R ') S (O) kN (R ')-,-N (R ')-N=,-C (R ')=N-,-C (R ')=N-N (R ')-,-C (R ')=N-N=,-C (R ') 2-N=N-and-C (R ') 2-N (R ')-N (R ')-, wherein R ' is H, alkyl independently of one another or is substituted alkyl;
R is H, alkyl, is substituted alkyl, cycloalkyl or is substituted cycloalkyl;
R 1Be H, amino protecting group, resin, amino acid, polypeptide or polynucleotide; And
R 2Be OH, ester protecting group, resin, amino acid, polypeptide or polynucleotide;
Its restricted condition is for when A is phenylene, and B exists; And when A is-(CH 2) 4In-time, B is not-NHC (O) (CH 2CH 2)-; And when A and B did not exist, R was not a methyl.These alpha-non-natural amino acids can be the form of salt, maybe can incorporate in non-natural amino acid polypeptides, polymkeric substance, polysaccharide or the polynucleotide and according to circumstances through posttranslational modification.
In addition, comprise the have formula amino acid of structure of (III):
Figure A20058004432800941
Wherein:
B is the connexon that is selected from the group that is made up of following each group: low-grade alkylidene, be substituted low-grade alkylidene, lower alkenylene, be substituted lower alkenylene, the assorted alkyl in rudimentary Asia, be substituted the assorted alkyl in rudimentary Asia ,-O-,-O-(alkylidene group or be substituted alkylidene group)-,-S-,-S-(alkylidene group or be substituted alkylidene group)-,-S (O) k-(wherein k is 1,2 or 3) ,-S (O) k(alkylidene group or be substituted alkylidene group)-,-C (O)-,-NS (O) 2-,-OS (O) 2,-C (O)-(alkylidene group or be substituted alkylidene group)-,-C (S)-,-C (S)-(alkylidene group or be substituted alkylidene group)-,-N (R ')-,-NR '-(alkylidene group or be substituted alkylidene group)-,-C (O) N (R ')-,-CON (R ')-(alkylidene group or be substituted alkylidene group)-,-CSN (R ')-,-CSN (R ')-(alkylidene group or be substituted alkylidene group)-,-N (R ') CO-(alkylidene group or be substituted alkylidene group)-,-N (R ') C (O) O-,-S (O) kN (R ')-,-N (R ') C (O) N (R ')-,-N (R ') C (S) N (R ')-,-N (R ') S (O) kN (R ')-,-N (R ')-N=,-C (R ')=N-,-C (R ')=N-N (R ')-,-C (R ')=N-N=,-C (R ') 2-N=N-and-C (R ') 2-N (R ')-N (R ')-, wherein R ' is H, alkyl independently of one another or is substituted alkyl;
R is H, alkyl, is substituted alkyl, cycloalkyl or is substituted cycloalkyl;
R 1Be H, amino protecting group, resin, amino acid, polypeptide or polynucleotide; And
R 2Be OH, ester protecting group, resin, amino acid, polypeptide or polynucleotide;
R aBe selected from the group that forms by following each group: H, halogen, alkyl independently of one another, be substituted alkyl ,-N (R ') 2,-C (O) kR ' (wherein k is 1,2 or 3) ,-C (O) N (R ') 2,-OR ' and-S (O) kR ', wherein R ' is H, alkyl independently of one another or is substituted alkyl.These alpha-non-natural amino acids can be the form of salt, maybe can incorporate in non-natural amino acid polypeptides, polymkeric substance, polysaccharide or the polynucleotide and according to circumstances through posttranslational modification.
In addition, comprise following amino acid:
These alpha-non-natural amino acids can be according to circumstances amino group through protection, carboxyl through the group of protection and/or be the form of salt, maybe can incorporate in non-natural amino acid polypeptides, polymkeric substance, polysaccharide or the polynucleotide and according to circumstances through posttranslational modification.
In addition, comprise following amino acid with structure of formula (IV):
Figure A20058004432800952
Wherein
-NS (O) 2-,-OS (O) 2-, optionally, and be the connexon that is selected from the group that forms by following each group when exist: low-grade alkylidene, be substituted low-grade alkylidene, lower alkenylene, be substituted lower alkenylene, alkyl is mixed in rudimentary Asia, be substituted rudimentary Asia mix alkyl ,-O-,-O-(alkylidene group or be substituted alkylidene group)-,-S-,-S-(alkylidene group or be substituted alkylidene group)-,-S (O) k-(wherein k is 1,2 or 3) ,-S (O) k(alkylidene group or be substituted alkylidene group)-,-C (O)-,-C (O)-(alkylidene group or be substituted alkylidene group)-,-C (S)-,-C (S)-(alkylidene group or be substituted alkylidene group)-,-N (R ')-,-NR '-(alkylidene group or be substituted alkylidene group)-,-C (O) N (R ')-,-CON (R ')-(alkylidene group or be substituted alkylidene group)-,-CSN (R ')-,-CSN (R ')-(alkylidene group or be substituted alkylidene group)-,-N (R ') CO-(alkylidene group or be substituted alkylidene group)-,-N (R ') C (O) O-,-S (O) kN (R ')-,-N (R ') C (O) N (R ')-,-N (R ') C (S) N (R ')-,-N (R ') S (O) kN (R ')-,-N (R ')-N=,-C (R ')=N-,-C (R ')=N-N (R ')-,-C (R ')=N-N=,-C (R ') 2-N=N-and-C (R ') 2-N (R ')-N (R ')-, wherein R ' is H, alkyl independently of one another or is substituted alkyl;
R is H, alkyl, is substituted alkyl, cycloalkyl or is substituted cycloalkyl;
R 1Be H, amino protecting group, resin, amino acid, polypeptide or polynucleotide; And
R 2Be OH, ester protecting group, resin, amino acid, polypeptide or polynucleotide;
R aBe selected from the group that forms by following each group: H, halogen, alkyl independently of one another, be substituted alkyl ,-N (R ') 2,-C (O) kR ' (wherein k is 1,2 or 3) ,-C (O) N (R ') 2,-OR ' and-S (O) kR ', wherein R ' is H, alkyl independently of one another or is substituted alkyl; And n is 0 to 8;
Its restricted condition is for as A being-(CH 2) 4In-time, B is not-NHC (O) (CH 2CH 2)-.These alpha-non-natural amino acids can be the form of salt, maybe can incorporate in non-natural amino acid polypeptides, polymkeric substance, polysaccharide or the polynucleotide and according to circumstances through posttranslational modification.
In addition, comprise following amino acid:
Figure A20058004432800961
Figure A20058004432800962
And
Figure A20058004432800963
Wherein these compounds amino according to circumstances through protection, according to circumstances carboxyl through protection, according to circumstances amino through protection and carboxyl through protection; or be its salt, maybe can incorporate in non-natural amino acid polypeptides, polymkeric substance, polysaccharide or the polynucleotide and according to circumstances through posttranslational modification.
In addition, comprise following amino acid with structure of formula (VIII):
Figure A20058004432800971
Wherein,
A is optionally, and when exist, be low-grade alkylidene, be substituted low-grade alkylidene, rudimentary cycloalkylidene, be substituted rudimentary cycloalkylidene, lower alkenylene, be substituted lower alkenylene, alkynylene, the assorted alkyl in rudimentary Asia, be substituted inferior mix alkyl, rudimentary inferior Heterocyclylalkyl, be substituted rudimentary inferior Heterocyclylalkyl, arylidene, be substituted arylidene, inferior heteroaryl, be substituted inferior heteroaryl, alkarylene, be substituted alkarylene, inferior aralkyl or be substituted inferior aralkyl;
B is optionally, and is the connexon that is selected from the group that is made up of following each group when exist: low-grade alkylidene, be substituted low-grade alkylidene, lower alkenylene, be substituted lower alkenylene, alkyl is mixed in rudimentary Asia, be substituted rudimentary Asia mix alkyl ,-O-,-O-(alkylidene group or be substituted alkylidene group)-,-S-,-S-(alkylidene group or be substituted alkylidene group)-,-S (O) k-(wherein k is 1,2 or 3) ,-S (O) k(alkylidene group or be substituted alkylidene group)-,-C (O)-,-NS (O) 2-,-OS (O) 2-,-C (O)-(alkylidene group or be substituted alkylidene group)-,-C (S)-,-C (S)-(alkylidene group or be substituted alkylidene group)-,-N (R ')-,-NR '-(alkylidene group or be substituted alkylidene group)-,-C (O) N (R ')-,-CON (R ')-(alkylidene group or be substituted alkylidene group)-,-CSN (R ')-,-CSN (R ')-(alkylidene group or be substituted alkylidene group)-,-N (R ') CO-(alkylidene group or be substituted alkylidene group)-,-N (R ') C (O) O-,-S (O) kN (R ')-,-N (R ') C (O) N (R ')-,-N (R ') C (S) N (R ')-,-N (R ') S (O) kN (R ')-,-N (R ')-N=,-C (R ')=N-,-C (R ')=N-N (R ')-,-C (R ')=N-N=,-C (R ') 2-N=N-and-C (R ') 2-N (R ')-N (R ')-, wherein R ' is H, alkyl independently of one another or is substituted alkyl;
R 1Be H, amino protecting group, resin, amino acid, polypeptide or polynucleotide; And
R 2Be OH, ester protecting group, resin, amino acid, polypeptide or polynucleotide.
These alpha-non-natural amino acids can be the form of salt, maybe can incorporate in non-natural amino acid polypeptides, polymkeric substance, polysaccharide or the polynucleotide and according to circumstances through posttranslational modification.
In addition, comprise following amino acid with structure of formula (IX):
Figure A20058004432800981
Wherein,
B is optionally, and is the connexon that is selected from the group that is made up of following each group when exist: low-grade alkylidene, be substituted low-grade alkylidene, lower alkenylene, be substituted lower alkenylene, alkyl is mixed in rudimentary Asia, be substituted rudimentary Asia mix alkyl ,-O-,-O-(alkylidene group or be substituted alkylidene group)-,-S-,-S-(alkylidene group or be substituted alkylidene group)-,-S (O) k-(wherein k is 1,2 or 3) ,-S (O) k(alkylidene group or be substituted alkylidene group)-,-C (O)-,-NS (O) 2-,-OS (C)) 2-,-C (O)-(alkylidene group or be substituted alkylidene group)-,-C (S)-,-C (S)-(alkylidene group or be substituted alkylidene group)-,-N (R ')-,-NR '-(alkylidene group or be substituted alkylidene group)-,-C (O) N (R ')-,-CON (R ')-(alkylidene group or be substituted alkylidene group)-,-CSN (R ')-,-CSN (R ')-(alkylidene group or be substituted alkylidene group)-,-N (R ') CO-(alkylidene group or be substituted alkylidene group)-,-N (R ') C (O) O-,-S (O) kN (R ')-,-N (R ') C (O) N (R ')-,-N (R ') C (S) N (R ')-,-N (R ') S (O) kN (R ') ,-N (R ')-N=,-C (R ')=N-,-C (R ')=N-N (R ')-,-C (R ')=N-N=,-C (R ') 2-N=N-and-C (R ') 2-N (R ')-N (R ')-, wherein R ' is H, alkyl independently of one another or is substituted alkyl;
R is H, alkyl, is substituted alkyl, cycloalkyl or is substituted cycloalkyl;
R 1Be H, amino protecting group, resin, amino acid, polypeptide or polynucleotide; And
R 2Be OH, ester protecting group, resin, amino acid, polypeptide or polynucleotide;
R wherein aBe selected from the group that forms by following each group: H, halogen, alkyl independently of one another, be substituted alkyl ,-N (R ') 2,-C (O) kR ' (wherein k is 1,2 or 3) ,-C (O) N (R ') 2,-OR ' and-S (O) kR ', wherein R ' is H, alkyl independently of one another or is substituted alkyl.
These alpha-non-natural amino acids can be the form of salt, maybe can incorporate in non-natural amino acid polypeptides, polymkeric substance, polysaccharide or the polynucleotide and according to circumstances through posttranslational modification.
In addition, comprise following amino acid:
Figure A20058004432800991
Wherein these compounds amino according to circumstances through protection, according to circumstances carboxyl through protection, according to circumstances amino through protection and carboxyl through protection; or be its salt, maybe can incorporate in non-natural amino acid polypeptides, polymkeric substance, polysaccharide or the polynucleotide and according to circumstances through posttranslational modification.
In addition, comprise following amino acid with structure of formula (X):
Figure A20058004432800992
Wherein,
B is optionally, and is the connexon that is selected from the group that is made up of following each group when exist: low-grade alkylidene, be substituted low-grade alkylidene, lower alkenylene, be substituted lower alkenylene, alkyl is mixed in rudimentary Asia, be substituted rudimentary Asia mix alkyl ,-O-,-O-(alkylidene group or be substituted alkylidene group)-,-S-,-S-(alkylidene group or be substituted alkylidene group)-,-S (O) k-(wherein k is 1,2 or 3) ,-S (O) k(alkylidene group or be substituted alkylidene group)-,-C (O)-,-NS (O) 2-,-OS (O) 2-,-C (O)-(alkylidene group or be substituted alkylidene group)-,-C (S)-,-C (S)-(alkylidene group or be substituted alkylidene group)-,-N (R ')-,-NR '-(alkylidene group or be substituted alkylidene group)-,-C (O) N (R ') ,-CON (R ')-(alkylidene group or be substituted alkylidene group)-,-CSN (R ')-,-CSN (R ')-(alkylidene group or be substituted alkylidene group)-,-N (R ') CO-(alkylidene group or be substituted alkylidene group)-,-N (R ') C (O) O-,-S (O) kN (R ')-,-N (R ') C (O) N (R ')-,-N (R ') C (S) N (R ')-,-N (R ') S (O) kN (R ')-,-N (R ')-N=,-C (R ')=N-,-C (R ')=N-N (R ')-,-C (R ')=N-N=,-C (R ') 2-N=N-and-C (R ') 2-N (R ')-N (R ')-, wherein R ' is H, alkyl independently of one another or is substituted alkyl;
R is H, alkyl, is substituted alkyl, cycloalkyl or is substituted cycloalkyl;
R 1Be H, amino protecting group, resin, amino acid, polypeptide or polynucleotide; And
R 2Be OH, ester protecting group, resin, amino acid, polypeptide or polynucleotide;
R aBe selected from the group that forms by following each group: H, halogen, alkyl independently of one another, be substituted alkyl ,-N (R ') 2,-C (O) kR ' (wherein k is 1,2 or 3) ,-C (O) N (R ') 2,-OR ' and-S (O) kR ', wherein R ' is H, alkyl independently of one another or is substituted alkyl; And n is 0 to 8.
These alpha-non-natural amino acids can be the form of salt, maybe can incorporate in non-natural amino acid polypeptides, polymkeric substance, polysaccharide or the polynucleotide and according to circumstances through posttranslational modification.
In addition, comprise following amino acid:
Figure A20058004432801001
And
Figure A20058004432801002
Wherein these compounds amino according to circumstances through protection, according to circumstances carboxyl through protection, according to circumstances amino through protection and carboxyl through protection; or be its salt, maybe can incorporate in non-natural amino acid polypeptides, polymkeric substance, polysaccharide or the polynucleotide and according to circumstances through posttranslational modification.
Except mono carbonyl structure, the alpha-non-natural amino acid of Miao Shuing can comprise such as dicarbapentaborane, class dicarbapentaborane, through sheltering dicarbapentaborane and through the group of protection dicarbapentaborane herein.
For instance, comprise following amino acid with structure of formula V:
Figure A20058004432801003
Wherein,
A is optionally, and when exist, be low-grade alkylidene, be substituted low-grade alkylidene, rudimentary cycloalkylidene, be substituted rudimentary cycloalkylidene, lower alkenylene, be substituted lower alkenylene, alkynylene, the assorted alkyl in rudimentary Asia, be substituted inferior mix alkyl, rudimentary inferior Heterocyclylalkyl, be substituted rudimentary inferior Heterocyclylalkyl, arylidene, be substituted arylidene, inferior heteroaryl, be substituted inferior heteroaryl, alkarylene, be substituted alkarylene, inferior aralkyl or be substituted inferior aralkyl;
B is optionally, and is the connexon that is selected from the group that is made up of following each group when exist: low-grade alkylidene, be substituted low-grade alkylidene, lower alkenylene, be substituted lower alkenylene, alkyl is mixed in rudimentary Asia, be substituted rudimentary Asia mix alkyl ,-O-,-O-(alkylidene group or be substituted alkylidene group)-,-S-,-S-(alkylidene group or be substituted alkylidene group)-,-S (O) k-(wherein k is 1,2 or 3) ,-S (O) k(alkylidene group or be substituted alkylidene group)-,-C (O)-,-NS (O) 2-,-OS (O) 2-,-C (O)-(alkylidene group or be substituted alkylidene group)-,-C (S)-,-C (S)-(alkylidene group or be substituted alkylidene group)-,-N (R ')-,-NR '-(alkylidene group or be substituted alkylidene group)-,-C (O) N (R ')-,-CON (R ')-(alkylidene group or be substituted alkylidene group)-,-CSN (R ')-,-CSN (R ')-(alkylidene group or be substituted alkylidene group)-,-N (R ') CO-(alkylidene group or be substituted alkylidene group)-,-N (R ') C (O) O-,-S (O) kN (R ')-,-N (R ') C (O) N (R ')-,-N (R ') C (S) N (R ')-,-N (R ') S (O) kN (R ')-,-N (R ')-N=,-C (R ')=N-,-C (R ')=N-N (R ')-,-C (R ')=N-N=,-C (R ') 2-N=N-and-C (R ') 2-N (R ')-N (R ')-, wherein R ' is H, alkyl independently of one another or is substituted alkyl;
R is H, alkyl, is substituted alkyl, cycloalkyl or is substituted cycloalkyl;
R 1Be H, amino protecting group, resin, amino acid, polypeptide or polynucleotide; And
R 2Be OH, ester protecting group, resin, amino acid, polypeptide or polynucleotide.
These alpha-non-natural amino acids can be the form of salt, maybe can incorporate in non-natural amino acid polypeptides, polymkeric substance, polysaccharide or the polynucleotide and according to circumstances through posttranslational modification.
In addition, comprise following amino acid with structure of formula (VI):
Figure A20058004432801011
Wherein,
B is optionally, and is the connexon that is selected from the group that is made up of following each group when exist: low-grade alkylidene, be substituted low-grade alkylidene, lower alkenylene, be substituted lower alkenylene, alkyl is mixed in rudimentary Asia, be substituted rudimentary Asia mix alkyl ,-O-,-O-(alkylidene group or be substituted alkylidene group)-,-S-,-S-(alkylidene group or be substituted alkylidene group)-,-S (O) k-(wherein k is 1,2 or 3) ,-S (O) k(alkylidene group or be substituted alkylidene group)-,-C (O)-,-NS (O) 2-,-OS (O) 2-,-C (O)-(alkylidene group or be substituted alkylidene group)-,-C (S)-,-C (S)-(alkylidene group or be substituted alkylidene group)-,-N (R ')-,-NR '-(alkylidene group or be substituted alkylidene group)-,-C (O) N (R ')-,-CON (R ')-(alkylidene group or be substituted alkylidene group)-,-CSN (R ')-,-CSN (R ')-(alkylidene group or be substituted alkylidene group)-,-N (R ') CO-(alkylidene group or be substituted alkylidene group)-,-N (R ') C (O) O-,-S (O) kN (R ')-,-N (R ') C (O) N (R ')-,-N (R ') C (S) N (R ')-,-N (R ') S (O) kN (R ')-,-N (R ')-N=,-C (R ')=N-,-C (R ')=N-N (R ')-,-C (R ')=N-N=,-C (R ') 2-N=N-and-C (R ') 2-N (R ')-N (R ')-, wherein R ' is H, alkyl independently of one another or is substituted alkyl;
R is H, alkyl, is substituted alkyl, cycloalkyl or is substituted cycloalkyl;
R 1Be H, amino protecting group, resin, amino acid, polypeptide or polynucleotide; And
R 2Be OH, ester protecting group, resin, amino acid, polypeptide or polynucleotide;
R wherein aBe selected from the group that forms by following each group: H, halogen, alkyl independently of one another, be substituted alkyl ,-N (R ') 2,-C (O) kR ' (wherein k is 1,2 or 3) ,-C (O) N (R ') 2,-OR ' and-S (O) kR ', wherein R ' is H, alkyl independently of one another or is substituted alkyl.
These alpha-non-natural amino acids can be the form of salt, maybe can incorporate in non-natural amino acid polypeptides, polymkeric substance, polysaccharide or the polynucleotide and according to circumstances through posttranslational modification.
In addition, comprise following amino acid:
Figure A20058004432801021
Wherein these compounds amino according to circumstances through protection and carboxyl through protection, or be its salt.These alpha-non-natural amino acids can be the form of salt, maybe can incorporate in non-natural amino acid polypeptides, polymkeric substance, polysaccharide or the polynucleotide and according to circumstances through posttranslational modification.
In addition, comprise following amino acid with structure of formula (VII):
Figure A20058004432801022
Wherein,
B is optionally, and is the connexon that is selected from the group that is made up of following each group when exist: low-grade alkylidene, be substituted low-grade alkylidene, lower alkenylene, be substituted lower alkenylene, alkyl is mixed in rudimentary Asia, be substituted rudimentary Asia mix alkyl ,-O-,-O-(alkylidene group or be substituted alkylidene group)-,-S-,-S-(alkylidene group or be substituted alkylidene group)-,-S (O) k-(wherein k is 1,2 or 3) ,-S (O) k(alkylidene group or be substituted alkylidene group)-,-C (O)-,-NS (O) 2-,-OS (O) 2-,-C (O)-(alkylidene group or be substituted alkylidene group)-,-C (S)-,-C (S)-(alkylidene group or be substituted alkylidene group)-,-N (R ')-,-NR '-(alkylidene group or be substituted alkylidene group)-,-C (O) N (R ') ,-CON (R ')-(alkylidene group or be substituted alkylidene group)-,-CSN (R ')-,-CSN (R ')-(alkylidene group or be substituted alkylidene group)-,-N (R ') CO-(alkylidene group or be substituted alkylidene group)-,-N (R ') C (O) O-,-S (O) kN (R ')-,-N (R ') C (O) N (R ')-,-N (R ') C (S) N (R ')-,-N (R ') S (O) kN (R ')-,-N (R ')-N=,-C (R ')=N-,-C (R ')=N-N (R ')-,-C (R ')=N-N=,-C (R ') 2-N=N-and-C (R ') 2-N (R ')-N (R ')-, wherein R ' is H, alkyl independently of one another or is substituted alkyl;
R is H, alkyl, is substituted alkyl, cycloalkyl or is substituted cycloalkyl;
R 1Be H, amino protecting group, resin, amino acid, polypeptide or polynucleotide; And
R 2Be OH, ester protecting group, resin, amino acid, polypeptide or polynucleotide;
R aBe selected from the group that forms by following each group: H, halogen, alkyl independently of one another, be substituted alkyl ,-N (R ') 2,-C (O) kR ' (wherein k is 1,2 or 3) ,-C (O) N (R ') 2,-OR ' and-S (O) kR ', wherein R ' is H, alkyl independently of one another or is substituted alkyl; And n is 0 to 8.
These alpha-non-natural amino acids can be the form of salt, maybe can incorporate in non-natural amino acid polypeptides, polymkeric substance, polysaccharide or the polynucleotide and according to circumstances through posttranslational modification.
In addition, comprise following amino acid:
Figure A20058004432801031
And
Figure A20058004432801032
，
Wherein these compounds amino according to circumstances through protection and carboxyl through protection, or be its salt, maybe can incorporate in non-natural amino acid polypeptides, polymkeric substance, polysaccharide or the polynucleotide and according to circumstances through posttranslational modification.
In addition, comprise following amino acid with structure of formula (XXX):
Figure A20058004432801041
Wherein:
A is optionally, and when exist, be low-grade alkylidene, be substituted low-grade alkylidene, rudimentary cycloalkylidene, be substituted rudimentary cycloalkylidene, lower alkenylene, be substituted lower alkenylene, alkynylene, the assorted alkyl in rudimentary Asia, be substituted inferior mix alkyl, rudimentary inferior Heterocyclylalkyl, be substituted rudimentary inferior Heterocyclylalkyl, arylidene, be substituted arylidene, inferior heteroaryl, be substituted inferior heteroaryl, alkarylene, be substituted alkarylene, inferior aralkyl or be substituted inferior aralkyl;
R is H, alkyl, is substituted alkyl, cycloalkyl or is substituted cycloalkyl;
R 1Be H, amino protecting group, resin, amino acid, polypeptide or polynucleotide; And
R 2Be OH, ester protecting group, resin, amino acid, polypeptide or polynucleotide;
X 1Be C, S or S (O); And L is alkylidene group, is substituted alkylidene group, N (R ') (alkylidene group) or N (R ') (being substituted alkylidene group) that wherein R ' is H, alkyl, is substituted alkyl, cycloalkyl or is substituted cycloalkyl.
These alpha-non-natural amino acids can be the form of salt, maybe can incorporate in non-natural amino acid polypeptides, polymkeric substance, polysaccharide or the polynucleotide and according to circumstances through posttranslational modification.
In addition, comprise following amino acid with structure of formula (XXX-A):
Figure A20058004432801042
Wherein:
A is optionally, and when exist, be low-grade alkylidene, be substituted low-grade alkylidene, rudimentary cycloalkylidene, be substituted rudimentary cycloalkylidene, lower alkenylene, be substituted lower alkenylene, alkynylene, the assorted alkyl in rudimentary Asia, be substituted inferior mix alkyl, rudimentary inferior Heterocyclylalkyl, be substituted rudimentary inferior Heterocyclylalkyl, arylidene, be substituted arylidene, inferior heteroaryl, be substituted inferior heteroaryl, alkarylene, be substituted alkarylene, inferior aralkyl or be substituted inferior aralkyl;
R is H, alkyl, is substituted alkyl, cycloalkyl or is substituted cycloalkyl;
R 1Be H, amino protecting group, resin, amino acid, polypeptide or polynucleotide; And
R 2Be OH, ester protecting group, resin, amino acid, polypeptide or polynucleotide;
L is alkylidene group, is substituted alkylidene group, N (R ') (alkylidene group) or N (R ') (being substituted alkylidene group) that wherein R ' is H, alkyl, is substituted alkyl, cycloalkyl or is substituted cycloalkyl.
These alpha-non-natural amino acids can be the form of salt, maybe can incorporate in non-natural amino acid polypeptides, polymkeric substance, polysaccharide or the polynucleotide and according to circumstances through posttranslational modification.
In addition, comprise following amino acid with structure of formula (XXX-B):
Figure A20058004432801051
Wherein:
A is optionally, and when exist, be low-grade alkylidene, be substituted low-grade alkylidene, rudimentary cycloalkylidene, be substituted rudimentary cycloalkylidene, lower alkenylene, be substituted lower alkenylene, alkynylene, the assorted alkyl in rudimentary Asia, be substituted inferior mix alkyl, rudimentary inferior Heterocyclylalkyl, be substituted rudimentary inferior Heterocyclylalkyl, arylidene, be substituted arylidene, inferior heteroaryl, be substituted inferior heteroaryl, alkarylene, be substituted alkarylene, inferior aralkyl or be substituted inferior aralkyl;
R is H, alkyl, is substituted alkyl, cycloalkyl or is substituted cycloalkyl;
R 1Be H, amino protecting group, resin, amino acid, polypeptide or polynucleotide;
And R 2Be OH, ester protecting group, resin, amino acid, polypeptide or polynucleotide:
L is alkylidene group, is substituted alkylidene group, N (R ') (alkylidene group) or N (R ') (being substituted alkylidene group) that wherein R ' is H, alkyl, is substituted alkyl, cycloalkyl or is substituted cycloalkyl.
These alpha-non-natural amino acids can be the form of salt, maybe can incorporate in non-natural amino acid polypeptides, polymkeric substance, polysaccharide or the polynucleotide and according to circumstances through posttranslational modification.
In addition, comprise following amino acid with structure of formula (XXXI):
Wherein:
A is optionally, and when exist, be low-grade alkylidene, be substituted low-grade alkylidene, rudimentary cycloalkylidene, be substituted rudimentary cycloalkylidene, lower alkenylene, be substituted lower alkenylene, alkynylene, the assorted alkyl in rudimentary Asia, be substituted inferior mix alkyl, rudimentary inferior Heterocyclylalkyl, be substituted rudimentary inferior Heterocyclylalkyl, arylidene, be substituted arylidene, inferior heteroaryl, be substituted inferior heteroaryl, alkarylene, be substituted alkarylene, inferior aralkyl or be substituted inferior aralkyl;
R is H, alkyl, is substituted alkyl, cycloalkyl or is substituted cycloalkyl;
R 1Be H, amino protecting group, resin, amino acid, polypeptide or polynucleotide; And
R 2Be OH, ester protecting group, resin, amino acid, polypeptide or polynucleotide;
X 1Be C, S or S (O); And n is 0,1,2,3,4 or 5; And each CR 8R 9R on the group 8And R 9Be selected from the group that forms by H, alkoxyl group, alkylamine, halogen, alkyl, aryl independently of one another, or any R 8And R 9Can form jointly=O or cycloalkyl, or any and adjacent R 8Group can form cycloalkyl jointly.
These alpha-non-natural amino acids can be the form of salt, maybe can incorporate in non-natural amino acid polypeptides, polymkeric substance, polysaccharide or the polynucleotide and according to circumstances through posttranslational modification.
In addition, comprise following amino acid with structure of formula (XXXI-A):
Figure A20058004432801062
Wherein:
A is optionally, and when exist, be low-grade alkylidene, be substituted low-grade alkylidene, rudimentary cycloalkylidene, be substituted rudimentary cycloalkylidene, lower alkenylene, be substituted lower alkenylene, alkynylene, the assorted alkyl in rudimentary Asia, be substituted inferior mix alkyl, rudimentary inferior Heterocyclylalkyl, be substituted rudimentary inferior Heterocyclylalkyl, arylidene, be substituted arylidene, inferior heteroaryl, be substituted inferior heteroaryl, alkarylene, be substituted alkarylene, inferior aralkyl or be substituted inferior aralkyl;
R is H, alkyl, is substituted alkyl, cycloalkyl or is substituted cycloalkyl;
R 1Be H, amino protecting group, resin, amino acid, polypeptide or polynucleotide; And
R 2Be OH, ester protecting group, resin, amino acid, polypeptide or polynucleotide;
N is 0,1,2,3,4 or 5; And each CR 8R 9R on the group 8And R 9Be selected from the group that forms by H, alkoxyl group, alkylamine, halogen, alkyl, aryl independently of one another, or any R 8And R 9Can form jointly=O or cycloalkyl, or any and adjacent R 8Group can form cycloalkyl jointly.
These alpha-non-natural amino acids can be the form of salt, maybe can incorporate in non-natural amino acid polypeptides, polymkeric substance, polysaccharide or the polynucleotide and according to circumstances through posttranslational modification.
In addition, comprise following amino acid with structure of formula (XXXI-B):
Figure A20058004432801071
Wherein:
A is optionally, and when exist, be low-grade alkylidene, be substituted low-grade alkylidene, rudimentary cycloalkylidene, be substituted rudimentary cycloalkylidene, lower alkenylene, be substituted lower alkenylene, alkynylene, the assorted alkyl in rudimentary Asia, be substituted inferior mix alkyl, rudimentary inferior Heterocyclylalkyl, be substituted rudimentary inferior Heterocyclylalkyl, arylidene, be substituted arylidene, inferior heteroaryl, be substituted inferior heteroaryl, alkarylene, be substituted alkarylene, inferior aralkyl or be substituted inferior aralkyl;
R is H, alkyl, is substituted alkyl, cycloalkyl or is substituted cycloalkyl;
R 1Be H, amino protecting group, resin, amino acid, polypeptide or polynucleotide; And
R 2Be OH, ester protecting group, resin, amino acid, polypeptide or polynucleotide;
N is 0,1,2,3,4 or 5; And each CR 8R 9R on the group 8And R 9Be selected from the group that forms by H, alkoxyl group, alkylamine, halogen, alkyl, aryl independently of one another, or any R 8And R 9Can form jointly=O or cycloalkyl, or any and adjacent R 8Group can form cycloalkyl jointly.
These alpha-non-natural amino acids can be the form of salt, maybe can incorporate in non-natural amino acid polypeptides, polymkeric substance, polysaccharide or the polynucleotide and according to circumstances through posttranslational modification.
In addition, comprise following amino acid with structure of formula (XXXII):
Figure A20058004432801081
Wherein:
A is optionally, and when exist, be low-grade alkylidene, be substituted low-grade alkylidene, rudimentary cycloalkylidene, be substituted rudimentary cycloalkylidene, lower alkenylene, be substituted lower alkenylene, alkynylene, the assorted alkyl in rudimentary Asia, be substituted inferior mix alkyl, rudimentary inferior Heterocyclylalkyl, be substituted rudimentary inferior Heterocyclylalkyl, arylidene, be substituted arylidene, inferior heteroaryl, be substituted inferior heteroaryl, alkarylene, be substituted alkarylene, inferior aralkyl or be substituted inferior aralkyl;
R is H, alkyl, is substituted alkyl, cycloalkyl or is substituted cycloalkyl;
R 1Be H, amino protecting group, resin, amino acid, polypeptide or polynucleotide; And
R 2Be OH, ester protecting group, resin, amino acid, polypeptide or polynucleotide;
X 1Be C, S or S (O); And L is alkylidene group, is substituted alkylidene group, N (R ') (alkylidene group) or N (R ') (being substituted alkylidene group) that wherein R ' is H, alkyl, is substituted alkyl, cycloalkyl or is substituted cycloalkyl.
These alpha-non-natural amino acids can be the form of salt, maybe can incorporate in non-natural amino acid polypeptides, polymkeric substance, polysaccharide or the polynucleotide and according to circumstances through posttranslational modification.
In addition, comprise following amino acid with structure of formula (XXXII-A):
Figure A20058004432801082
Wherein:
A is optionally, and when exist, be low-grade alkylidene, be substituted low-grade alkylidene, rudimentary cycloalkylidene, be substituted rudimentary cycloalkylidene, lower alkenylene, be substituted lower alkenylene, alkynylene, the assorted alkyl in rudimentary Asia, be substituted inferior mix alkyl, rudimentary inferior Heterocyclylalkyl, be substituted rudimentary inferior Heterocyclylalkyl, arylidene, be substituted arylidene, inferior heteroaryl, be substituted inferior heteroaryl, alkarylene, be substituted alkarylene, inferior aralkyl or be substituted inferior aralkyl;
R is H, alkyl, is substituted alkyl, cycloalkyl or is substituted cycloalkyl;
R 1Be H, amino protecting group, resin, amino acid, polypeptide or polynucleotide; And
R 2Be OH, ester protecting group, resin, amino acid, polypeptide or polynucleotide;
L is alkylidene group, is substituted alkylidene group, N (R ') (alkylidene group) or N (R ') (being substituted alkylidene group) that wherein R ' is H, alkyl, is substituted alkyl, cycloalkyl or is substituted cycloalkyl.
These alpha-non-natural amino acids can be the form of salt, maybe can incorporate in non-natural amino acid polypeptides, polymkeric substance, polysaccharide or the polynucleotide and according to circumstances through posttranslational modification.
In addition, comprise following amino acid with structure of formula (XXXII-B):
Wherein:
A is optionally, and when exist, be low-grade alkylidene, be substituted low-grade alkylidene, rudimentary cycloalkylidene, be substituted rudimentary cycloalkylidene, lower alkenylene, be substituted lower alkenylene, alkynylene, the assorted alkyl in rudimentary Asia, be substituted inferior mix alkyl, rudimentary inferior Heterocyclylalkyl, be substituted rudimentary inferior Heterocyclylalkyl, arylidene, be substituted arylidene, inferior heteroaryl, be substituted inferior heteroaryl, alkarylene, be substituted alkarylene, inferior aralkyl or be substituted inferior aralkyl;
R is H, alkyl, is substituted alkyl, cycloalkyl or is substituted cycloalkyl;
R 1Be H, amino protecting group, resin, amino acid, polypeptide or polynucleotide; And
R 2Be OH, ester protecting group, resin, amino acid, polypeptide or polynucleotide;
L is alkylidene group, is substituted alkylidene group, N (R ') (alkylidene group) or N (R ') (being substituted alkylidene group) that wherein R ' is H, alkyl, is substituted alkyl, cycloalkyl or is substituted cycloalkyl.
These alpha-non-natural amino acids can be the form of salt, maybe can incorporate in non-natural amino acid polypeptides, polymkeric substance, polysaccharide or the polynucleotide and according to circumstances through posttranslational modification.
In addition, comprise the have formula amino acid of structure of (XXXX):
Figure A20058004432801101
Wherein:
A is optionally, and when exist, be low-grade alkylidene, be substituted low-grade alkylidene, rudimentary cycloalkylidene, be substituted rudimentary cycloalkylidene, lower alkenylene, be substituted lower alkenylene, alkynylene, the assorted alkyl in rudimentary Asia, be substituted inferior mix alkyl, rudimentary inferior Heterocyclylalkyl, be substituted rudimentary inferior Heterocyclylalkyl, arylidene, be substituted arylidene, inferior heteroaryl, be substituted inferior heteroaryl, alkarylene, be substituted alkarylene, inferior aralkyl or be substituted inferior aralkyl;
M is-C (R 3)-,
Figure A20058004432801102
Wherein (a) indication and A group bond and (b) indication and carbonyl bond out of the ordinary, R 3With R 4Be independently selected from H, halogen, alkyl, be substituted alkyl, cycloalkyl or be substituted cycloalkyl, or R 3With R 4Or two R 3Group or two R 4Group forms cycloalkyl or Heterocyclylalkyl according to circumstances;
R is H, halogen, alkyl, is substituted alkyl, cycloalkyl or is substituted cycloalkyl;
T 3Be a key, C (R) (R), O or S, and R is H, halogen, alkyl, is substituted alkyl, cycloalkyl or is substituted cycloalkyl;
R 1Be H, amino protecting group, resin, amino acid, polypeptide or polynucleotide; And
R 2Be OH, ester protecting group, resin, amino acid, polypeptide or polynucleotide.
These alpha-non-natural amino acids can be the form of salt, maybe can incorporate in non-natural amino acid polypeptides, polymkeric substance, polysaccharide or the polynucleotide and according to circumstances through posttranslational modification.
In addition, comprise the have formula amino acid of structure of (XXXXI):
Figure A20058004432801111
Wherein:
M is-C (R 3)-,
Figure A20058004432801112
Figure A20058004432801113
Wherein (a) indication and A group bond and (b) indication and carbonyl bond out of the ordinary, R 3With R 4Be independently selected from H, halogen, alkyl, be substituted alkyl, cycloalkyl or be substituted cycloalkyl, or R 3With R 4Or two R 3Group or two R 4Group forms cycloalkyl or Heterocyclylalkyl according to circumstances;
R is H, halogen, alkyl, is substituted alkyl, cycloalkyl or is substituted cycloalkyl;
T 3Be a key, C (R) (R), O or S, and R is H, halogen, alkyl, is substituted alkyl, cycloalkyl or is substituted cycloalkyl;
R 1Be H, amino protecting group, resin, amino acid, polypeptide or polynucleotide; And
R 2Be OH, ester protecting group, resin, amino acid, polypeptide or polynucleotide;
R aBe selected from the group that forms by following each group: H, halogen, alkyl independently of one another, be substituted alkyl ,-N (R ') 2,-C (O) kR ' (wherein k is 1,2 or 3) ,-C (O) N (R ') 2,-OR ' and-S (O) kR ', wherein R ' is H, alkyl independently of one another or is substituted alkyl.
These alpha-non-natural amino acids can be the form of salt, maybe can incorporate in non-natural amino acid polypeptides, polymkeric substance, polysaccharide or the polynucleotide and according to circumstances through posttranslational modification.
In addition, comprise the have formula amino acid of structure of (XXXXII):
Wherein:
R is H, halogen, alkyl, is substituted alkyl, cycloalkyl or is substituted cycloalkyl; And
T 3Be O or S.
These alpha-non-natural amino acids can be the form of salt, maybe can incorporate in non-natural amino acid polypeptides, polymkeric substance, polysaccharide or the polynucleotide and according to circumstances through posttranslational modification.
In addition, comprise the have formula amino acid of structure of (XXXXIII):
Figure A20058004432801122
Wherein:
R is H, halogen, alkyl, is substituted alkyl, cycloalkyl or is substituted cycloalkyl.
In addition, comprise following amino acid with structure of formula (XXXXIII):
Figure A20058004432801123
These alpha-non-natural amino acids can be the form of salt, maybe can incorporate in non-natural amino acid polypeptides, polymkeric substance, polysaccharide or the polynucleotide and according to circumstances through posttranslational modification.
Carbonyl or dicarbapentaborane functional group can react to be formed on corresponding oxime key stable under the physiological condition with the reagent selectivity ground that contains azanol in the aqueous solution under mild conditions.Referring to (for example) Jencks, W.P., J.Am.Chem.Soc.81,475-481 (1959); Shao, J. and Tam, J.P., J.Am.Chem.Soc.117 (14): 3893-3899 (1995).In addition, unique reactivity of carbonyl or dicarbapentaborane is allowed the selective modification in the presence of other amino acid side chains.Referring to (for example) Cornish, people such as V.W., J.Am.Chem.Soc.118:8150-8151 (1996); Geoghegan, K.F. and Stroh, J.G., Bioconjug.Chem.3:138-146 (1992); Mahal, people such as L.K., Science 276:1125-1128 (1997).
To the synthetic Zhang that incorporates into by reference that is described in of ethanoyl-(+/-)-phenylalanine and an ethanoyl-(+/-)-phenylalanine, people such as Z. are among the Biochemistry 42:6735-6746 (2003).Can similar preparation other contain the amino acid of carbonyl or dicarbapentaborane.Non-limiting exemplary synthetic being of the alpha-non-natural amino acid that comprises herein in addition, is shown among Fig. 4, Figure 24-Figure 34 and Figure 36-Figure 39.
In certain embodiments, the polypeptide that comprises alpha-non-natural amino acid through chemically modified to produce reactive carbonyl or dicarbapentaborane functional group.For example, be applicable to that the aldehyde functional group who engages reaction can produce by having contiguous functional group amino and hydroxyl.Wherein bioactive molecules is a polypeptide, for example, can use terminal Serine of N-or Threonine (it can exist usually maybe and can expose by chemical digestion or enzymatic digestion) to use periodate to produce the aldehyde functional group under the mild oxidation cracking condition.Referring to people such as (for example) Gaertner, Bioconjug.Chem.3:262-268 (1992); Geoghegan, K. and Stroh, J., Bioconjug.Chem.3:138-146 (1992); People such as Gaertner, J.Biol.Chem.269:7224-7230 (1994).Yet known method is confined to the amino acid at peptide or proteinic N-end in the affiliated field.
In addition, for example having the form that contiguous hydroxyl and amino alpha-non-natural amino acid can " through sheltering " aldehyde functional group incorporates in the polypeptide.For instance, the 5-oxylysine has in abutting connection with the hydroxyl of ε amine.The reaction conditions that produces aldehyde is usually included in the sodium metaperiodate of interpolation molar excess under the mild conditions to avoid other site oxidations in polypeptide.The pH value of oxidizing reaction is generally about 7.0.Typical reaction comprises the sodium metaperiodate that adds about 1.5 molar excess in the buffered soln of polypeptide, then cultivates in the dark about 10 minutes.Referring to No. the 6th, 423,685, (for example) United States Patent (USP).
B. alpha-non-natural amino acid: contain the amino acid whose structure of azanol and synthetic
The alpha-non-natural amino acid that contains azanol (being also referred to as the amino oxygen base) group allow with multiple electrophilic group reaction with form joiner (including (but not limited to) with PEG or other water-soluble polymerss).As hydrazine, hydrazides and semicarbazides, the enhanced nucleophilicity of amino oxygen base make its effectively and optionally with the multiple molecular reaction that contains carbonyl or dicarbapentaborane (including (but not limited to) ketone, aldehyde or have similar chemically reactive other functional groups).Referring to (for example) Shao, J. and Tarn, J., J.Am.Chem.Soc.117:3893-3899 (1995); H.Hang and C.Bertozzi, Ace.Chem.Res.34 (9): 727-736 (2001).And with the reaction result of diazanyl be corresponding hydrazone, yet, oxime usually by amino oxygen base and the group that contains carbonyl or dicarbapentaborane (such as, ketone, aldehyde or have similar chemically reactive other functional groups) reaction produces.
Therefore; in this article among some embodiment of Miao Shuing for having the alpha-non-natural amino acid of the side chain that comprises following group, described side chain comprises azanol base, class azanol base (its have on the reactivity that is similar to the azanol base and the structure be similar to the azanol base), through sheltering azanol base (it can be easy to change into the azanol base) or through protection azanol base (it has the reactivity that is similar to the azanol base after going to protect).These amino acid comprise the amino acid of the structure with following formula:
Wherein:
A is optionally, and when exist, be low-grade alkylidene, be substituted low-grade alkylidene, rudimentary cycloalkylidene, be substituted rudimentary cycloalkylidene, lower alkenylene, be substituted lower alkenylene, alkynylene, the assorted alkyl in rudimentary Asia, be substituted inferior mix alkyl, rudimentary inferior Heterocyclylalkyl, be substituted rudimentary inferior Heterocyclylalkyl, arylidene, be substituted arylidene, inferior heteroaryl, be substituted inferior heteroaryl, alkarylene, be substituted alkarylene, inferior aralkyl or be substituted inferior aralkyl;
B is optionally, and is the connexon that is selected from the group that is made up of following each group when exist: low-grade alkylidene, be substituted low-grade alkylidene, lower alkenylene, be substituted lower alkenylene, alkyl is mixed in rudimentary Asia, be substituted rudimentary Asia mix alkyl ,-O-,-O-(alkylidene group or be substituted alkylidene group)-,-S-,-S-(alkylidene group or be substituted alkylidene group)-,-S (O) k-(wherein k is 1,2 or 3) ,-S (O) k(alkylidene group or be substituted alkylidene group)-,-C (O)-,-NS (O) 2-,-OS (O) 2-,-C (O)-(alkylidene group or be substituted alkylidene group)-,-C (S)-,-C (S)-(alkylidene group or be substituted alkylidene group)-,-N (R ')-,-NR '-(alkylidene group or be substituted alkylidene group)-,-C (O) N (R ')-,-CON (R ')-(alkylidene group or be substituted alkylidene group)-,-CSN (R ')-,-CSN (R ')-(alkylidene group or be substituted alkylidene group)-,-N (R ') CO-(alkylidene group or be substituted alkylidene group)-,-N (R ') C (O) O-,-S (O) kN (R ')-,-N (R ') C (O) N (R ')-,-N (R ') C (S) N (R ')-,-N (R ') S (O) kN (R ')-,-N (R ')-N=,-C (R ')=N-,-C (R ')=N-N (R ')-,-C (R ')=N-N=,-C (R ') 2-N=N-and-C (R ') 2-N (R ')-N (R ')-, wherein R ' is H, alkyl independently of one another or is substituted alkyl;
K is-NR 6R 7Or-N=CR 6R 7
R 1Be H, amino protecting group, resin, amino acid, polypeptide or polynucleotide; And
R 2Be OH, ester protecting group, resin, amino acid, polypeptide or polynucleotide;
R 3With R 4Be H, halogen, low alkyl group or be substituted low alkyl group independently of one another, or R 3With R 4Or two R 3Group forms cycloalkyl or Heterocyclylalkyl according to circumstances;
R 6With R 7Be selected from the group that forms by following each group independently of one another: H, alkyl, be substituted alkyl, thiazolinyl, be substituted thiazolinyl, alkoxyl group, be substituted alkoxyl group, polyoxyalkylene, be substituted polyoxyalkylene, aryl, be substituted aryl, heteroaryl, be substituted heteroaryl, alkaryl, be substituted alkaryl, aralkyl, and be substituted aralkyl ,-C (O) R " ,-C (O) 2R " ,-C (O) N (R ") 2, R wherein " independently of one another for hydrogen, alkyl, be substituted alkyl, thiazolinyl, be substituted thiazolinyl, alkoxyl group, be substituted alkoxyl group, aryl, be substituted aryl, heteroaryl, alkaryl, be substituted alkaryl, aralkyl or be substituted aralkyl; Or R 6Or R 7Be L-X, wherein
X is selected from the group that is made up of following each thing: mark; Dyestuff; Polymkeric substance; Water-soluble polymers; The derivative of polyoxyethylene glycol; Photocrosslinking agent; Cytotoxic compound; Medicine; Affinity marker; The photoaffinity mark; Reactive compounds; Resin; Second protein or polypeptide or polypeptide analog; Antibody or antibody fragment; Metal chelator; Cofactor; Lipid acid; Carbohydrate; Polynucleotide; DNA; RNA; Antisense polynucleotide; Carbohydrate, water-soluble branch-shape polymer, cyclodextrin, biomaterial; Nanoparticle; Spin labeling; Fluorophore, metallic part; Radioactive segment; Novel functional group; With the covalently or non-covalently interactional group of other molecules; The light cage covers part; But photoisomerization part; Vitamin H; The vitamin H analogue; Be combined with the part of heavy atom; But the group of chemical cracking; But the group of photodestruciton; The side chain that prolongs; The sugar of carbon bond connection; Redox active agent; Amino thioic acid sulfoacid; The toxicity part; Through isotope-labeled part; The biophysics probe; The phosphorescence group; Chemiluminescent groups; The electron dense group; The magnetic group; Insert group; Chromophoric group; Energy transfer agent; Biologically active agent; Detectable label; With and any combination; And
L is optionally, and is the connexon that is selected from the group that is made up of following each group when existing: alkylidene group, be substituted alkylidene group, alkenylene, be substituted alkenylene ,-O-,-O-(alkylidene group or be substituted alkylidene group)-,-S-,-S-(alkylidene group or be substituted alkylidene group)-,-S (O) k-(wherein k is 1,2 or 3) ,-S (O) k(alkylidene group or be substituted alkylidene group)-,-C (O)-,-C (O)-(alkylidene group or be substituted alkylidene group)-,-C (S)-,-C (S)-(alkylidene group or be substituted alkylidene group)-,-N (R ')-,-NR '-(alkylidene group or be substituted alkylidene group)-,-C (O) N (R ')-,-CON (R ')-(alkylidene group or be substituted alkylidene group)-,-CSN (R ')-,-CSN (R ')-(alkylidene group or be substituted alkylidene group)-,-N (R ') CO-(alkylidene group or be substituted alkylidene group)-,-N (R ') C (O) O-,-S (O) kN (R ')-,-N (R ') C (O) N (R ')-,-N (R ') C (S) N (R ')-,-N (R ') S (O) kN (R ')-,-N (R ')-N=,-C (R ')=N-,-C (R ')=N-N (R ')-,-C (R ')=N-N=,-C (R ') 2-N=N-and-C (R ') 2-N (R ')-N (R ')-, wherein R ' is H, alkyl independently of one another or is substituted alkyl.
These alpha-non-natural amino acids can be the form of salt, maybe can incorporate in non-natural amino acid polypeptides, polymkeric substance, polysaccharide or the polynucleotide and according to circumstances through posttranslational modification.
In some embodiment of formula (XIV) compound, A is phenylene or is substituted phenylene.In some embodiment of formula (XIV) compound, B for-(alkylidene group or be substituted alkylidene group)-,-O-(alkylidene group or be substituted alkylidene group)-,-S-(alkylidene group or be substituted alkylidene group)-or-C (O)-(alkylidene group or be substituted alkylidene group)-.In some embodiment of formula (XIV) compound, B is-O (CH 2) 2,-S (CH 2) 2-,-NH (CH 2) 2-,-CO (CH 2) 2-or-(CH 2) n-, wherein n is 1 to 4.In some embodiment of formula (XIV) compound, R 1Be H, tertbutyloxycarbonyl (Boc), 9-fluorenyl methoxy carbonyl (Fmoc), N-ethanoyl, tetrafluoro ethanoyl (TFA) or carbobenzoxy-(Cbz) (Cbz).In some embodiment of formula (XIV) compound, R wherein 1Be resin, amino acid, polypeptide or polynucleotide.In some embodiment of formula (XIV) compound, R wherein 2Be OH, O-methyl, O-ethyl or the O-tertiary butyl.In some embodiment of formula (XIV) compound, R wherein 2Be resin, amino acid, polypeptide or polynucleotide.In some embodiment of formula (XIV) compound, R wherein 2Be polynucleotide.In some embodiment of formula (XIV) compound, R wherein 2Be Yeast Nucleic Acid (RNA).In some embodiment of formula (XIV) compound, R wherein 2Be tRNA.In some embodiment of formula (XIV) compound, wherein the tRNA specific recognition is selected codon.In some embodiment of formula (XIV) compound, wherein selecting codon is to be selected from by amber codon, ocher codon, opal codon, unique codon, rare codon, non-natural codon, five base codons and the molecular group of four base passwords.In some embodiment of formula (XIV) compound, R wherein 2For suppressing tRNA.In some embodiment of formula (XIV) compound, R 6With R 7Be selected from the group that forms by following each group independently of one another: H, alkyl, be substituted alkyl, alkoxyl group, be substituted alkoxyl group, polyoxyalkylene, be substituted polyoxyalkylene, aryl, be substituted aryl, heteroaryl, be substituted heteroaryl, alkaryl, be substituted alkaryl, aralkyl and be substituted aralkyl.In some embodiment of formula (XIV) compound, R 6With R 7Be selected from independently of one another the group that forms by following each group: H, methyl, phenyl and-[(alkylidene group or be substituted alkylidene group)-O-(hydrogen, alkyl or be substituted alkyl)] x, wherein x is 1-50.In some embodiment of formula (XIV) compound, K is-NR 6R 7
In some embodiment of formula (XIV) compound, X is the biologically active agent that is selected from the group that is made up of peptide, protein, enzyme, antibody, medicine, dyestuff, lipid, nucleosides, oligonucleotide, cell, virus, liposome, particulate and micella.In some embodiment of formula (XIV) compound, X is the medicine that is selected from the group that is made up of microbiotic, mycocide, antiviral agent, antiphlogistic, antineoplastic agent, cardiovascular agents, antianxiety agent, hormone, somatomedin and steroid agent.In some embodiment of formula (XIV) compound, X is the enzyme that is selected from the group that is made up of horseradish peroxidase, alkaline phosphatase, beta-galactosidase enzymes and glucose oxidase.In some embodiment of formula (XIV) compound, X is the detectable label that is selected from the group that is grouped into by fluorescence part, phosphorescence part, chemiluminescent moiety, chelating moiety, electron dense part, magnetic part, insertion portion, radioactive segment, chromophoric group part and energy transfer portion.
In certain embodiments, formula (XIV) compound was stablized 1 month in the aqueous solution under appropriate acidic conditions at least.In certain embodiments, formula (XIV) compound stable at least 2 weeks under appropriate acidic conditions.In certain embodiments, formula (XIV) compound was stablized 5 days under appropriate acidic conditions at least.In certain embodiments, described acidic conditions is pH 2 to 8.
These amino acid comprise the have formula amino acid of structure of (XV):
Figure A20058004432801171
Wherein
A is optionally, and when exist, be low-grade alkylidene, be substituted low-grade alkylidene, rudimentary cycloalkylidene, be substituted rudimentary cycloalkylidene, lower alkenylene, be substituted lower alkenylene, alkynylene, the assorted alkyl in rudimentary Asia, be substituted inferior mix alkyl, rudimentary inferior Heterocyclylalkyl, be substituted rudimentary inferior Heterocyclylalkyl, arylidene, be substituted arylidene, inferior heteroaryl, be substituted inferior heteroaryl, alkarylene, be substituted alkarylene, inferior aralkyl or be substituted inferior aralkyl;
B is optionally, and is the connexon that is selected from the group that is made up of following each group when exist: low-grade alkylidene, be substituted low-grade alkylidene, lower alkenylene, be substituted lower alkenylene, alkyl is mixed in rudimentary Asia, be substituted rudimentary Asia mix alkyl ,-O-,-O-(alkylidene group or be substituted alkylidene group)-,-S-,-S-(alkylidene group or be substituted alkylidene group)-,-S (O) k-(wherein k is 1,2 or 3) ,-S (O) k(alkylidene group or be substituted alkylidene group)-,-C (O)-,-NS (O) 2-,-OS (O) 2,-C (O)-(alkylidene group or be substituted alkylidene group)-,-C (S)-,-C (S)-(alkylidene group or be substituted alkylidene group)-,-N (R ')-,-NR '-(alkylidene group or be substituted alkylidene group)-,-C (O) N (R ')-,-CON (R ')-(alkylidene group or be substituted alkylidene group)-,-CSN (R ')-,-CSN (R ')-(alkylidene group or be substituted alkylidene group)-,-N (R ') CO-(alkylidene group or be substituted alkylidene group)-,-N (R ') C (O) O-,-S (O) kN (R ')-,-N (R ') C (O) N (R ')-,-N (R ') C (S) N (R ')-,-N (R ') S (O) kN (R ')-,-N (R ')-N=,-C (R ')=N-,-C (R ')=N-N (R ')-,-C (R ')=N-N=,-C (R ') 2-N=N-and-C (R ') 2-N (R ')-N (R ')-, wherein R ' is H, alkyl independently of one another or is substituted alkyl;
R 1Be H, amino protecting group, resin, amino acid, polypeptide or polynucleotide; And
R 2Be OH, ester protecting group, resin, amino acid, polypeptide or polynucleotide;
R 3With R 4Be H, halogen, low alkyl group or be substituted low alkyl group independently of one another, or R 3With R 4Or two R 3Group forms cycloalkyl or Heterocyclylalkyl according to circumstances.
These alpha-non-natural amino acids can be the form of salt, maybe can incorporate in non-natural amino acid polypeptides, polymkeric substance, polysaccharide or the polynucleotide and according to circumstances through posttranslational modification.
Non-limiting, representative amino acid has following structure:
Figure A20058004432801181
These alpha-non-natural amino acids can be the form of salt, maybe can incorporate in non-natural amino acid polypeptides, polymkeric substance, polysaccharide or the polynucleotide and according to circumstances through posttranslational modification.
The amino acid that contains the amino oxygen base can be prepared by the amino acid precursor that is easy to obtain (homoserine, Serine and Threonine).Referring to (for example) M.Carrasco and R.Brown, J.Org.Chem.68:8853-8858 (2003).Some amino acid (such as L-2-amino-4-(amino oxygen base) butyric acid) that contains the amino oxygen base separates (Rosenthal, people such as G., Life Sci.60:1635-1641 (1997)) from natural origin.Other amino acid that contain the amino oxygen base can similarly prepare.In addition, non-limiting exemplary synthetic being of the alpha-non-natural amino acid of describing herein is shown among Fig. 5.
C. alpha-non-natural amino acid: contain the amino acid whose chemosynthesis of oxime
The alpha-non-natural amino acid that contains oximido is allowed the new alpha-non-natural amino acid that comprises new oximido with the plurality of reagents reaction that contains some reactive carbonyl or dicarbapentaborane (including (but not limited to) ketone, aldehyde or have similar reactive other groups) with formation.This oxime permutoid reaction allows that non-natural amino acid polypeptides is further functionalized.In addition, the original alpha-non-natural amino acid itself that contains oximido can be suitable, as long as the oxime key is stable (for example, the chemical synthesis process of describing) in vivo, in vitro and herein in that amino acid is incorporated under the necessary condition in the polypeptide.
Therefore; in this article among some embodiment of Miao Shuing for having the alpha-non-natural amino acid of the side chain that comprises following group, described side chain comprises oximido, class oximido (its have on the reactivity that is similar to oximido and the structure be similar to oximido), through sheltering oximido (it can be easy to change into oximido) or through protection oximido (it has the reactivity that is similar to oximido after going to protect).These amino acid comprise the have formula amino acid of structure of (XI):
Figure A20058004432801182
Wherein:
A is optionally, and when exist, be low-grade alkylidene, be substituted low-grade alkylidene, rudimentary cycloalkylidene, be substituted rudimentary cycloalkylidene, lower alkenylene, be substituted lower alkenylene, alkynylene, the assorted alkyl in rudimentary Asia, be substituted inferior mix alkyl, rudimentary inferior Heterocyclylalkyl, be substituted rudimentary inferior Heterocyclylalkyl, arylidene, be substituted arylidene, inferior heteroaryl, be substituted inferior heteroaryl, alkarylene, be substituted alkarylene, inferior aralkyl or be substituted inferior aralkyl;
B is optionally, and is the connexon that is selected from the group that is made up of following each group when exist: low-grade alkylidene, be substituted low-grade alkylidene, lower alkenylene, be substituted lower alkenylene, alkyl is mixed in rudimentary Asia, be substituted rudimentary Asia mix alkyl ,-O-,-O-(alkylidene group or be substituted alkylidene group)-,-S-,-S-(alkylidene group or be substituted alkylidene group)-,-S (O) k-(wherein k is 1,2 or 3) ,-S (O) k(alkylidene group or be substituted alkylidene group)-,-C (O)-,-NS (O) 2-,-OS (O) 2-,-C (O)-(alkylidene group or be substituted alkylidene group)-,-C (S)-,-C (S)-(alkylidene group or be substituted alkylidene group)-,-N (R ')-,-NR '-(alkylidene group or be substituted alkylidene group)-,-C (O) N (R ')-,-CON (R ')-(alkylidene group or be substituted alkylidene group)-,-CSN (R ')-,-CSN (R ')-(alkylidene group or be substituted alkylidene group)-,-N (R ') CO-(alkylidene group or be substituted alkylidene group)-,-N (R ') C (O) O-,-S (O) kN (R ')-,-N (R ') C (O) N (R ')-,-N (R ') C (S) N (R ')-,-N (R ') S (O) kN (R ')-,-N (R ')-N=,-C (R ')=N-,-C (R ')=N-N (R ')-,-C (R ')=N-N=,-C (R ') 2-N=N-and-C (R ') 2-N (R ')-N (R ')-, wherein R ' is H, alkyl independently of one another or is substituted alkyl;
R is H, alkyl, is substituted alkyl, cycloalkyl or is substituted cycloalkyl;
R 1Be H, amino protecting group, resin, amino acid, polypeptide or polynucleotide; And
R 2Be OH, ester protecting group, resin, amino acid, polypeptide or polynucleotide;
R 3With R 4Be H, halogen, low alkyl group or be substituted low alkyl group independently of one another, or R 3With R 4Or two R 3Group forms cycloalkyl or Heterocyclylalkyl according to circumstances;
R 5For H, alkyl, be substituted alkyl, thiazolinyl, be substituted thiazolinyl, alkynyl, be substituted alkynyl, alkoxyl group, be substituted alkoxyl group, alkyl alkoxy, be substituted alkyl alkoxy, polyoxyalkylene, be substituted polyoxyalkylene, aryl, be substituted aryl, heteroaryl, be substituted heteroaryl, alkaryl, be substituted alkaryl, aralkyl, be substituted aralkyl ,-(alkylidene group or be substituted alkylidene group)-ON (R ") 2,-(alkylidene group or be substituted alkylidene group)-C (O) SR " ,-(alkylidene group or be substituted alkylidene group)-S-S-(aryl or be substituted aryl) ,-C (O) R " ,-C (O) 2R " or-C (O) N (R ") 2, R wherein " independently of one another for hydrogen, alkyl, be substituted alkyl, thiazolinyl, be substituted thiazolinyl, alkoxyl group, be substituted alkoxyl group, aryl, be substituted aryl, heteroaryl, alkaryl, be substituted alkaryl, aralkyl or be substituted aralkyl;
Or R 5Be L-X, wherein
X is selected from the group that is made up of following each thing: mark; Dyestuff; Polymkeric substance; Water-soluble polymers; The derivative of polyoxyethylene glycol; Photocrosslinking agent; Cytotoxic compound; Medicine; Affinity marker; The photoaffinity mark; Reactive compounds; Resin; Second protein or polypeptide or polypeptide analog; Antibody or antibody fragment; Metal chelator; Cofactor; Lipid acid; Carbohydrate; Polynucleotide; DNA; RNA; Antisense polynucleotide; Carbohydrate, water-soluble branch-shape polymer, cyclodextrin, biomaterial; Nanoparticle; Spin labeling; Fluorophore, metallic part; Radioactive segment; Novel functional group; With the covalently or non-covalently interactional group of other molecules; The light cage covers part; But photoisomerization part; Vitamin H; The vitamin H analogue; Be combined with the part of heavy atom; But the group of chemical cracking; But the group of photodestruciton; The side chain that prolongs; The sugar of carbon bond connection; Redox active agent; Amino thioic acid sulfoacid; The toxicity part; Through isotope-labeled part; The biophysics probe; The phosphorescence group; Chemiluminescent groups; The electron dense group; The magnetic group; Insert group; Chromophoric group; Energy transfer agent; Biologically active agent; Detectable label; With and any combination; And L is optionally, and is the connexon that is selected from the group that is made up of following each group when existing: alkylidene group, be substituted alkylidene group, alkenylene, be substituted alkenylene ,-O-,-O-(alkylidene group or be substituted alkylidene group)-,-S-,-S-(alkylidene group or be substituted alkylidene group)-,-S (O) k-(wherein k is 1,2 or 3) ,-S (O) k(alkylidene group or be substituted alkylidene group)-,-C (O)-,-C (O)-(alkylidene group or be substituted alkylidene group)-,-C (S)-,-C (S)-(alkylidene group or be substituted alkylidene group)-,-N (R ')-,-NR '-(alkylidene group or be substituted alkylidene group)-,-C (O) N (R ')-,-CON (R ')-(alkylidene group or be substituted alkylidene group)-,-CSN (R ')-,-CSN (R ')-(alkylidene group or be substituted alkylidene group)-,-N (R ') CO-(alkylidene group or be substituted alkylidene group)-,-N (R ') C (O) O-,-(alkylidene group or be substituted alkylidene group)-O-N=CR '-,-(alkylidene group or be substituted alkylidene group)-C (O) NR '-(alkylidene group or be substituted alkylidene group)-,-(alkylidene group or be substituted alkylidene group)-S (O) k(alkylidene group or be substituted alkylidene group)-S-,-(alkylidene group or be substituted alkylidene group)-S-S-,-S (O) kN (R ')-,-N (R ') C (O) N (R ')-,-N (R ') C (S) N (R ')-,-N (R ') S (O) kN (R ')-,-N (R ')-N=,-C (R ')=N-,-C (R ')=N-N (R ')-,-C (R ')=N-N=,-C (R ') 2-N=N-and-C (R ') 2-N (R ')-N (R ')-, wherein R ' is H, alkyl independently of one another or is substituted alkyl;
Its restricted condition is not for when A and B exist, and R is not a methyl.
These alpha-non-natural amino acids can be the form of salt, maybe can incorporate in non-natural amino acid polypeptides, polymkeric substance, polysaccharide or the polynucleotide and according to circumstances through posttranslational modification.
In some embodiment of formula (XI) compound, B for-O-(alkylidene group or be substituted alkylidene group)-.In some embodiment of formula (XI) compound, B is-O (CH 2)-.In some embodiment of formula (XI) compound, R is C 1-6Alkyl.In some embodiment of formula (XI) compound, R is-CH 3In some embodiment of formula (XI) compound, R 1Be H, tertbutyloxycarbonyl (Boc), 9-fluorenyl methoxy carbonyl (Fmoc), N-ethanoyl, tetrafluoro ethanoyl (TFA) or carbobenzoxy-(Cbz) (Cbz).In some embodiment of formula (XI) compound, R 1Be resin, amino acid, polypeptide or polynucleotide.In some embodiment of formula (XI) compound, R 2Be OH, O-methyl, O-ethyl or the O-tertiary butyl.In some embodiment of formula (XI) compound, R 2Be resin, amino acid, polypeptide or polynucleotide.In some embodiment of formula (XI) compound, R 2Be polynucleotide.
In some embodiment of formula (XI) compound, R 2Be Yeast Nucleic Acid (RNA).In some embodiment of formula (XI) compound, R 2Be tRNA.In some embodiment of formula (XI) compound, the tRNA specific recognition is selected codon.In some embodiment of formula (XI) compound, selecting codon is to be selected from by amber codon, ocher codon, opal codon, unique codon, rare codon, non-natural codon, five base codons and the molecular group of four base passwords.In some embodiment of formula (XI) compound, R 2For suppressing tRNA.In some embodiment of formula (XI) compound, R 5For alkyl alkoxy, be substituted alkyl alkoxy, polyoxyalkylene, be substituted polyoxyalkylene or-C (O) 2R ".In some embodiment of formula (XI) compound, R 5For-[(alkylidene group or be substituted alkylidene group)-O-(hydrogen, alkyl or be substituted alkyl)] x, wherein x is 1-50.In some embodiment of formula (XI) compound, R 5For-(CH 2CH 2)-O-CH 3Or-COOH.
In certain embodiments, formula (I) compound was stablized 1 month in the aqueous solution under appropriate acidic conditions at least.In certain embodiments, formula (I) compound stable at least 2 weeks under appropriate acidic conditions.In certain embodiments, formula (I) compound was stablized 5 days under appropriate acidic conditions at least.In certain embodiments, described acidic conditions is pH 2 to 8.
The amino acid of formula (XI) comprises the have formula amino acid of structure of (XII):
Figure A20058004432801211
Wherein,
B is optionally, and is the connexon that is selected from the group who is made up of following each group when exist: low-grade alkylidene, be substituted low-grade alkylidene, lower alkenylene, be substituted lower alkenylene, alkyl is mixed in rudimentary Asia, be substituted rudimentary Asia mix alkyl ,-O-,-O-(alkylidene group or be substituted alkylidene group)-,-S-,-S-(alkylidene group or be substituted alkylidene group)-,-S (O) k-(wherein k is 1,2 or 3) ,-S (O) k(alkylidene group or be substituted alkylidene group)-,-C (O)-,-NS (O) 2-,-OS (O) 2-,-C (O)-(alkylidene group or be substituted alkylidene group)-,-C (S)-,-C (S)-(alkylidene group or be substituted alkylidene group)-,-N (R ')-,-NR '-(alkylidene group or be substituted alkylidene group)-,-C (O) N (R ')-,-CON (R ')-(alkylidene group or be substituted alkylidene group)-,-CSN (R ')-,-CSN (R ')-(alkylidene group or be substituted alkylidene group)-,-N (R ') CO-(alkylidene group or be substituted alkylidene group)-,-N (R ') C (O) O-,-S (O) kN (R-')-,-N (R ') C (O) N (R ')-,-N (R ') C (S) N (R ')-,-N (R ') S (O) kN (R ')-,-N (R ')-N=,-C (R ')=N-,-C (R ')=N-N (R ')-,-C (R ')=N-N=,-C (R ') 2-N=N-and-C (R ') 2-N (R ')-N (R ')-, wherein R ' is H, alkyl independently of one another or is substituted alkyl;
R is H, alkyl, is substituted alkyl, cycloalkyl or is substituted cycloalkyl;
R 1Be H, amino protecting group, resin, amino acid, polypeptide or polynucleotide; And
R 2Be OH, ester protecting group, resin, amino acid, polypeptide or polynucleotide;
R 5For H, alkyl, be substituted alkyl, thiazolinyl, be substituted thiazolinyl, alkynyl, be substituted alkynyl, alkoxyl group, be substituted alkoxyl group, alkyl alkoxy, be substituted alkyl alkoxy, polyoxyalkylene, be substituted polyoxyalkylene, aryl, be substituted aryl, heteroaryl, be substituted heteroaryl, alkaryl, be substituted alkaryl, aralkyl, be substituted aralkyl ,-(alkylidene group or be substituted alkylidene group)-ON (R ") 2,-(alkylidene group or be substituted alkylidene group)-C (O) SR " ,-(alkylidene group or be substituted alkylidene group)-S-S-(aryl or be substituted aryl) ,-C (O) R " ,-C (O) 2R " or-C (O) N (R ") 2R wherein " independently of one another for hydrogen, alkyl, be substituted alkyl, thiazolinyl, be substituted thiazolinyl, alkoxyl group, be substituted alkoxyl group, aryl, be substituted aryl, heteroaryl, alkaryl, be substituted alkaryl, aralkyl or be substituted aralkyl;
Or R 5Be L-X, wherein
X is selected from the group that is made up of following each thing: mark; Dyestuff; Polymkeric substance; Water-soluble polymers; The derivative of polyoxyethylene glycol; Photocrosslinking agent; Cytotoxic compound; Medicine; Affinity marker; The photoaffinity mark; Reactive compounds; Resin; Second protein or polypeptide or polypeptide analog; Antibody or antibody fragment; Metal chelator; Cofactor; Lipid acid; Carbohydrate; Polynucleotide; DNA; RNA; Antisense polynucleotide; Carbohydrate, water-soluble branch-shape polymer, cyclodextrin, biomaterial; Nanoparticle; Spin labeling; Fluorophore, metallic part; Radioactive segment; Novel functional group; With the covalently or non-covalently interactional group of other molecules; The light cage covers part; But photoisomerization part; Vitamin H; The vitamin H analogue; Be combined with the part of heavy atom; But the group of chemical cracking; But the group of photodestruciton; The side chain that prolongs; The sugar of carbon bond connection; Redox active agent; Amino thioic acid sulfoacid; The toxicity part; Through isotope-labeled part; The biophysics probe; The phosphorescence group; Chemiluminescent groups; The electron dense group; The magnetic group; Insert group; Chromophoric group; Energy transfer agent; Biologically active agent; Detectable label; With and any combination; And L is optionally, and is the connexon that is selected from the group that is made up of following each group when existing: alkylidene group, be substituted alkylidene group, alkenylene, be substituted alkenylene ,-O-,-O-(alkylidene group or be substituted alkylidene group)-,-S-,-S-(alkylidene group or be substituted alkylidene group)-,-S (O) k-(wherein k is 1,2 or 3) ,-S (O) k(alkylidene group or be substituted alkylidene group)-,-C (O)-,-C (O)-(alkylidene group or be substituted alkylidene group)-,-C (S)-,-C (S)-(alkylidene group or be substituted alkylidene group)-,-N (R ')-,-NR '-(alkylidene group or be substituted alkylidene group)-,-C (O) N (R ')-,-CON (R ')-(alkylidene group or be substituted alkylidene group)-,-CSN (R ')-,-CSN (R ')-(alkylidene group or be substituted alkylidene group)-,-N (R ') CO-(alkylidene group or be substituted alkylidene group)-,-N (R ') C (O) O-,-(alkylidene group or be substituted alkylidene group)-O-N=CR '-,-(alkylidene group or be substituted alkylidene group)-C (O) NR '-(alkylidene group or be substituted alkylidene group)-,-(alkylidene group or be substituted alkylidene group)-S (O) k-(alkylidene group or be substituted alkylidene group)-S-,-(alkylidene group or be substituted alkylidene group)-S-S-,-S (O) kN (R ')-,-N (R ') C (O) N (R ')-,-N (R ') C (S) N (R ')-,-N (R ') S (O) kN (R ')-,-N (R ')-N=,-C (R ')=N-,-C (R ')=N-N (R ')-,-C (R ')=N-N=,-C (R ') 2-N=N-and-C (R ') 2-N (R ')-N (R ')-, wherein R ' is H, alkyl independently of one another or is substituted alkyl.
These alpha-non-natural amino acids can be the form of salt, maybe can incorporate in non-natural amino acid polypeptides, polymkeric substance, polysaccharide or the polynucleotide and according to circumstances through posttranslational modification.
These amino acid comprise the amino acid of (XIII) structure that has formula:
Figure A20058004432801231
Wherein,
R is H, alkyl, is substituted alkyl, cycloalkyl or is substituted cycloalkyl;
R 1Be H, amino protecting group, resin, amino acid, polypeptide or polynucleotide; And
R 2Be OH, ester protecting group, resin, amino acid, polypeptide or polynucleotide;
R 5For H, alkyl, be substituted alkyl, thiazolinyl, be substituted thiazolinyl, alkynyl, be substituted alkynyl, alkoxyl group, be substituted alkoxyl group, alkyl alkoxy, be substituted alkyl alkoxy, polyoxyalkylene, be substituted polyoxyalkylene, aryl, be substituted aryl, heteroaryl, be substituted heteroaryl, alkaryl, be substituted alkaryl, aralkyl, be substituted aralkyl ,-(alkylidene group or be substituted alkylidene group)-ON (R ") 2,-(alkylidene group or be substituted alkylidene group)-C (O) SR " ,-(alkylidene group or be substituted alkylidene group)-S-S-(aryl or be substituted aryl) ,-C (O) R " ,-C (O) 2R " or-C (O) N (R ") 2, R wherein " independently of one another for hydrogen, alkyl, be substituted alkyl, thiazolinyl, be substituted thiazolinyl, alkoxyl group, be substituted alkoxyl group, aryl, be substituted aryl, heteroaryl, alkaryl, be substituted alkaryl, aralkyl or be substituted aralkyl;
Or R 5Be L-X, wherein
X is selected from the group that is made up of following each thing: mark; Dyestuff; Polymkeric substance; Water-soluble polymers; The derivative of polyoxyethylene glycol; Photocrosslinking agent; Cytotoxic compound; Medicine; Affinity marker; The photoaffinity mark; Reactive compounds; Resin; Second protein or polypeptide or polypeptide analog; Antibody or antibody fragment; Metal chelator; Cofactor; Lipid acid; Carbohydrate; Polynucleotide; DNA; RNA; Antisense polynucleotide; Carbohydrate, water-soluble branch-shape polymer, cyclodextrin, biomaterial; Nanoparticle; Spin labeling; Fluorophore, metallic part; Radioactive segment; Novel functional group; With the covalently or non-covalently interactional group of other molecules; The light cage covers part; But photoisomerization part; Vitamin H; The vitamin H analogue; Be combined with the part of heavy atom; But the group of chemical cracking; But the group of photodestruciton; The side chain that prolongs; The sugar of carbon bond connection; Redox active agent; Amino thioic acid sulfoacid; The toxicity part; Through isotope-labeled part; The biophysics probe; The phosphorescence group; Chemiluminescent groups; The electron dense group; The magnetic group; Insert group; Chromophoric group; Energy transfer agent; Biologically active agent; Detectable label; With and any combination; And L is optionally, and is the connexon that is selected from the group that is made up of following each group when existing: alkylidene group, be substituted alkylidene group, alkenylene, be substituted alkenylene ,-O-,-O-(alkylidene group or be substituted alkylidene group)-,-S-,-S-(alkylidene group or be substituted alkylidene group)-,-S (O) k-(wherein k is 1,2 or 3) ,-S (O) k(alkylidene group or be substituted alkylidene group)-,-C (O)-,-C (O)-(alkylidene group or be substituted alkylidene group)-,-C (S)-,-C (S)-(alkylidene group or be substituted alkylidene group)-,-N (R ')-,-NR '-(alkylidene group or be substituted alkylidene group)-,-C (O) N (R ')-,-CON (R ')-(alkylidene group or be substituted alkylidene group)-,-CSN (R ')-,-CSN (R ')-(alkylidene group or be substituted alkylidene group)-,-N (R ') CO-(alkylidene group or be substituted alkylidene group)-,-N (R ') C (O) O-,-(alkylidene group or be substituted alkylidene group)-O-N=CR '-,-(alkylidene group or be substituted alkylidene group)-C (O) NR '-(alkylidene group or be substituted alkylidene group)-,-(alkylidene group or be substituted alkylidene group)-S (O) k-(alkylidene group or be substituted alkylidene group)-S-,-(alkylidene group or be substituted alkylidene group)-S-S-,-S (O) kN (R ')-,-N (R ') C (O) N (R ')-,-N (R ') C (S) N (R ')-,-N (R ') S (O) kN (R ')-,-N (R ')-N=,-C (R ')=N-,-C (R ')=N-N (R ')-,-C (R ')=N-N=,-C (R ') 2-N=N-and-C (R ') 2-N (R ')-N (R ')-, wherein R ' is H, alkyl independently of one another or is substituted alkyl.
These alpha-non-natural amino acids can be the form of salt, maybe can incorporate in non-natural amino acid polypeptides, polymkeric substance, polysaccharide or the polynucleotide and according to circumstances through posttranslational modification.
These amino acid whose other limiting examples comprise the amino acid with following structure:
Figure A20058004432801241
These alpha-non-natural amino acids can be the form of salt, maybe can incorporate in non-natural amino acid polypeptides, polymkeric substance, polysaccharide or the polynucleotide and according to circumstances through posttranslational modification.
In addition, these amino acid comprise the have formula amino acid of structure of (XIV):
Wherein:
A is optionally, and when exist, be low-grade alkylidene, be substituted low-grade alkylidene, rudimentary cycloalkylidene, be substituted rudimentary cycloalkylidene, lower alkenylene, be substituted lower alkenylene, alkynylene, the assorted alkyl in rudimentary Asia, be substituted inferior mix alkyl, rudimentary inferior Heterocyclylalkyl, be substituted rudimentary inferior Heterocyclylalkyl, arylidene, be substituted arylidene, inferior heteroaryl, be substituted inferior heteroaryl, alkarylene, be substituted alkarylene, inferior aralkyl or be substituted inferior aralkyl;
B is optionally, and is the connexon that is selected from the group that is made up of following each group when exist: low-grade alkylidene, be substituted low-grade alkylidene, lower alkenylene, be substituted lower alkenylene, alkyl is mixed in rudimentary Asia, be substituted rudimentary Asia mix alkyl ,-O-,-O-(alkylidene group or be substituted alkylidene group)-,-S-,-S-(alkylidene group or be substituted alkylidene group)-,-S (O) k-(wherein k is 1,2 or 3) ,-S (O) k(alkylidene group or be substituted alkylidene group)-,-C (O)-,-NS (O) 2-,-OS (O) 2-,-C (O)-(alkylidene group or be substituted alkylidene group)-,-C (S)-,-C (S)-(alkylidene group or be substituted alkylidene group)-,-N (R ')-,-NR '-(alkylidene group or be substituted alkylidene group)-,-C (O) N (R ')-,-CON (R ')-(alkylidene group or be substituted alkylidene group)-,-CSN (R ')-,-CSN (R ')-(alkylidene group or be substituted alkylidene group)-,-N (R ') CO-(alkylidene group or be substituted alkylidene group)-,-N (R ') C (O) O-,-S (O) kN (R ')-,-N (R ') C (O) N (R ')-,-N (R ') C (S) N (R ')-,-N (R ') S (O) kN (R ')-,-N (R ')-N=,-C (R ')=N-,-C (R ')=N-N (R ')-,-C (R ')=N-N=,-C (R ') 2-N=N-and-C (R ') 2-N (R ')-N (R ')-, wherein R ' is H, alkyl independently of one another or is substituted alkyl;
K is-NR 6R 7Or-N=CR 6R 7
R 1Be H, amino protecting group, resin, amino acid, polypeptide or polynucleotide; And
R 2Be OH, ester protecting group, resin, amino acid, polypeptide or polynucleotide;
R 3With R 4Be H, halogen, low alkyl group or be substituted low alkyl group independently of one another, or R 3With R 4Or two R 3Group forms cycloalkyl or Heterocyclylalkyl according to circumstances;
R 6With R 7Be selected from the group that forms by following each group independently of one another: H, alkyl, be substituted alkyl, thiazolinyl, be substituted thiazolinyl, alkoxyl group, be substituted alkoxyl group, polyoxyalkylene, be substituted polyoxyalkylene, aryl, be substituted aryl, heteroaryl, be substituted heteroaryl, alkaryl, be substituted alkaryl, aralkyl, and be substituted aralkyl ,-C (O) R " ,-C (O) 2R " ,-C (O) N (R ") 2, R wherein " independently of one another for hydrogen, alkyl, be substituted alkyl, thiazolinyl, be substituted thiazolinyl, alkoxyl group, be substituted alkoxyl group, aryl, be substituted aryl, heteroaryl, alkaryl, be substituted alkaryl, aralkyl or be substituted aralkyl; Or R 6Or R 7Be L-X, wherein
X is selected from the group that is made up of following each thing: mark; Dyestuff; Polymkeric substance; Water-soluble polymers; The derivative of polyoxyethylene glycol; Photocrosslinking agent; Cytotoxic compound; Medicine; Affinity marker; The photoaffinity mark; Reactive compounds; Resin; Second protein or polypeptide or polypeptide analog; Antibody or antibody fragment; Metal chelator; Cofactor; Lipid acid; Carbohydrate; Polynucleotide; DNA; RNA; Antisense polynucleotide; Carbohydrate, water-soluble branch-shape polymer, cyclodextrin, biomaterial; Nanoparticle; Spin labeling; Fluorophore, metallic part; Radioactive segment; Novel functional group; With the covalently or non-covalently interactional group of other molecules; The light cage covers part; But photoisomerization part; Vitamin H; The vitamin H analogue; Be combined with the part of heavy atom; But the group of chemical cracking; But the group of photodestruciton; The side chain that prolongs; The sugar of carbon bond connection; Redox active agent; Amino thioic acid sulfoacid; The toxicity part; Through isotope-labeled part; The biophysics probe; The phosphorescence group; Chemiluminescent groups; The electron dense group; The magnetic group; Insert group; Chromophoric group; Energy transfer agent; Biologically active agent; Detectable label; With and any combination; And L is optionally, and is the connexon that is selected from the group that is made up of following each group when existing: alkylidene group, be substituted alkylidene group, alkenylene, be substituted alkenylene ,-O-,-O-(alkylidene group or be substituted alkylidene group)-,-S-,-S-(alkylidene group or be substituted alkylidene group)-,-S (O) k-(wherein k is 1,2 or 3) ,-S (O) k(alkylidene group or be substituted alkylidene group)-,-C (O)-,-C (O)-(alkylidene group or be substituted alkylidene group)-,-C (S)-,-C (S)-(alkylidene group or be substituted alkylidene group)-,-N (R ')-,-NR '-(alkylidene group or be substituted alkylidene group)-,-C (O) N (R ')-,-CON (R ')-(alkylidene group or be substituted alkylidene group)-,-CSN (R ')-,-CSN (R ')-(alkylidene group or be substituted alkylidene group)-,-N (R ') CO-(alkylidene group or be substituted alkylidene group)-,-N (R ') C (O) O-,-S (O) kN (R ')-,-N (R ') C (O) N (R ')-,-N (R ') C (S) N (R ')-,-N (R ') S (O) kN (R ')-,-N (R ')-N=,-C (R ')=N-,-C (R ')=N-N (R ')-,-C (R ')=N-N=,-C (R ') 2N=N-and-C (R ') 2-N (R ')-N (R ')-, wherein R ' is H, alkyl independently of one another or is substituted alkyl.
These alpha-non-natural amino acids can be the form of salt, maybe can incorporate in non-natural amino acid polypeptides, polymkeric substance, polysaccharide or the polynucleotide and according to circumstances through posttranslational modification.
These amino acid further comprise the amino acid of the structure with formula (XVI):
Figure A20058004432801261
Wherein:
A is optionally, and when exist, be low-grade alkylidene, be substituted low-grade alkylidene, rudimentary cycloalkylidene, be substituted rudimentary cycloalkylidene, lower alkenylene, be substituted lower alkenylene, alkynylene, the assorted alkyl in rudimentary Asia, be substituted inferior mix alkyl, rudimentary inferior Heterocyclylalkyl, be substituted rudimentary inferior Heterocyclylalkyl, arylidene, be substituted arylidene, inferior heteroaryl, be substituted inferior heteroaryl, alkarylene, be substituted alkarylene, inferior aralkyl or be substituted inferior aralkyl;
B is optionally, and is the connexon that is selected from the group that is made up of following each group when exist: low-grade alkylidene, be substituted low-grade alkylidene, lower alkenylene, be substituted lower alkenylene, alkyl is mixed in rudimentary Asia, be substituted rudimentary Asia mix alkyl ,-O-,-O-(alkylidene group or be substituted alkylidene group)-,-S-,-S-(alkylidene group or be substituted alkylidene group)-,-S (O) k-(wherein k is 1,2 or 3) ,-S (O) k(alkylidene group or be substituted alkylidene group)-,-C (O)-,-NS (O) 2-,-OS (O) 2-,-C (O)-(alkylidene group or be substituted alkylidene group)-,-C (S)-,-C (S)-(alkylidene group or be substituted alkylidene group)-,-N (R ')-,-NR '-(alkylidene group or be substituted alkylidene group)-,-C (O) N (R ')-,-CON (R ')-(alkylidene group or be substituted alkylidene group)-,-CSN (R ')-,-CSN (R ')-(alkylidene group or be substituted alkylidene group)-,-N (R ') CO-(alkylidene group or be substituted alkylidene group)-,-N (R ') C (O) O-,-S (O) kN (R ')-,-N (R ') C (O) N (R ')-,-N (R ') C (S) N (R ')-,-N (R ') S (O) kN (R ')-,-N (R ')-N=,-C (R ')=N-,-C (R ')=N-N (R ')-,-C (R ')=N-N=,-C (R ') 2-N=N-and-C (R ') 2-N (R ')-N (R ')-, wherein R ' is H, alkyl independently of one another or is substituted alkyl;
R 1Be H, amino protecting group, resin, amino acid, polypeptide or polynucleotide; And
R 2Be OH, ester protecting group, resin, amino acid, polypeptide or polynucleotide;
R 3With R 4Be H, halogen, low alkyl group or be substituted low alkyl group independently of one another, or R 3With R 4Or two R 3Group forms cycloalkyl or Heterocyclylalkyl according to circumstances;
R 6With R 7Be selected from the group that forms by following each group independently of one another: H, alkyl, be substituted alkyl, thiazolinyl, be substituted thiazolinyl, alkoxyl group, be substituted alkoxyl group, polyoxyalkylene, be substituted polyoxyalkylene, aryl, be substituted aryl, heteroaryl, be substituted heteroaryl, alkaryl, be substituted alkaryl, aralkyl, and be substituted aralkyl ,-C (O) R " ,-C (O) 2R " ,-C (O) N (R ") 2, R wherein " independently of one another for hydrogen, alkyl, be substituted alkyl, thiazolinyl, be substituted thiazolinyl, alkoxyl group, be substituted alkoxyl group, aryl, be substituted aryl, heteroaryl, alkaryl, be substituted alkaryl, aralkyl or be substituted aralkyl; Or R 6Or R 7Be L-X, wherein
X is selected from the group that is made up of following each thing: mark; Dyestuff; Polymkeric substance; Water-soluble polymers; The derivative of polyoxyethylene glycol; Photocrosslinking agent; Cytotoxic compound; Medicine; Affinity marker; The photoaffinity mark; Reactive compounds; Resin; Second protein or polypeptide or polypeptide analog; Antibody or antibody fragment; Metal chelator; Cofactor; Lipid acid; Carbohydrate; Polynucleotide; DNA; RNA; Antisense polynucleotide; Carbohydrate, water-soluble branch-shape polymer, cyclodextrin, biomaterial; Nanoparticle; Spin labeling; Fluorophore, metallic part; Radioactive segment; Novel functional group; With the covalently or non-covalently interactional group of other molecules; The light cage covers part; But photoisomerization part; Vitamin H; The vitamin H analogue; Be combined with the part of heavy atom; But the group of chemical cracking; But the group of photodestruciton; The side chain that prolongs; The sugar of carbon bond connection; Redox active agent; Amino thioic acid sulfoacid; The toxicity part; Through isotope-labeled part; The biophysics probe; The phosphorescence group; Chemiluminescent groups; The electron dense group; The magnetic group; Insert group; Chromophoric group; Energy transfer agent; Biologically active agent; Detectable label; With and any combination; And L is optionally, and is the connexon that is selected from the group that is made up of following each group when existing: alkylidene group, be substituted alkylidene group, alkenylene, be substituted alkenylene ,-O-,-O-(alkylidene group or be substituted alkylidene group)-,-S-,-S-(alkylidene group or be substituted alkylidene group)-,-S (O) k-(wherein k is 1,2 or 3) ,-S (O) k(alkylidene group or be substituted alkylidene group)-,-C (O)-,-C (O)-(alkylidene group or be substituted alkylidene group)-,-C (S)-,-C (S)-(alkylidene group or be substituted alkylidene group)-,-N (R ')-,-NR '-(alkylidene group or be substituted alkylidene group)-,-C (O) N (R ')-,-CON (R ')-(alkylidene group or be substituted alkylidene group)-,-CSN (R ')-,-CSN (R ')-(alkylidene group or be substituted alkylidene group)-,-N (R ') CO-(alkylidene group or be substituted alkylidene group)-,-N (R ') C (O) O-,-S (O) kN (R ')-,-N (R ') C (O) N (R ')-,-N (R ') C (S) N (R ')-,-N (R ') S (O) kN (R ')-,-N (R ')-N=,-C (R ')=N-,-C (R ')=N-N (R ')-,-C (R ')=N-N=,-C (R ') 2-N=N-and-C (R ') 2-N (R ')-N (R ')-, wherein R ' is H, alkyl independently of one another or is substituted alkyl.
These alpha-non-natural amino acids can be the form of salt, maybe can incorporate in non-natural amino acid polypeptides, polymkeric substance, polysaccharide or the polynucleotide and according to circumstances through posttranslational modification.
In addition, these amino acid comprise the have formula amino acid of structure of (XVII):
Figure A20058004432801281
Wherein:
B is optionally, and is the connexon that is selected from the group that is made up of following each group when exist: low-grade alkylidene, be substituted low-grade alkylidene, lower alkenylene, be substituted lower alkenylene, alkyl is mixed in rudimentary Asia, be substituted rudimentary Asia mix alkyl ,-O-,-O-(alkylidene group or be substituted alkylidene group)-,-S-,-S-(alkylidene group or be substituted alkylidene group)-,-S (O) k-(wherein k is 1,2 or 3) ,-S (O) k(alkylidene group or be substituted alkylidene group)-,-C (O)-,-NS (O) 2-,-OS (O) 2-,-C (O)-(alkylidene group or be substituted alkylidene group)-,-C (S)-,-C (S)-(alkylidene group or be substituted alkylidene group)-,-N (R ')-,-NR '-(alkylidene group or be substituted alkylidene group)-,-C (O) N (R ')-,-CON (R ')-(alkylidene group or be substituted alkylidene group)-,-CSN (R ')-,-CSN (R ')-(alkylidene group or be substituted alkylidene group)-,-N (R ') CO-(alkylidene group or be substituted alkylidene group)-,-N (R ') C (O) O-,-S (O) kN (R ')-,-N (R ') C (O) N (R ')-,-N (R ') C (S) N (R ')-,-N (R ') S (O) kN (R ')-,-N (R ')-N=,-C (R ')=N-,-C (R ')=N-N (R ')-,-C (R ')=N-N=,-C (R ') 2-N=N-and-C (R ') 2-N (R ')-N (R ')-, wherein R ' is H, alkyl independently of one another or is substituted alkyl;
R 1Be H, amino protecting group, resin, amino acid, polypeptide or polynucleotide; And
R 2Be OH, ester protecting group, resin, amino acid, polypeptide or polynucleotide;
R 6With R 7Be selected from the group that forms by following each group independently of one another: H, alkyl, be substituted alkyl, thiazolinyl, be substituted thiazolinyl, alkoxyl group, be substituted alkoxyl group, polyoxyalkylene, be substituted polyoxyalkylene, aryl, be substituted aryl, heteroaryl, be substituted heteroaryl, alkaryl, be substituted alkaryl, aralkyl, and be substituted aralkyl ,-C (O) R " ,-C (O) 2R " ,-C (O) N (R ") 2, R wherein " independently of one another for hydrogen, alkyl, be substituted alkyl, thiazolinyl, be substituted thiazolinyl, alkoxyl group, be substituted alkoxyl group, aryl, be substituted aryl, heteroaryl, alkaryl, be substituted alkaryl, aralkyl or be substituted aralkyl; Or R 6Or R 7Be L-X, wherein
X is selected from the group that is made up of following each thing: mark; Dyestuff; Polymkeric substance; Water-soluble polymers; The derivative of polyoxyethylene glycol; Photocrosslinking agent; Cytotoxic compound; Medicine; Affinity marker; The photoaffinity mark; Reactive compounds; Resin; Second protein or polypeptide or polypeptide analog; Antibody or antibody fragment; Metal chelator; Cofactor; Lipid acid; Carbohydrate; Polynucleotide; DNA; RNA; Antisense polynucleotide; Carbohydrate, water-soluble branch-shape polymer, cyclodextrin, biomaterial; Nanoparticle; Spin labeling; Fluorophore, metallic part; Radioactive segment; Novel functional group; With the covalently or non-covalently interactional group of other molecules; The light cage covers part; But photoisomerization part; Vitamin H; The vitamin H analogue; Be combined with the part of heavy atom; But the group of chemical cracking; But the group of photodestruciton; The side chain that prolongs; The sugar of carbon bond connection; Redox active agent; Amino thioic acid sulfoacid; The toxicity part; Through isotope-labeled part; The biophysics probe; The phosphorescence group; Chemiluminescent groups; The electron dense group; The magnetic group; Insert group; Chromophoric group; Energy transfer agent; Biologically active agent; Detectable label; With and any combination; And L is optionally, and is the connexon that is selected from the group that is made up of following each group when existing: alkylidene group, be substituted alkylidene group, alkenylene, be substituted alkenylene ,-O-,-O-(alkylidene group or be substituted alkylidene group)-,-S-,-S-(alkylidene group or be substituted alkylidene group)-,-S (O) k-(wherein k is 1,2 or 3) ,-S (O) k(alkylidene group or be substituted alkylidene group)-,-C (O)-,-C (O)-(alkylidene group or be substituted alkylidene group)-,-C (S)-,-C (S)-(alkylidene group or be substituted alkylidene group)-,-N (R ')-,-NR '-(alkylidene group or be substituted alkylidene group)-,-C (O) N (R ')-,-CON (R ')-(alkylidene group or be substituted alkylidene group)-,-CSN (R ')-,-CSN (R ')-(alkylidene group or be substituted alkylidene group)-,-N (R ') CO-(alkylidene group or be substituted alkylidene group)-,-N (R ') C (O) O-,-S (O) kN (R ') ,-N (R ') C (O) N (R ')-,-N (R ') C (S) N (R ')-,-N (R ') S (O) kN (R ')-,-N (R ')-N=,-C (R ')=N-,-C (R ')=N-N (R ')-,-C (R ')=N-N=,-C (R ') 2-N=N-and-C (R ') 2-N (R ')-N (R ')-, wherein R ' is H, alkyl independently of one another or is substituted alkyl.
These amino acid whose limiting examples comprise the amino acid with following structure:
Figure A20058004432801291
And
Figure A20058004432801293
These alpha-non-natural amino acids can be the form of salt, maybe can incorporate in non-natural amino acid polypeptides, polymkeric substance, polysaccharide or the polynucleotide and according to circumstances through posttranslational modification.
In addition, these amino acid comprise the have formula amino acid of structure of (XVIII):
Figure A20058004432801301
Wherein:
B is optionally, and is the connexon that is selected from the group that is made up of following each group when exist: low-grade alkylidene, be substituted low-grade alkylidene, lower alkenylene, be substituted lower alkenylene, alkyl is mixed in rudimentary Asia, be substituted rudimentary Asia mix alkyl ,-O-,-O-(alkylidene group or be substituted alkylidene group)-,-S-,-S-(alkylidene group or be substituted alkylidene group)-,-S (O) k-
(wherein k is 1,2 or 3) ,-S (O) k(alkylidene group or be substituted alkylidene group)-,-C (O)-,-NS (O) 2-,-OS (O) 2-,-C (O)-(alkylidene group or be substituted alkylidene group)-,-C (S)-,-C (S)-(alkylidene group or be substituted alkylidene group)-,-N (R ')-,-NR '-(alkylidene group or be substituted alkylidene group)-,-C (O) N (R ')-,-CON (R ')-(alkylidene group or be substituted alkylidene group)-,-CSN (R ')-,-CSN (R ')-(alkylidene group or be substituted alkylidene group)-,-N (R ') CO-(alkylidene group or be substituted alkylidene group)-,-N (R ') C (O) O-,-S (O) kN (R ')-,-N (R ') C (O) N (R ')-,-N (R ') C (S) N (R ')-,-N (R ') S (O) kN (R ')-,-N (R ')-N=-C (R ')=N-,-C (R ')=N-N (R ')-,-C (R ')=N-N=,-C (R ') 2-N=N-and-C (R ') 2-N (R ')-N (R ')-, wherein R ' is H, alkyl independently of one another or is substituted alkyl;
R 1Be H, amino protecting group, resin, amino acid, polypeptide or polynucleotide; And
R 2Be OH, ester protecting group, resin, amino acid, polypeptide or polynucleotide;
R 6With R 7Be selected from the group that forms by following each group independently of one another: H, alkyl, be substituted alkyl, thiazolinyl, be substituted thiazolinyl, alkoxyl group, be substituted alkoxyl group, polyoxyalkylene, be substituted polyoxyalkylene, aryl, be substituted aryl, heteroaryl, be substituted heteroaryl, alkaryl, be substituted alkaryl, aralkyl, and be substituted aralkyl ,-C (O) R " ,-C (O) 2R " ,-C (O) N (R ") 2, R wherein " independently of one another for hydrogen, alkyl, be substituted alkyl, thiazolinyl, be substituted thiazolinyl, alkoxyl group, be substituted alkoxyl group, aryl, be substituted aryl, heteroaryl, alkaryl, be substituted alkaryl, aralkyl or be substituted aralkyl; Or R 6Or R 7Be L-X, wherein
X is selected from the group that is made up of following each thing: mark; Dyestuff; Polymkeric substance; Water-soluble polymers; The derivative of polyoxyethylene glycol; Photocrosslinking agent; Cytotoxic compound; Medicine; Affinity marker; The photoaffinity mark; Reactive compounds; Resin; Second protein or polypeptide or polypeptide analog; Antibody or antibody fragment; Metal chelator; Cofactor; Lipid acid; Carbohydrate; Polynucleotide; DNA; RNA; Antisense polynucleotide; Carbohydrate, water-soluble branch-shape polymer, cyclodextrin, biomaterial; Nanoparticle; Spin labeling; Fluorophore, metallic part; Radioactive segment; Novel functional group; With the covalently or non-covalently interactional group of other molecules; The light cage covers part; But photoisomerization part; Vitamin H; The vitamin H analogue; Be combined with the part of heavy atom; But the group of chemical cracking; But the group of photodestruciton; The side chain that prolongs; The sugar of carbon bond connection; Redox active agent; Amino thioic acid sulfoacid; The toxicity part; Through isotope-labeled part; The biophysics probe; The phosphorescence group; Chemiluminescent groups; The electron dense group; The magnetic group; Insert group; Chromophoric group; Energy transfer agent; Biologically active agent; Detectable label; With and any combination; And L is optionally, and is the connexon that is selected from the group that is made up of following each group when existing: alkylidene group, be substituted alkylidene group, alkenylene, be substituted alkenylene ,-O-,-O-(alkylidene group or be substituted alkylidene group)-,-S-,-S-(alkylidene group or be substituted alkylidene group)-,-S (O) k-(wherein k is 1,2 or 3) ,-S (O) k(alkylidene group or be substituted alkylidene group)-,-C (O)-,-C (O)-(alkylidene group or be substituted alkylidene group)-,-C (S)-,-C (S)-(alkylidene group or be substituted alkylidene group)-,-N (R ')-,-NR '-(alkylidene group or be substituted alkylidene group)-,-C (O) N (R ')-,-CON (R ')-(alkylidene group or be substituted alkylidene group)-,-CSN (R ')-,-CSN (R ')-(alkylidene group or be substituted alkylidene group)-,-N (R ') CO-(alkylidene group or be substituted alkylidene group)-,-N (R ') C (O) O-,-S (O) kN (R ')-,-N (R ') C (O) N (R ')-,-N (R ') C (S) N (R ')-,-N (R ') S (O) kN (R ')-,-N (R ')-N=,-C (R ')=N-,-C (R ')=N-N (R ')-,-C (R ')=N-N=,-C (R ') 2-N=N-and-C (R ') 2-N (R ')-N (R ')-, wherein R ' is H, alkyl independently of one another or is substituted alkyl; And
R aBe selected from the group that forms by following each group: H, halogen, alkyl independently of one another, be substituted alkyl ,-N (R ') 2,-C (O) kR ' (wherein k is 1,2 or 3) ,-C (O) N (R ') 2,-OR ' and-S (O) kR '; Wherein R ' is independently of one another for H, alkyl or be substituted alkyl and n is 0 to 8.
These alpha-non-natural amino acids can be the form of salt, maybe can incorporate in non-natural amino acid polypeptides, polymkeric substance, polysaccharide or the polynucleotide and according to circumstances through posttranslational modification.
These amino acid whose limiting examples comprise the amino acid with following structure:
Figure A20058004432801311
And
These alpha-non-natural amino acids can be the form of salt, maybe can incorporate in non-natural amino acid polypeptides, polymkeric substance, polysaccharide or the polynucleotide and according to circumstances through posttranslational modification.
Alpha-non-natural amino acid based on oxime can synthesize by the method for having described in the affiliated field or by the method for describing herein, and it comprises: (a) make alpha-non-natural amino acid that contains azanol and the reagent react that contains carbonyl or dicarbapentaborane; (b) make alpha-non-natural amino acid that contains carbonyl or dicarbapentaborane and the reagent react that contains azanol; Or (c) make alpha-non-natural amino acid that contains oxime and the reagent that some contains carbonyl or dicarbapentaborane (comprise and for example contain ketone reagent or contain aldehyde reagent) reaction.Representativeness, the limiting examples of Fig. 5 and these synthetic methods of Fig. 6 oblatio.
D. the cell of alpha-non-natural amino acid absorbs
When design and selection alpha-non-natural amino acid (including (but not limited to) being used for incorporating into polypeptide), eukaryotic alpha-non-natural amino acid is absorbed as a problem of common consideration.For instance, the high charge density of a-amino acid hints that these compounds unlikely see through cell.Natural amino acid is to be absorbed in the eukaryotic cell by the set based on proteinic transmission system.Can assess the quick screening which alpha-non-natural amino acid (if present) absorbs by cell (limiting examples of the test that example 15 herein and 16 explanations can be carried out alpha-non-natural amino acid).Be the toxicological detection in the U.S. Patent Publication case of " Protein Arrays " No. 2004/198637 (this case is all to be incorporated herein by reference) referring to (for example) title, and Liu, D.R. and Schultz, P.G. (1999) Progress toward theevolution of an organism with an expanded genetic code. PNAS United States96:4780-4785.Be easy to by various calibrating analyses although absorb, the replacement scheme that design meets the alpha-non-natural amino acid of cell absorption approach in vivo produces amino acid whose biosynthetic pathway for providing.
Usually, absorbing the alpha-non-natural amino acid that produces by cell as described herein is to produce to be enough to the carrying out biosynthetic concentration of effective protein (including (but not limited to) the n cell amount), but does not reach such as influencing other amino acid whose concentration or exhausting the degree of cell resource.The typical concentration of Chan Shenging is that about 10mM is to about 0.05mM in this way.
E. the biosynthesizing of alpha-non-natural amino acid
Many biosynthetic pathways have been present in the cell that is used for producing amino acid and other compounds.Though the biosynthetic means of specific alpha-non-natural amino acid may not exist in nature (including (but not limited to) cell), the method and composition of Miao Shuing provides these methods herein.For instance, the biosynthetic pathway of alpha-non-natural amino acid can produce in host cell by adding new enzyme or modifying existing host cell approach.Other new enzymes comprise naturally occurring enzyme or the artificial enzyme that develops.For instance, the biosynthesizing of p-Aminophenylalanine (being the example institute oblatio among the WO 2002/085923 of " In vivo incorporation ofunnatural amino acids " as title) depends on the combination of interpolation from the known enzyme of other biological body.The gene of these enzymes can be by introducing in the eukaryotic cell with the plasmid transformant that comprises these genes.When expressing in cell, these genes provide the enzymatic route of synthetic required compound.The example of the type of the enzyme that adds according to circumstances is provided herein.Other enzyme sequences see in (for example) gene pool (Genbank).The artificial enzyme that develops can add in the cell in the same manner.In this way, the cell mechanism of manipulated cell and source are to produce alpha-non-natural amino acid.
Several different methods can be used for preparing and is used for biosynthetic pathway or the novel enzymes of the existing approach that develops.For instance, including (but not limited to) as by Maxygen, Inc. (can be in the World Wide Web (world wide web) go up with Www.maxygen.comAcquisition) reorganization of the recurrence of exploitation can be used for developing novel enzyme and approach.Referring to (for example) Stemmer (1994), Rapidevolution of a protein in vitro by DNA shuffling, Nature370 (4): 389-391; And Stemmer, (1994), DNA shuffling by random fragmentation and reassembly:In vitro recombination formolecular evolution, Proc.Natl.Acad.Sci.USA.,91:10747-10751.By Genencor (can be on the World Wide Web with Genencor.comAcquisition) the Similarly DesignPath of exploitation TMBe used for the metabolic pathway engineering design according to circumstances, it is including (but not limited to) being used for engineering design produces alpha-non-natural amino acid in cell approach.This technology use new gene combination (including (but not limited to) differentiate by functional genomics, molecular evolution and design those) transform the existing approach in the host organisms.Diversa Corporation (can be on the World Wide Web with Diversa.comObtain) technology in the storehouse of quick screening gene and gene approach also is provided, it is used for producing by biosynthesizing the new way of alpha-non-natural amino acid including (but not limited to) generation.
Usually, with the alpha-non-natural amino acid that produces through the biosynthetic pathway of engineering design as described herein is to produce to be enough to the carrying out biosynthetic concentration of effective protein (including (but not limited to) the n cell amount), but does not reach such as influencing other amino acid concentrations or exhausting the degree of cell resource.The typical concentration that in vivo produces is that about 10mM is to about 0.05mM in this way.In case with the plasmid transformant that comprises the gene that is used to produce the required enzyme of particular approach and produce alpha-non-natural amino acid, then use according to circumstances and in vivo select to grow into the generation of one-step optimization alpha-non-natural amino acid with cell at ribosomal protein is synthetic.
F. other synthetic methods
Method of describing in the field under the alpha-non-natural amino acid of Miao Shuing can use herein or the technology of using description herein or next synthetic by its combination.As auxiliary, following table provides can be through combination to produce required functional group's various initial electrophilic reagent and nucleophilic reagent.The information that is provided is intended for illustrative and and the unrestricted synthetic technology of describing herein.
Table 1: covalent linkage with and the example of precursor
The covalent linkage product Electrophilic reagent Nucleophilic reagent
Carboxylic acid amides Acibenzolar Amine/aniline
Carboxylic acid amides Acyl azide Amine/aniline
Carboxylic acid amides Acyl halide Amine/aniline
Ester Acyl halide Alcohol/phenol
Ester The acyl group nitrile Alcohol/phenol
Carboxylic acid amides The acyl group nitrile Amine/aniline
Imines Aldehyde Amine/aniline
Hydrazone Aldehydes or ketones Hydrazine
Oxime Aldehydes or ketones Azanol
Alkylamine Alkyl halide Amine/aniline
Ester Alkyl halide Carboxylic acid
Thioether Alkyl halide Mercaptan
Ether Alkyl halide Alcohol/phenol
Thioether Alkyl sulfonate esters Mercaptan
Ester Alkyl sulfonate esters Carboxylic acid
Ether Alkyl sulfonate esters Alcohol/phenol
Ester Acid anhydride Alcohol/phenol
Carboxylic acid amides Acid anhydride Amine/aniline
Thiophenol Aryl halide Mercaptan
Arylamines Aryl halide Amine
Thioether Azindine Mercaptan
Boric acid ester Borate Glycol
Carboxylic acid amides Carboxylic acid Amine/aniline
Ester Carboxylic acid Alcohol
Hydrazine Hydrazides Carboxylic acid
N-acylurea or acid anhydride Carbodiimide Carboxylic acid
Ester Diazoalkane Carboxylic acid
Thioether Epoxide Mercaptan
Thioether The halogen ethanamide Mercaptan
Aminotriazine The halogen triazine Amine/aniline
Triazinyl ether The halogen triazine Alcohol/phenol
Amidine Imide ester Amine/aniline
Urea Isocyanic ester Amine/aniline
Carbamate Isocyanic ester Alcohol/phenol
Thiocarbamide Lsothiocyanates Amine/aniline
Thioether Maleimide Mercaptan
Phosphorous acid ester Phosphoramidate Alcohol
Silyl ether Silylation halogenide Alcohol
Alkylamine Sulphonate Amine/aniline
Thioether Sulphonate Mercaptan
Ester Sulphonate Carboxylic acid
Ether Sulphonate Alcohol
Sulphonamide Alkylsulfonyl halogenide Amine/aniline
Sulphonate Alkylsulfonyl halogenide Phenol/alcohol
In general, the carbon electrophilic reagent is for the attack sensitivity of complementary nucleophilic reagent (comprising carbon nucleophile), and wherein the nucleophilic reagent of attack provides electron pair to form new key between nucleophilic reagent and carbon electrophilic reagent to the carbon electrophilic reagent.
The limiting examples of carbon nucleophile is including (but not limited to) alkyl Grignard reagent (alkyl Grignard), thiazolinyl Grignard reagent, aryl grignard reagent and alkynyl Grignard reagent, organolithium, organic zinc, tin alkyl reagent, thiazolinyl tin reagent, tin aryl SnAr2 reagent and alkynyl tin reagent (organic stannane), alkyl borane reagent, thiazolinyl borane reagent, aryl borane reagent and alkynyl borane reagent (organo-borane and organic borate); These carbon nucleophiles have in water or polar organic solvent advantage stable on kinetics.Other limiting examples of carbon nucleophile comprise phosphorus inner salt, enol and enolate reagent; These carbon nucleophiles have and are easy to the advantage that the precursor known by the technician in the synthetic organic chemistry field produces relatively.When using jointly with the carbon electrophilic reagent, carbon nucleophile forms new carbon-carbon bond between carbon nucleophile and carbon electrophilic reagent.
Be suitable for the limiting examples of the non-carbon nucleophile of carbon electrophilic reagent coupling including (but not limited to) primary amine and secondary amine, mercaptan, thiolate and thioether, alcohol, alkoxide, trinitride, semicarbazides with and analogue.When using jointly with the carbon electrophilic reagent, these non-carbon nucleophiles produce heteroatomic bond (C-X-C) usually, and wherein X is a heteroatoms, and it is including (but not limited to) oxygen, sulphur or nitrogen.
VI. the polypeptide that has alpha-non-natural amino acid
For simplicity, the form of the compound of describing in this part, character and other features are general and/or describe with particular instance.Yet, the generality that the form of describing in this part, character and other features should not only limit to provide in this part is described or particular instance, but same well suited all compounds in the category that is in formula I-XVIII, XXX-XXXIV (A and B) and XXXX-XXXXIII of the form of describing in this part, character and other features, it comprises interior any minor or the specific compound of category of the specification sheets, claim and graphic middle formula I-XVIII, XXX-XXXIV (A and B) that describes and the XXXX-XXXXIII that are in herein.
Composition of Miao Shuing and method provide at least one alpha-non-natural amino acid are incorporated in the polypeptide herein.Alpha-non-natural amino acid can be present in any position on the polypeptide, and it comprises any terminal position or any interior location of polypeptide.With respect to the natural amino acid polypeptide that exists of homology, preferred alpha-non-natural amino acid does not destroy the activity and/or the tertiary structure of polypeptide, and this destruction of removing nonactive and/or tertiary structure is for incorporating alpha-non-natural amino acid into a purpose in the polypeptide.In addition, with respect to the natural amino acid polypeptide that exists of homology, incorporate in the polypeptide to a certain extent alpha-non-natural amino acid the activity of modified polypeptide into and (for example, handle the therapeutic efficiency of polypeptide; The security overview of improvement polypeptide; Pharmacokinetics, pharmacology and/or the drug effect of regulating polypeptide (for example, increase water-soluble, biological usability; Increase serum half-life; Increase the treatment transformation period; Regulate immunogenicity; Regulate biological activity; Or prolong cycling time); Provide other functional groups to polypeptide; In polypeptide, incorporate label, mark or detectable signal into; The separating property of facilitation polypeptide; And the destruction of not causing activity and/or tertiary structure fully and any combination of aforementioned modification) and/or tertiary structure.These modifications to activity and/or tertiary structure are generally these targets incorporating into of realization, although with respect to the natural amino acid polypeptide that exists of homology, alpha-non-natural amino acid incorporated in the polypeptide into also can the activity and/or the tertiary structure of polypeptide do not exerted an influence.Correspondingly, think non-natural amino acid polypeptides, comprise non-natural amino acid polypeptides composition, be used to make these polypeptide and peptide composition method, be used for purifying, separate and the method for these polypeptide of sign and peptide composition, and the method for using these polypeptide and peptide composition is in the category of this disclosure.In addition, the non-natural amino acid polypeptides of describing herein also can be connected on another polypeptide (including (for example) non-natural amino acid polypeptides or the natural amino acid polypeptide that exists).
The non-natural amino acid polypeptides of Miao Shuing can be by biosynthesizing or abiotic synthetic preparation herein.Biosynthesizing means any method of utilizing translation system (cell or acellular), and it comprises in the following component of use at least one: polynucleotide, codon, tRNA and rrna.Abiotic synthesizing means any method of not utilizing translation system: this method can be further divided into the method for utilizing solid-state peptide synthetic method, solid-phase peptide synthetic method; Utilize the method for at least a enzyme; And the method for not utilizing at least a enzyme; In addition, any part in this segmentation part can be overlapping and many methods can utilize the combination of these segmentation parts.
The method of Miao Shuing, composition, strategy and technology are not limited to polypeptide or proteinic particular type, kind or family herein.Really, any in fact polypeptide can comprise the alpha-non-natural amino acid that at least one is described herein.Only for instance, polypeptide can with the treatment protein homology that is selected from the group that forms by following each thing: α-1 antitrypsin, angiostatin, AHF, antibody, lipophorin, apoprotein, atrionatriuretic factor, atrial natriuretic polypeptins, atrial natriuretic peptide, the C-X-C chemokine, T39765, NAP-2, ENA-78, gro-a, gro-b, gro-c, IP-10, GCP-2, NAP-4, SDF-1, PF4, MIG, calcitonin, the c-kit ligand, cytokine, the CC chemokine, MCP-1, MCP-2, monocyte chemoattractant protein-3, monocyte inflammatory protein-1 α, monocyte inflammatory protein-i β, RANTES, 1309, R83915, R91733, HCC1, T58847, D31065, T64262, CD40, the CD40 ligand, the c-kit ligand, collagen protein, G CFS (CSF), complement factor 5a, complement inhibitor, complement receptor 1, cytokine, epithelium neutrophilic granulocyte activation peptide-78, MIP-16, MCP-1, Urogastron (EGF), epithelium neutrophilic granulocyte activation peptide, erythropoietin (EPO), exfoliative toxin,or exfoliatin, factors IX, factor VII, Factor IX, factor X, fibroblast growth factor (FGF), Fibrinogen, fibronectin, four-helix bundle albumen, G-CSF, glp-1, GM-CSF, glucocerebrosidase, gonad-stimulating hormone, somatomedin, growth factor receptors, grf, Shh, hemochrome, pHGF (hGF), r-hirudin, human growth hormone (hGH), the human serum albumin, ICAM-1, the ICAM-1 acceptor, LFA-1, the LFA-1 acceptor, Regular Insulin, insulin-like growth factor (IGF), IGF-I, IGF-II, Interferon, rabbit (IFN), IFN-α, IFN-β, IFN-γ, interleukin-(IL), IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, keratinocyte growth factor (KGF), lactoferrin, leukaemia inhibitory factor, luciferase, neural plain, neutrophil inhibitory factor (nif) (NIF), oncostatin M, bone morphogenic protein, oncoprotein, paracitonin, Rat parathyroid hormone 1-34, PD-ECSF, PDGF, peptide hormone, PTN, a-protein, protein G, pth, pyrogenic exotoxin A, pyrogenic exotoxin B, pyrogenic exotoxin C, pyy, Relaxin, feritin, SCF, the atom synthetic proteins, soluble complement acceptor I, solubility I-CAM 1, soluble interleukin receptor, soluble TNF acceptor, somatomedin, Somat, tethelin, streptokinase, superantigen, staphyloentero-toxin, SEA, SEB, SEC1, SEC2, SEC3, SED, SEE, steroid hormone receptor, superoxide-dismutase, the toxic shock syndrome toxin, thymosin, the histiotype plasminogen activation factor, tumor growth factor (TGF), tumour necrosis factor, tumor necrosis factor alpha, tumor necrosis factor, Tumor Necrosis Factor Receptors (TNFR), VLA-4 albumen, VCAM-1 albumen, vascular endothelial growth factor (VEGF), urokinase, mos, ras, raf, met, p53, tat, fos, myc, jun, myb, rel, estrogen receptor, PgR, the testosterone acceptor, aldosterone receptor, ldl receptor and Kendall compound.In relevant or other embodiment, non-natural amino acid polypeptides also can with any polypeptide member homology of tethelin supergene family.
Non-natural amino acid polypeptides can use without further modifying as further modification of warp as described in the elsewhere in this disclosure or non-natural amino acid polypeptides.Can be multiple purpose and alpha-non-natural amino acid is incorporated in the polypeptide, it is including (but not limited to) the change of customization protein structure and/or function, change size, acidity, nucleophilicity, hydrogen bond, hydrophobicity, the accessibility of proteolytic enzyme target site, targeting moiety (including (but not limited to), for the polypeptide array) etc.The polypeptide that comprises alpha-non-natural amino acid can have enhanced or even complete new catalysis or bio-physical property.Only for instance, following character can be transformed by alpha-non-natural amino acid is contained in the polypeptide: toxicity, bio distribution, textural property, spectral quality, chemistry and/or spectrochemical property, catalytic capability, transformation period (including (but not limited to) serum half-life), with the ability of other molecular reactions (including (but not limited to) covalently or non-covalently) with and similarity.Composition with the polypeptide that comprises at least one alpha-non-natural amino acid is applicable to and (comprises, but be not limited to) novel treatment, diagnosis, katalaze enzyme, industrial enzyme, conjugated protein (including (but not limited to) antibody), and research (including (but not limited to) the research to protein structure and function).Referring to (for example) Dougherty, (2000) Unnatural Amino Acids asProbes of Protein Structure and Function, Current Opinion in Chemical Biology,4:645-652.
In addition, the side chain of the alpha-non-natural amino acid component of polypeptide can provide multiple other functional groups to polypeptide; Only for instance, and be not in a limitative way, the side chain of the alpha-non-natural amino acid part of polypeptide can comprise any one in the following material: mark; Dyestuff; Polymkeric substance; Water-soluble polymers; The derivative of polyoxyethylene glycol; Photocrosslinking agent; Cytotoxin compounds; Medicine; Affinity marker; The photoaffinity mark; Reactive compounds; Resin; Second protein or polypeptide or polypeptide analog; Antibody or antibody fragment; Metal chelator; Cofactor; Lipid acid; Carbohydrate; Polynucleotide; DNA; RNA; Antisense polynucleotide; Carbohydrate, water-soluble branch-shape polymer, cyclodextrin, biomaterial; Nanoparticle; Spin labeling; Fluorophore, metallic part; Radioactive segment; Novel functional group; With the covalently or non-covalently interactional group of other molecules; The light cage covers part; But the part that actinic radiation excites; Ligand; But photoisomerization part; Vitamin H; The vitamin H analogue; Be combined with the part of heavy atom; But the group of chemical cracking; But the group of photodestruciton; The side chain that prolongs; The sugar of carbon bond connection; Redox active agent; Amino thioic acid sulfoacid; The toxicity part; Through isotope-labeled part; The biophysics probe; The phosphorescence group; Chemiluminescent groups; The electron dense group; The magnetic group; Insert group; Chromophoric group; Energy transfer agent; Biologically active agent; Detectable label; Small molecules; Inhibition Yeast Nucleic Acid, radioactive nuleus thuja acid; Neutron capture agent; The derivative of vitamin H; Quantum dot; Nanometer is transmitted plain; Radioactivity is transmitted plain; Abzyme, activation composite activating agent, virus, adjuvant, aglycone, anaphylactogen, angiostatin, hormone antagonist, antioxidant, fit, guide RNA, Saponin/TSM, shuttle vectors, macromole, plan epitope, acceptor, reverse micelle with and any combination.
On the one hand, composition comprises at least a polypeptide with at least one (including (but not limited to) more than at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine or at least ten or ten) alpha-non-natural amino acid.These alpha-non-natural amino acids can be identical or different.In addition, in the polypeptide that comprises the similar and different alpha-non-natural amino acid more than 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20 or 20, may there be different loci more than 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20 or 20.On the other hand, composition comprises the polypeptide that at least one (but being less than all) specific amino acids of existing in the polypeptide wherein replaces through alpha-non-natural amino acid.For given polypeptide with an above alpha-non-natural amino acid, these alpha-non-natural amino acids can be identical or different (such as, only for instance, polypeptide can comprise two or more dissimilar alpha-non-natural amino acid, maybe can comprise two identical alpha-non-natural amino acids).For the given polypeptide with two above alpha-non-natural amino acids, alpha-non-natural amino acid can be identical, different or be the combination of alpha-non-natural amino acid and at least a different alpha-non-natural amino acids of the identical type of a plurality of numbers.
Although herein the embodiment of the non-natural amino acid polypeptides of Miao Shuing can pass through the solid-phase peptide synthetic method (such as, only for instance, on solid resin), solution phase peptide synthetic method and/or do not come chemosynthesis by means of enzyme, but other embodiment of the non-natural amino acid polypeptides of Miao Shuing allow by cytolemma, cell extract or lysate system herein, or by system in vivo (such as, only for instance, use prokaryotic cell prokaryocyte or eukaryotic cell mechanism) synthesize.In other or extra embodiment, a kind of key feature of the non-natural amino acid polypeptides of Miao Shuing is that it can utilize rrna to synthesize herein.Among other of the non-natural amino acid polypeptides of Miao Shuing or the extra embodiment, non-natural amino acid polypeptides can come synthetic by the combination (including (but not limited to) solid resin, not by means of enzyme, by means of rrna and/or by the in vivo combination of system) of method in this article.
By rrna and/or in vivo the synthetic non-natural amino acid polypeptides of system have with on solid resin or not by means of the non-natural amino acid polypeptides of enzymic synthesis different advantage and feature.These advantages comprise different Impurity Distribution with feature: utilize rrna and/or in vivo the system of system should have the impurity that comes from the biosystem of being utilized; it comprises host cell proteins matter, membrane portions and lipid, and from utilizing solid resin and/or can not comprising other chemical that use in organic solvent, protecting group, resin material, coupling reagent and the synthesis program by means of the Impurity Distribution of the system of enzyme.In addition, by use rrna and/or in vivo the isotopic pattern of system's synthetic non-natural amino acid polypeptides can reflect the isotopic pattern of the raw material that cell utilizes; On the other hand, on solid resin and/or not by means of the isotopic pattern of the non-natural amino acid polypeptides of enzymic synthesis can reflect synthetic in the amino acid whose isotopic pattern of utilization.In addition, by use rrna and/or in vivo system's synthetic alpha-non-natural amino acid can not contain amino acid whose D-isomer in fact and/or can be easy to inner cysteine amino acids is incorporated in the structure of polypeptide, and/or may seldom provide internal amino acid disappearance polypeptide.On the other hand, by solid resin and/or do not use the non-natural amino acid polypeptides of enzymic synthesis can have amino acid whose D-isomer and/or the inside cysteine amino acids of lower aq and/or the internal amino acid disappearance polypeptide of higher percent of high level.In addition, the those skilled in the art should be able to distinguish by use rrna and/or in vivo system's synthetic non-natural amino acid polypeptides with by solid resin and/or do not use the non-natural amino acid polypeptides of enzymic synthesis.
VII. the composition and the method that comprise nucleic acid and oligonucleotide
A. be used for general recombinant nucleic acid method herein
In this article among numerous embodiment of the method and composition of Miao Shuing, separate nucleic acid, the clone of the polypeptide (comprising for example GH polypeptide) that coding is paid close attention to and use the recombination method transformation usually.These embodiment are used for (comprise, but be not limited to) protein expression or use in generation derives from the process of variant polypeptides, derivative, expression cassette or other sequences.In certain embodiments, the sequence of coded polypeptide is that operability is connected on the allogeneic promoter.
Coding comprises that the nucleotide sequence of the polypeptide of alpha-non-natural amino acid can be synthetic according to the aminoacid sequence of parent polypeptide, and changes nucleotide sequence subsequently with the introducing (that is, incorporate into or replaces) that realizes the related amino acid residue or remove (that is, disappearance or replacement).Nucleotide sequence can form easily by rite-directed mutagenesis according to conventional methods and modify.Perhaps, nucleotide sequence can prepare by chemosynthesis, it is including (but not limited to) passing through to use oligonucleotide synthesizer, and wherein oligonucleotide is according to required amino acid sequence of polypeptide design, and preferably selects recombinant polypeptide will result from those codons of host cell preference wherein.For instance, encode some small oligonucleotides of part of required polypeptide can come synthetic and assembling by PCR, connection or connection chain reaction.Referring to people such as (for example) Barany, Proc.Natl.Acad.Sci.88:189-193 (1991); U.S.6,521,427, it is to be incorporated herein by reference.
The alpha-non-natural amino acid method and composition of Miao Shuing utilizes the routine techniques in the genetic recombination field herein.The basic works of the universal method of the alpha-non-natural amino acid method and composition that exposure is used for describing herein comprises people such as Sambrook, Molecular Cloning, A Laboratory Manual (the 3rd edition, 2001); Kriegler, Gene Transfer andExpression:A Laboratory Manual (1990); And Current Protocols in Molecular Biology (people such as Ausubel compiles, 1994).
The general works of describing Protocols in Molecular Biology comprises Berger and Kimmel, Guide to Molecular CloningTechniques, Methods in Enzymology, the 152nd volume, Academic Press, Inc., San Diego, CA (Berger); People such as Sambrook, Molecular Cloning-A Laboratory Manual (the 2nd edition), the 1-3 volume, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989 (" Sambrook ") and Current Protocols in Molecular Biology, people such as F.M.Ausubel compile, Current Protocols, GreenePublishing Associates, Inc. and John Wiley ﹠amp; Sons, the joint venture of Inc., (augment 1999 whole years) (" Ausubel ")).These works are described sudden change and are formed, the purposes of carrier, promotor, and many other related subjects, its relate to including (but not limited to) comprise be used to prepare the proteinic selection codon that comprises alpha-non-natural amino acid, quadrature tRNA, quadrature synthetic enzyme with and the right gene or the generation of polynucleotide.
Think in the alpha-non-natural amino acid method and composition that various types of sudden changes are formed for describing herein and realize multiple purpose, it is including (but not limited to) being used to produce novel synthetic enzyme or tRNA, be used to make the sudden change of tRNA molecule, be used to make the mutant polynucleotide in coding synthetic enzyme, tRNA storehouse, be used to produce the synthetic enzyme storehouse, be used for produce selecting codon, be used for inserting the selection codon of the alpha-non-natural amino acid of protein that coding pays close attention to or polypeptide.Its including (but not limited to) rite-directed mutagenesis formation, random point mutation formation, homologous recombination, DNA reorganization or other recurrence sudden change formation methods, chimeric construct, use contain the sudden change formation, oligonucleotide directed mutagenesis of the template of uridylic, the dna mutation of modifying through thiophosphatephosphorothioate forms, uses the sudden change of notched double-stranded DNA or its analogue to form, or its any combination.The sudden change of the host strain that other appropriate methodologies comprise a mispairing reparation, use to lack repair forms, restriction-selections and restriction-purifying, deletion mutantion formation, synthesize by total gene and to bring out sudden change, double-strand break reparation, with and similar approach.Be also contained in the alpha-non-natural amino acid method and composition of describing herein including (but not limited to) comprising that chimeric sudden change of constructing body forms.In one embodiment, sudden change forms and can be instructed by the natural natural Given information (including (but not limited to) sequence comparison, physical properties, crystalline structure or its analogue) of molecule that exists of molecule or variation or sudden change that exists.
These and other dependent programs of works of Cun Zaiing and case description herein.Other information see the following discloses case and the reference wherein quoted in: people such as Ling, Approaches to DNA mutagenesis:an overview, AnalBiochem.254 (2): 157-178 (1997); People such as Dale, Oligonucleotide-directed random mutagenesisusing the phosphorothioate method, Methods Mol.Biol.57:369-374 (1996); Smith, In vitromutagenesis, Ann.Rev.Genet.19:423-462 (1985); Botstein and Shortle, Strategies andapplications of in vitro mutagenesis, Science 229:1193-1201 (1985); Carter, Site-directedmutagenesis, Biochem.J.237:1-7 (1986); Kunkel, The efficiency of oligonucleotide directedmutagenesis is at Nucleic Acids ﹠amp; Among the Molecular Biology (D.M.J. compiles for Eckstein, F. and Lilley, Springer Verlag, Berlin)) (1987); Kunkel, Rapid and efficient site-specific mutagenesiswithout phenotypic selection, Proc.Natl.Acad.Sci.USA 82:488-492 (1985); People such as Kunkel, Rapid and efficient site-specific mutagenesis without phenotypic selection, Methods inEnzymol.154,367-382 (1987); People such as Bass, Mutant Trp repressors with new DNA-bindingspecificities, Science 242:240-245 (1988); Methods in Enzymol.100:468-500 (1983); Methods in Enzymol.154:329-350 (1987); Zoller and Smith, Oligonucleotide-directedmutagenesis using Ml3-derived vectors:an efficient and general procedure for theproduction of point mutations in any DNA fragment, Nucleic Acids Res.10:6487-6500 (1982); Zoller and Smith, Oligonucleotide-directed mutagenesis of DNA fragments cloned intoMl3 vectors, Methods in Enzymol.100:468-500 (1983); Zoller and Smith, Oligonucleotide-directed mutagenesis:a simple method using two oligonucleotide primersand a single-stranded DNA template, Methods in Enzymol.154:329-350 (1987); People such as Taylor, The use of phosphorothioate-modified DNA in restriction enzyme reactions to preparenicked DNA, Nucl.Acids Res.13:8749-8764 (1985); People such as Taylor, The rapid generation ofoligonucleotide-directed mutations at high frequency using phosphorothioate-modified DNA, Nucl.Acids Res.13:8765-8785 (1985); Nakamaye and Eckstein, Inhibition of restrictionendonuclease Nci I cleavage by phosphorothioate groups and its application tooligonucleotide-directed mutagenesis, Nucl.Acids Res.14:9679-9698 (1986); People such as Sayers, 5 '-3 ' Exonucleases in phosphorothioate-based oligonucleotide-directed mutagenesis, Nucl.Acids Res.16:791-802 (1988); People such as Sayers, Strand specific cleavage ofphosphorothioate-containing DNA by reaction with restriction endonucleases in the presenceofethidium bromide, (1988) Nucl.Acids Res.16:803-814; People such as Kramer, The gapped duplexDNA approach to oligonucleotide-directed mutation construction, Nucl.Acids Res.12:9441-9456 (1984); Kramer and Fritz, Oligonucleotide-directed construction of mutations viagapped duplex DNA, Methods in Enzymol.154:350-367 (1987); People such as Kramer, Improvedenzymatic in vitro reactions in the gapped duplex DNA approach to oligonucleotide-directedconstruction of mutations, Nucl.Acids Res.16:7207 (1988); People such as Fritz, Oligonucleotide-directed construction of mutations:a gapped duplex DNA procedure withoutenzymatic reactions in vitro, Nucl.Acids Res.16:6987-6999 (1988); People such as Kramer, PointMismatch Repair, Cell 38:879-887 (1984); People such as Carter, Improved oligonucleotidesite-directed mutagenesis using Ml3 vectors, Nucl.Acids Res.13:4431-4443 (1985); Carter, Improved oligonucleotide-directed mutagenesis using Ml3 vectors, Methods in Enzymol.154:382-403 (1987); Eghtedarzadeh and Henikoff, Use of oligonucleotides to generate largedeletions, Nucl.Acids Res.14:5115 (1986); People such as Wells, Importance of hydrogen-bondformation in stabilizing the transition state of subtilisin, Phil.Trans.R.Soc.Lond.A 317:415-423 (1986); People such as Nambiar, Total synthesis and cloning of a gene coding for theribonuclease Sprotein, Science 223:1299-1301 (1984); Sakmar and Khorana, Total synthesisand expression of a gene for the a-subunit of bovine rod outer segment guaninenucleotide-binding protein (transducin), Nucl.Acids Res.14:6361-6372 (1988); People such as Wells, Cassette mutagenesis:an efficient method for generation of multiple mutations at definedsites, Gene 34:315-323 (1985); People such as Grundstrom, Oligonucleotide-directed mutagenesisbymicroscale ' shot-gun ' gene synthesis, Nucl.Acids Res.13:3305-3316 (1985); Mandecki, Oligonucleotide-directed double-strand break repair in plasmids of Escherichia coli:amethod for site-specific mutagenesis, Proc.Natl.Acad.Sci.USA, 83:7177-7181 (1986); Arnold, Protein engineering for unusual environments, Current Opinion in Biotechnology4:450-455 (1993); People such as Sieber, Nature Biotechnology, 19:456-460 (2001) .W.P.C.Stemmer, Nature 370,389-91 (1994); And I.A.Lorimer, I.Pastan, Nucleic Acids Res.23,3067-8 (1995).Other details about many these methods are found in Methods in Enzymology the 154th volume, and it is also described with the effective control of various sudden change formation methods to the problem of generation fault.
Herein the method and composition of Miao Shuing also comprise use eukaryotic host cell, non-eukaryotic host cell and organism by quadrature tRNA/RS in vivo incorporating alpha-non-natural amino acid into.Host cell is through corresponding to the polynucleotide of the polypeptide of describing herein with comprise and construct body (including (but not limited to) carrier corresponding to the polypeptide herein described, it can be (for example) cloning vector and expression vector) genetically engineered (including (but not limited to) conversion, transduction or transfection) corresponding to the polynucleotide of the polypeptide of describing herein.For instance, quadrature tRNA, quadrature tRNA synthetic enzyme and the proteinic coding region operability of desire deutero-are connected on the genetic expression controlling elements that has function in required host cell.Carrier can be (for example) plasmid, clay, phage, bacterium, virus, exposed polynucleotide and engages the form of polynucleotide.(comprise electroporation (people such as Fromm with standard method; Proc.Natl.Acad.Sci.USA 82; 5824 (1985)), by viral vector infection, by the small-particle high speed ballistic penetration that in the matrix of beads or particle, has nucleic acid) carrier is introduced in cell and/or the microorganism; or cause the surface and go up (people such as Klein; Nature 327,70-73 (1987)) and/or its analogue.
Through the host cell of through engineering approaches design can in through improvement to be suitable for such as (for example) screening step, activation promotor or to select to cultivate in the conventional nutritional medium of behavior of transformant.These cells can be cultivated according to circumstances and be transgenic organism.Other are including (but not limited to) about cellular segregation and cultivation (for example, about separate nucleic acid subsequently) suitable reference comprise Freshney (1994) Culture of Animal Cells, a Manual of Basic Technique, the 3rd edition, Wiley-Liss, New York and the reference of wherein quoting; People such as Payne. (1992) Plant Cell and TissueCulture in Liquid Systems John Wiley; Sons, Inc.New York, NY; Gamborg and Phillips (volume) (1995) Plant Cell, Tissue and Organ Culture; Fundamental Methods Springer Lab Manual, Springer-Verlag (Berlin Heidelberg New York) and Atlas and Parks (volume) The Handbook ofMicrobiological Media (1993) CRC Press, Boca Raton, FL.
Can obtain target nucleic acid is introduced some kinds of well-known process in the cell, wherein in any one method and composition that can be used for describing herein.It comprises: with recipient cell and the fusion of bacterium protoplastis, the electroporation that contain DNA, projectile bombardment, and with viral vector infection (further discussing in this article) etc.Bacterial cell can be used for increasing and contains the number of constructing the plasmid of body herein corresponding to the DNA of the polypeptide of describing.Make the plasmid of bacterial growth to log stage and the bacterium can separate (referring to .Sambrook for example) by known several different methods in the affiliated field.In addition, numerous test kits can be buied on the market to be used for from the bacterium plasmid purification, (referring to (for example) EasyPrep TM, FlexiPrep TM, both are all from Pharmacia Biotech; StrataClean TM, from Stratagene; And QIAprep TM, from Qiagen).Through separate and the plasmid of purifying with after further handle to produce other plasmids, be used for transfectional cell or incorporate the related vector that is used for the infection biological body into.Typical carrier contains transcribes with translation termination, transcribes and translation initiation sequence, and is applicable to the promotor of regulating the specific objective expression of nucleic acids.Carrier comprises the general expression cassette that contains at least one independent terminator sequence according to circumstances, the admissible sequence box is in eukaryote or prokaryotic organism or the sequence (including (but not limited to) shuttle vectors) of duplicating among both and be used for prokaryotic system and the selective marker of eukaryotic system.Carrier is suitable for prokaryotic organism, eukaryote or preferably duplicates and integrate among both.Referring to, Gillam and Smith, Gene 8:81 (1979); People such as Roberts, Nature, 328:731 (1987); Schneider, people such as E., Protein Expr.Purif.6 (1): 10-14 (1995); Ausubel, Sambrook, Berger (all as mentioned).Be applicable to that clone's bacterium and the catalogue of phage are provided by (for example) ATCC, for example, people's (volume) such as The ATCC Catalogue of bacteria andbacteriophage (1992) Gherna that ATCC publishes.Be used for order-checking, clone and molecular biological otherwise other base programs and potential theoretical Consideration and also see people such as Watson. (1992) Recombinant DNASecond Edition Scientific American Books, among the NY.In addition, basically any nucleic acid is (and any in fact through labeling nucleic acid, standard or non-standard no matter) can order from any one customization or the standard of multiple commercial source, these commercial source are such as the Midland Certified Reagent Company (Midland, TX Mcrc.com), TheGreat American Gene Company (Ramona, CA, on the World Wide Web with Genco.comObtain), ExpressGenInc. (Chicago, IL, on the World Wide Web with Expressgen.comObtain), Operon Technologies Inc. (Alameda, CA) and many other sources.
B. select codon
Be covered by the genetic code subframe of the selection codon detailed description protein biosynthesizing mechanism in the method and composition of describing herein.For instance, select codon, rare codon or its analogue of codon including (but not limited to) unique three base codons, nonsense codon (such as terminator codon, it is including (but not limited to) amber codon (UAG) or opal codon (UGA)), non-natural codon, four or more base.There is broad range in the number that can introduce the selection codon in required gene or the polynucleotide, its in the single polynucleotide of at least a portion of the polypeptide paid close attention to of coding including (but not limited to) one or more, two or more, more than three, 4,5,6,7,8,9, more than 10 or 10.
In one embodiment, described method comprises that use is used in vivo incorporating into one or more alpha-non-natural amino acid as the selection codon of terminator codon.For instance, produce the O-tRNA of identification terminator codon (including (but not limited to) UAG) and its by O-RS with required alpha-non-natural amino acid aminoacylization.This O-tRNA also can't help the natural host's of existence aminoacyl-tRNA synthetase identification.Can use conventional rite-directed mutagenesis be formed in the polypeptide of being paid close attention to the site of paying close attention to introduce terminator codon (including (but not limited to) UAG).Referring to (for example) Sayers, people such as J.R.. (1988), 5 ', 3 ' Exonuclease in phosphorothioate-based oligomicleotide-directed mutagenesis.Nucleic AcidsRes, 16 (3): 791-802.When the nucleic acid of the polypeptide that O-RS, O-tRNA and coding are paid close attention in vivo made up, alpha-non-natural amino acid was responded the UAG codon and is incorporated into to obtain containing in specified location the polypeptide of alpha-non-natural amino acid.
Can carry out incorporating into of alpha-non-natural amino acid in vivo and do not cause the remarkable confusion of eukaryotic host cell.For instance, because the inhibition efficient of UAG codon depends on the competition between O-tRNA (suppressing tRNA including (but not limited to) amber) and the eucaryon releasing hormone (including (but not limited to) eRF) (it combines with terminator codon and causes that the peptide in the growth discharges from rrna), suppressing efficient can regulate by the expression level that increases (comprise, but be not limited to) O-tRNA and/or inhibition tRNA.
The selection codon also comprises the codon of prolongation, its codon including (but not limited to) four or more base (such as, the codon of four, five, six or six above bases).The example of four base codons including (but not limited to) AGGA, CUAG, UAGA, CCCU with and analogue.The example of five base codons including (but not limited to) AGGAC, CCCCU, CCCUC, CUAGA, CUACU, UAGGC with and analogue.The feature of the method and composition of Miao Shuing comprises use suppresses prolongation according to frameshit codon herein.The codon of four or more base can (comprise, but be not limited to) one or more alpha-non-natural amino acid and insert in the same protein.For instance, (for example has anticodon loop, have 8-10nt anticodon loop at least) the existence of mutant O-tRNA (including (but not limited to) specific frameshift suppressor tRNA) under, the codon of four or more base reads as single amino acid.In other embodiments, the anticodon loop decodable code, it is including (but not limited to) at least one four base codon, at least one five base codon or at least one hexabasic basic codon or more.Because there are 256 four possible base codons, multiple alpha-non-natural amino acid can use the codon coding of four or more base in same cell.Referring to people such as Anderson, (2002) Exploring the Limits of Codon and Anticodon Size, Chemistry and Biology, 9:237-244; Magliery, (2001) Expanding the Genetic Code:Selectionof Efficient Suppressors of Four-base Codons and Identification of " Shifty " Four-baseCodons with a Library Approach in Escherichia coli, J.Mol.Biol.307:755-769.
For instance, four base codons have been used for using in vitro biosynthetic means to incorporate alpha-non-natural amino acid into protein.Referring to people such as (for example) Ma, (1993) Biochemistry, 32:7939-7945; And people such as Hohsaka, (1999) J.Am.Chem.Soc, 121:34-40.With two chemical acylation frameshift suppressor tRNAs, use CGGG and AGGU the NBU derivative of 2-naphthyl L-Ala and Methionin to be incorporated in the strepto-affinity element simultaneously in vitro.Referring to people such as (for example) Hohsaka, (1999) J.Am.Chem.Soc, 121:12194-12195.In vivo in the research, people such as Moore research has the ability of the tRNALeu derivative inhibition UAGN codon (N can be U, A, G or C) of NCUA anticodon, and find that tetrad group UAGA can decode wherein only a little decoding in 0 or-1 framework with 13% to 26% efficient by the tRNALeu with UCUA anticodon.Referring to, people such as Moore, (2000) J.Mol.Biol., 298:195-205.In one embodiment, in the method and composition that can be used for describing herein based on the prolongation codon of rare codon or nonsense codon, it can reduce to read over frameshit in the missense of other unwanted site and suppresses.
For giving fixed system, select codon also can comprise one in the natural three base codons, wherein the endogenous system does not use (or seldom using) natural base codon.For instance, it comprises the system of the tRNA that lacks the natural three base codons of identification, and/or wherein three base codons are the system of rare codon.
Select codon to comprise the non-natural base pair according to circumstances.These non-natural base pairs further enlarge existing genetic symbol system.Extra base pair increases to 125 with the number of triplet codon by 64.The character of the 3rd base pair comprises stable and the selectivity base pairing, by incorporating among the DNA that polysaccharase carries out with the effective enzymatic of high frequency high fidelity, and effectively continue the primer prolongation in the synthetic back of nascent non-natural base pair.To the description of the non-natural base pair that can be suitable for method and composition including (for example) people such as Hirao, (2002) An unnatural base pair for incorporating amino acidanalogues into protein, Nature Biotechnology, 20:177-182, and also referring to Wu, people such as Y. (2002) J.Am.Chem.Soc.124:14626-14630.Other relevant open cases are listed in herein.
For in vivo using, but non-natural nucleoside be the film perviousness and through phosphorylation to form corresponding triguaiacyl phosphate.In addition, the genetic information of increase is stable and can't help cellular enzymes and destroy.The different hydrogen bond type of those hydrogen bond types in Benner and other people previous effort utilization and the typical Wo Sen-Ke Like base pair (Watson-Crick pair), wherein the most noticeable example is different-C: different-G is right.Referring to people such as (for example) Switzer, (1989) J.Am.Chem.Soc, 111:8322-8322; And people such as Piccirilli, (1990) Nature, 343:33-37; Kool, (2000) Curr.Opin.Chem.Biol., 4:602-608.These bases are usually to a certain extent with natural base mispairing and can not duplicate by enzymatic.Kool and co-worker confirm that hydrophobic between the base pile up interaction and can replace hydrogen bond to drive the formation of base pair.Referring to, Kool, (2000) Curr.Opin.Chem.Biol., 4:602-608; And Guckian and Kool, (1998) Angew.Chem.Int.Ed.Engl., 36 (24): 2825-2828.Satisfy in the process of non-natural base pair of all above-mentioned requirements making great efforts exploitation, Schultz, Romesberg and co-worker are systematically synthetic and studied the hydrophobic base of a series of non-naturals.Find that PICS: PICS self pairing is more stable than natural base pair, and it can effectively be incorporated among the DNA by the Klenow fragment (KF) of e. coli dna polymerase I into.Referring to people such as (for example) McMinn, (1999) J.Am.Chem.Soc, 121:11585-11586; And people such as Ogawa, (2000) J.Am.Chem.Soc, 122:3274-3278.3MN-3MN self pairing can be synthetic by KF for biological function enough efficient and selectivity.Referring to people such as (for example) Ogawa, (2000) J.Am.Chem.Soc, 122:8803-8804.Yet two kinds of bases are served as the chain terminator that further duplicates.Developed recently and can be used for to duplicate PICS self paired mutant DNA polymerases.In addition, 7AI self pairing can be replicated.Referring to people such as (for example) Tae, (2001) J.Am.Chem.Soc, 123:7439-7440.Also developed and stablized the novel metal base pair of paired Dipic:Py in conjunction with forming behind the Cu (II).Referring to, people such as Meggers, (2000) J.Am.Chem.Soc, 122:10714-10715.Because codon that prolongs and non-natural codon inherently with natural codon quadrature, so the alpha-non-natural amino acid method of Miao Shuing can utilize this character with the generation quadrature tRNA relevant with it herein.
The translation bypath system also can be used for alpha-non-natural amino acid is incorporated in the required polypeptide.In the translation bypath system, big sequence is incorporated in the gene, but be not translated as protein.Described sequence contains to serve as brings out the structure that rrna is crossed described sequence and continued the signal of translation in the insertion downstream.
In certain embodiments, protein or the polypeptide of being paid close attention in method of describing herein and/or the composition (or its part) is by nucleic acid encoding.Usually, nucleic acid comprises that at least one selects codon, at least two selection codons, at least three selection codons, at least four selection codons, at least five selection codons, at least six selection codons, at least seven selection codons, at least eight selection codons, at least nine selection codons, the selection codon more than ten or ten.
The gene of the protein paid close attention to of coding or polypeptide can instruct at " Mutagenesis and Other Molecular BiologyTechniques " and use the those skilled in the art to know down and the method for description is herein carried out mutagenesis with including (for example) the one or more selection codon that is used to incorporate into alpha-non-natural amino acid.For instance, to comprise one or more selection codon, provide incorporating into of one or more alpha-non-natural amino acid with the proteinic nucleic acid mutagenesis paid close attention to.The method and composition of Miao Shuing comprises any this variant herein, and it is including (but not limited to) any protein mutant form of (for example, comprising at least one alpha-non-natural amino acid).Similarly, the method and composition of Miao Shuing comprises corresponding nucleic herein, that is, have one or more codings or allow any nucleic acid of the selection codon of in vivo incorporating into of one or more alpha-non-natural amino acid.
Any desired location place that the nucleic acid molecule of the polypeptide (comprising (only for instance) GH polypeptide) that coding is paid close attention to can be easy to suddenly change with at polypeptide introduces halfcystine.Halfcystine is widely used in reactive molecule, water-soluble polymers, protein or multiple other molecules is introduced on the protein of being paid close attention to.Be suitable for halfcystine incorporate into method in the desired location of polypeptide in affiliated field for knowing, such as being described in United States Patent (USP) the 6th, those methods in 608, No. 183 (it is all to be incorporated herein by reference), and standard sudden change formation technology.This introducing and utilize the use of the technology of halfcystine and to utilize the technical tie-up of alpha-non-natural amino acid to use herein with the introducing of describing.
VIII. comprise the in vivo generation of the polypeptide of alpha-non-natural amino acid
The in vivo generation of the polypeptide of describing in this part for simplicity, that comprises alpha-non-natural amino acid is general and/or describe with particular instance.Yet, general description or particular instance that the in vivo generation of the polypeptide of describing in this part that comprises alpha-non-natural amino acid should not only limit to provide in this part, but the in vivo generation of the polypeptide of describing in this part that comprises alpha-non-natural amino acid is same well suited in being in formula I-XVIII, all compounds in the category of XXX-XXXIV (A and B) and XXXX-XXXXIII, it comprises the specification sheets that is in herein, claim and the graphic middle formula I-XVIII that describes, any minor or specific compound in the category of XXX-XXXIV (A and B) and XXXX-XXXXIII.
The polypeptide of Miao Shuing can use and modifiedly in vivo produce to add or to replace not at the natural tRNA that has an amino acids coding in the system and tRNA synthetic enzyme herein.
Produce to use and be not described in (for example) U.S. Patent Application Publication case 2003/0082575 (sequence number 10/126 in the natural tRNA of amino acids coding in the system and the method for tRNA synthetic enzyme of existing, 927) and 2003/0108885 (sequence number 10/126,931) in, it is all to be incorporated herein by reference.These methods comprise producing to be independent of translation system are the synthetic enzyme of endogenous (and therefore be known as sometimes " quadrature ") and the translating mechanism of tRNA functionating.In one embodiment, translation system comprises the polynucleotide of coded polypeptide; Polynucleotide can be the mRNA that transcribes from corresponding DNA, or mRNA can produce from rna virus vector; Polynucleotide comprises corresponding to the selection codon of specifying the site in advance of incorporating alpha-non-natural amino acid in addition.Translation system further comprise at and (suitably time) also comprise the tRNA of alpha-non-natural amino acid, wherein said tRNA has the aforementioned selection codon of specificity/specific recognition to aforementioned selection codon; In other embodiments, alpha-non-natural amino acid is through aminoacylization.Alpha-non-natural amino acid comprises and has herein formula I-XVIII, the XXX-XXXIV (A and B) that describe and any one those alpha-non-natural amino acids of structure among the XXXX-XXXXIII.In other or extra embodiment, translation system comprises that described tRNA is had specific amino acyl synthetase, and in other or embodiment in addition, translation system comprises quadrature tRNA and quadrature aminoacyl tRNA synthetase.In other or extra embodiment, translation system comprises at least one in following each thing: comprise aforementioned polynucleotide plasmid (such as, only for instance, form with DNA), comprise aforementioned polynucleotide genomic dna (such as, only for instance, form with DNA) or aforementioned polynucleotide be integrated in wherein genomic dna (the described stable integration that is integrated in other embodiments).In other or extra embodiment of translation system, selecting codon is to be selected from by amber codon, ocher codon, opal codon, unique codon, rare codon, non-natural codon, five base codons and the molecular group of four base passwords.In other or extra embodiment of translation system, tRNA is for suppressing tRNA.In other or extra embodiment, non-natural amino acid polypeptides is to be synthesized by rrna.
In other or extra embodiment, translation system comprises quadrature tRNA (O-tRNA) and quadrature aminoacyl tRNA synthetase (O-RS).Usually, in translation system O-RS preferentially with at least a selection codon of can't help other tRNA identifications in the system of at least a alpha-non-natural amino acid aminoacyl O-tRNA and O-tRNA identification.Therefore translation system is with in the polypeptide that produces in the alpha-non-natural amino acid insertion system, responding coded selection codon, and then alpha-non-natural amino acid " replacement " gone in the position in the encoded polypeptide.
Be used for the multiple quadrature tRNA and the aminoacyl tRNA synthetase of specific synthesizing amino acid insertion polypeptide are described in affiliated field, and be applicable to usually in the method for describing herein to produce the non-natural amino acid polypeptides of describing herein.For instance, ketone specificity O-tRNA/ aminoacyl-tRNA synthetase is described in Wang, people such as L., and Proc.Natl.Acad.Sci.USA 100 (1): 56-61 (2003) and Zhang, people such as Z. are among Biochem.42 (22): the 6735-6746 (2003).Exemplary O-RS or its part are by polynucleotide sequence encode and comprise the aminoacid sequence that discloses in the U.S. Patent Application Publication case 2003/0082575 and 2003/0108885 (its each is all to be incorporated herein by reference).Also be described in U.S. Patent Application Publication case 2003/0082575 (sequence number 10/126,927) and 2003/0108885 (sequence number 10/126,931) with the corresponding O-tRNA molecule of a use of O-RS, it is all to be incorporated herein by reference.In addition, people such as Mehl is at J.Am.Chem.Soc.2003; Among the 125:935-939 and people .Nature Biotechnology such as Santoro in October, 2002; Aminoacyl tRNA synthetase and tRNA molecule that 20:1044-1048 (it is all to be incorporated herein by reference) discusses the screening method and is used for p-Aminophenylalanine is incorporated into polypeptide.
Be applicable to that herein exemplary O-tRNA sequence in the method for describing is including (but not limited to) as disclosed nucleotide sequence SEQ ID NO:1-3 in the U.S. Patent Application Publication case 2003/0108885 (sequence number 10/126,931) (it is to be incorporated herein by reference).Specific alpha-non-natural amino acid is had other right case descriptions of specific O-tRNA/ aminoacyl-tRNA synthetase in U.S. Patent Application Publication case 2003/0082575 (sequence number 10/126,927), and it is all to be incorporated herein by reference.Be described in Chin in conjunction with containing keto amino acid with the O-RS and the O-tRNA that contain trinitride amino acid in yeast saccharomyces cerevisiae, people such as J.W. are among the Science 301:964-967 (2003).
The purposes of O-tRNA/ aminoacyl-tRNA synthetase comprises the specificity codon of selecting the coding alpha-non-natural amino acid.Although can use any codon, need usually to select seldom or the codon that never in the cell that the O-tRNA/ aminoacyl-tRNA synthetase is expressed therein, uses.Only for instance, exemplary codon comprises nonsense codon, such as the codon of terminator codon (amber codon, ocher codon and opal codon), four or more base and seldom use or obsolete other natural three base codons.
Can use known sudden change formation method in the affiliated field (form, limit and select the formation etc. that suddenlys change) to select codon to introduce in the appropriate location of polynucleotide coding sequence specificity including (but not limited to) rite-directed mutagenesis formation, cassette mutagenesis.
Generation can be used for incorporating into alpha-non-natural amino acid protein biosynthesizing mechanism (such as O-RS, O-tRNA and quadrature O-tRNA/O-RS to) the method for component be described in Wang, people such as L., Science 292:498-500 (2001); Chin, people such as J.W., J.Am.Chem.Soc.124:9026-9027 (2002); Zhang, people such as Z. are among the Biochemistry 42:6735-6746 (2003).The method and composition of in vivo incorporating into that is used for alpha-non-natural amino acid is described in the U.S. Patent Application Publication case 2003/0082575 (sequence number 10/126,927), and it is all to be incorporated herein by reference.The right method of quadrature tRNA-tRNA synthetic enzyme that is used for selecting being used for the in vivo translation system of organism also is described in U.S. Patent Application Publication case 2003/0082575 (sequence number 10/126,927) and 2003/0108885 (sequence number 10/126,931) in, it is all to be incorporated herein by reference.In addition, be used to incorporate into the quadrature RS and the tRNA of keto amino acid for the open case WO of the PCT of " Site Specific Incorporation of KetoAmino Acids into proteins " No. 04/035743 (it is all to incorporate into by reference) describes right for title.Title is used for non-naturally encoded amino acid is incorporated into eukaryotic host cell for the open case WO of the PCT of " Expanding the EukaryoticGenetic Code " No. 04/094593 (it is all to be incorporated herein by reference) describes quadrature RS and tRNA are right.
The method that is used to produce at least a reorganization quadrature aminoacyl-tRNA synthetase (O-RS) comprises: (a) produce (mutant according to circumstances) the RS storehouse that derives from from least a aminoacyl-tRNA synthetase (RS) of first organism, wherein first organism is including (but not limited to) the prokaryotic organism body, such as, only for instance, Methanococcus jannaschii, hot autotrophic methane bacteria, have a liking for the ancient green-ball bacterium of salt bacillus, intestinal bacteria, flicker, strong red-hot coccus, pick Yue Shi hot-bulb bacterium, the hot bacterium of quick gas, thermus thermophilus or its analogue, or most eukaryotes; (b) select in (and/or screening) RS (mutant RS according to circumstances) storehouse the member of aminoacyl quadrature tRNA (O-tRNA) in the presence of alpha-non-natural amino acid and natural amino acid, and then the pond of activity (mutant according to circumstances) RS is provided; And/or, (c) select in (selecting by negative according to circumstances) described pond under the situation that does not have alpha-non-natural amino acid the preferentially active RS (including (but not limited to) mutant RS) of aminoacyl O-tRNA, and then at least a reorganization O-RS is provided; Wherein at least a reorganization O-RS is preferentially with alpha-non-natural amino acid aminoacyl O-tRNA.
In one embodiment, RS is nonactive RS.Nonactive RS can produce by active RS is suddenlyd change.Only for instance, nonactive RS can by make at least about a kind, at least about 2 kinds, at least about 3 kinds, at least about 4 kinds, at least about 5 kinds, at least about 6 kinds or be that different aminoacids (including (but not limited to) L-Ala) produces at least about the amino acid mutation more than 10 kinds or 10 kinds.
Under mutant RS can use in the storehouse in the field known various technology produce, it is including (but not limited to) the appropriate design according to the three-dimensional RS structure of protein, or the sudden change of RS Nucleotide forms at random or in the appropriate design technology.Only for instance, mutant RS can by locus specificity sudden change, random mutation, the multifarious recombination mutation of generation, chimeric construct body, appropriate design and describe herein or affiliated field in known additive method produce.
In one embodiment, select in (and/or screening) RS (mutant RS according to circumstances) storehouse active member (it is including (but not limited to) those members of aminoacyl quadrature tRNA (O-tRNA) in the presence of alpha-non-natural amino acid and natural amino acid) including (but not limited to): positive selection or screening mark (including (but not limited to) antibiotics resistance gene or its analogue) and (mutant according to circumstances) RS storehouse are introduced in a plurality of cells, wherein positive selection and/or screening mark comprise at least a selection codon, and it is including (but not limited to) amber codon, ocher codon, the opal codon, unique codon, rare codon, the non-natural codon, five base codons and four base codons; In the presence of selective agent, make described a plurality of cell growth; Differentiate the cell of (or showing specific reaction) of survival in the presence of selection and/or screening agent by at least a selection codon that suppresses in positive selection or the screening mark, and then the subclass of the positive selection cell in the pond of containing activity (mutant according to circumstances) RS is provided.According to circumstances, selection and/or screening agent concentration can change.
On the one hand, positive selectable marker is that chloramphenicol acetyltransferase (CAT) gene and selection codon are the amber terminator codon in the CAT gene.According to circumstances, positive selectable marker is that β-Nei Xiananmei gene and selection codon are the amber terminator codon in the β-Nei Xiananmei gene.On the other hand, positive screening mark comprises fluorescence or luminous screening mark or based on the screening mark (including (but not limited to) cell surface marker) of affinity.
In one embodiment, negative select or the screening pond in active RS (mutant according to circumstances) (including (but not limited to) those the active RS of preferential aminoacyl O-tRNA under the situation that does not have alpha-non-natural amino acid) including (but not limited to): to select from the positive or feminine gender is selected in the pond of activity (mutant according to circumstances) RS of screening or the screening mark is introduced a plurality of cells of second organism, wherein negative selection or screening mark comprise at least a selection codon (including (but not limited to) antibiotics resistance gene, it is including (but not limited to) chloramphenicol acetyltransferase (CAT) gene); And, discriminating is survived in being supplemented with first substratum of alpha-non-natural amino acid and screening or selective agent or is showed specificity screening reaction, but in second substratum that is not supplemented with alpha-non-natural amino acid and selection or screening agent, can not survive or show the cell of specific reaction, and then survivaling cell or the screening cell with at least a reorganization O-RS is provided.Only for instance, the CAT authentication schemes serves as positive selection and/or the negative screening of determining suitable O-RS recombinant chou according to circumstances.For example, duplicate having or do not have containing on the CAT growth plate of (it comprises at least a selection codon) of one or more alpha-non-natural amino acids according to circumstances in the clone pond.Therefore be considered to contain reorganization O-RS at the bacterium colony that contains unique growth on the plate of alpha-non-natural amino acid.On the one hand, change the concentration of selecting (and/or screening) agent.In certain aspects, first organism is different with second organism.Therefore, first organism and/or second organism comprise according to circumstances: prokaryotic organism, eukaryote, Mammals, intestinal bacteria, fungi, yeast, archeobacteria, eubacterium, plant, insect, protobiont etc.In other embodiments, the screening mark comprises fluorescence or luminous screening mark or based on the screening mark of affinity.
In another embodiment, activity (mutant according to circumstances) RS in screening or selection (selecting) pond including (but not limited to) feminine gender including (but not limited to): separate the pond of selecting the active mutant RS in the step (b) from the positive; Feminine gender selected or screening mark (wherein negative select or the screening mark comprises that at least a selection codon is (including (but not limited to) the toxicity marker gene, it is including (but not limited to) rnase barnase gene, and it comprises at least a selection codon)) and the pond of active (mutant according to circumstances) RS introduce in a plurality of cells of second organism; And differentiate survival or displaying specificity screening reaction in first substratum that does not replenish alpha-non-natural amino acid, but in being supplemented with second substratum of alpha-non-natural amino acid, can not surviving or show the cell of specificity screening reaction, and then survivaling cell or the screening cell with at least a reorganization O-RS is provided, wherein at least a reorganization O-RS has specificity to alpha-non-natural amino acid.On the one hand, at least a selection codon comprises two or more approximately selection codons.These embodiment can comprise wherein at least a selection codon according to circumstances and comprise two or more selection codons, and wherein first organism different with second organism (including (but not limited to), each organism is according to circumstances including (but not limited to) prokaryotic organism, eukaryote, Mammals, intestinal bacteria, fungi, yeast, archeobacteria, eubacterium, plant, insect, protobiont etc.).Equally, some aspects comprise wherein that negative selection marker comprises rnase barnase gene (it comprises at least a selection codon).Other aspects comprise that screening mark wherein comprises fluorescence or luminous screening mark according to circumstances or based on the screening mark of affinity.Among the embodiment in this article, screening and/or selection comprise screening according to circumstances and/or select the variation of strict degree.
In another embodiment, the method that produces at least a reorganization quadrature aminoacyl-tRNA synthetase (O-RS) can further comprise: (d) separating at least one reorganization O-RS; (e) produce the second group of O-RS (according to circumstances through sudden change) that derives from least a reorganization O-RS; And, (f) repeating step (b) and (c) until the mutant O-RS of the ability that obtains to comprise preferential aminoacyl O-tRNA.According to circumstances, repeating step (d)-(f) (comprise, but be not limited to) is at least about twice.On the one hand, the second group of mutant O-RS that derives from least a reorganization O-RS can pass through sudden change formation (including (but not limited to) random mutation formation, locus specificity sudden change formation, reorganization or its combination) generation.
The strict degree of selection/screening step including (but not limited to) positive selection/screening step (b), negative selections/screening step (c) or positive and feminine gender selection/screening step (b) and (c) both, in aforesaid method, comprise the strict degree of change selection/screening according to circumstances.In another embodiment, positive selections/screening step (b), negative selections/screening step (c) or positive and feminine gender selection/screening step (b) and (c) both comprise the use reporter gene, wherein reporter gene be by fluorescent activation cell sorting technology (FACS) detection or wherein reporter gene be to pass through luminous detection.According to circumstances, reporter gene is shown on the cell surface, selects on phage indicating meter or its analogue and according to affinity that relates to alpha-non-natural amino acid or analogue or catalytic activity.In one embodiment, the mutant synthetic enzyme be shown on the cell surface, on phage indicating meter or its analogue.
The method that produces reorganization quadrature tRNA (O-tRNA) including (but not limited to): (a) produce the storehouse of the mutant tRNA that derives from least a tRNA (including (but not limited to) inhibition tRNA) from first organism; (b) select (selecting) or screening including (but not limited to) feminine gender under from the non-existent situation of the RS of first organism by storehouse from (mutant according to circumstances) tRNA of aminoacyl-tRNA synthetase (RS) aminoacylization of second organism, and then provide tRNA the pond of (mutant according to circumstances); And, (c) select or the pond of the described tRNA of screening (mutant according to circumstances) in by the member of quadrature RS (O-RS) aminoacylization of introducing, and then provide at least a reorganization O-tRNA; Wherein at least a reorganization O-tRNA identification selection codon and non-origin are from the effective identification of the RS of second organism and by the preferential aminoacylization of O-RS.In certain embodiments, at least a tRNA is the uniqueness three base codons that suppress tRNA and/or comprise natural and/or non-natural base, or is nonsense codon, rare codon, non-natural codon, the codon that comprises at least 4 bases, amber codon, ocher codon or opal terminator codon.In one embodiment, the raising of reorganization O-tRNA with orthogonality.Should be appreciated that in certain embodiments, O-tRNA is introduced into according to circumstances in first organism and need not to modify from second organism.In various embodiments, first organism and second organism are identical or different and be selected from (comprise, but be not limited to) prokaryotic organism (including (but not limited to) Methanococcus jannaschii, hot autotrophic methane bacteria, intestinal bacteria, have a liking for salt bacillus etc.), eukaryote, Mammals, fungi, yeast, archeobacteria, eubacterium, plant, insect, protobiont etc. according to circumstances.In addition, tRNA is according to circumstances through the alpha-non-natural amino acid aminoacylization in reorganization, and wherein alpha-non-natural amino acid is natively or passes through genetic manipulation biosynthesizing in vivo.Alpha-non-natural amino acid adds in the growth medium of the first organism or second organism according to circumstances at least, and wherein alpha-non-natural amino acid can reach suitable IC and incorporates in the non-natural amino acid polypeptides to allow.
On the one hand, select in (selecting) or the screening storehouse to comprise: in a plurality of cells of storehouse introducing with toxicity marker gene (its toxic marker gene comprises at least a selection codon (or cause the gene or the essential gene of organism of toxigenicity agent or static agent, wherein this marker gene comprises at least a selection codon)) and (mutant according to circumstances) tRNA from second organism by (mutant according to circumstances) tRNA of aminoacyl-tRNA synthetase (step (b)) aminoacylization including (but not limited to) feminine gender; And, select survivaling cell, wherein said survivaling cell contains the pond of (mutant according to circumstances) tRNA that comprises at least a quadrature tRNA or non-functional tRNA.For instance, survivaling cell can be selected by using the compa-ratios cell density to examine and determine.
On the other hand, the toxicity marker gene can comprise two or more selection codons.Among another embodiment of the method for Miao Shuing, the toxicity marker gene is a rnase barnase gene in this article, and wherein rnase barnase gene comprises at least one amber codon.According to circumstances, rnase barnase gene can comprise two or more amber codons.
In one embodiment, select or the pond of screening (mutant according to circumstances) tRNA in can comprise by the member of quadrature RS (O-RS) aminoacylization of introducing: the positive is selected or the screening marker gene (wherein positive mark's gene comprises that drug resistance gene is (including (but not limited to) the β-Nei Xiananmei gene, it comprises at least a selection codon, such as at least one amber terminator codon) or the essential gene of organism, or make toxic agents toxicide gene) and the pond of O-RS and (mutant according to circumstances) tRNA introduce in a plurality of cells from second organism; And; the survivaling cell or the screening cell of discriminating growth in the presence of selection or screening agent (including (but not limited to) microbiotic); and then provide cell pool with at least a reorganization tRNA; wherein at least a reorganization tRNA is by the O-RS aminoacylization and with in the translation product of aminoacid insertion by positive mark's genes encoding, to respond described at least a selection codon.In another embodiment, change the concentration of selection and/or screening agent.
The right method of specificity O-tRNA/O-RS that produces is provided.Method including (but not limited to): (a) produce the storehouse derive from from the mutant tRNA of at least a tRNA of first organism; (b) negative select or the screening storehouse under from the non-existent situation of the RS of first organism by (mutant according to circumstances) tRNA from aminoacyl-tRNA synthetase (RS) aminoacylization of second organism, and then provide the pond of (mutant according to circumstances) tRNA; (c) select or the pond of screening (mutant according to circumstances) tRNA in by the member of quadrature RS (O-RS) aminoacylization of introducing, and then provide at least a reorganization O-tRNA.Described at least a reorganization O-tRNA identification selection codon and non-origin are from the effective identification of the RS of second organism and by the preferential aminoacylization of O-RS.Described method also comprises (d) and produces the storehouse derive from from (mutant according to circumstances) RS of at least a aminoacyl-tRNA synthetase (RS) of the 3rd organism; (e) in the presence of alpha-non-natural amino acid and natural amino acid, select or the storehouse of screening mutant RS in the member of the described at least a reorganization O-tRNA of preferential aminoacylization, and then provide the pond of active (mutant according to circumstances) RS; And; (f) negative select or the screening pond under the situation that does not have alpha-non-natural amino acid activity (mutant according to circumstances) RS of the described at least a reorganization O-tRNA of preferential aminoacylization; and then providing at least a specificity O-tRNA/O-RS right, wherein said at least a specificity O-tRNA/O-RS is at least a to alpha-non-natural amino acid with have specific reorganization O-RS and an at least a reorganization O-tRNA to comprising.The specificity O-tRNA/O-RS that is produced by the method for describing herein is to being contained in the category of describing herein and method.For instance, specificity O-tRNA/O-RS to can comprise (comprise, but be not limited to) mutRNATyr-mutTyrRS to (such as mutRNATyr-SS12TyrRS to), mutRNALeu-mutLeuRS to, mutRNAThr-mutThrRS to, mutRNAGlu-mutGluRS to or its analogue.In addition, these methods comprise wherein first organism identical with the 3rd organism (including (but not limited to) Methanococcus jannaschii).
The right method of quadrature tRNA-tRNA synthetic enzyme that is used for selecting being used for the in vivo translation system of second organism is also contained in the method for describing herein.Method including (but not limited to): separate or the aminoacyl-tRNA synthetase (RS) that derives from first organism is introduced first group of cell from second organism with marker gene, tRNA and from first organism; Marker gene and tRNA are introduced in the duplicate groups of cells from second organism; And, the cell that in the duplicate groups of cells, can not produce this reaction that specificity screening reaction is showed in survivaling cell in the selection can not survive in the duplicate groups of cells first group or screening, wherein first group and duplicate groups of cells be select or the screening agent in the presence of grow, wherein survivaling cell or screening cell comprise that the quadrature tRNA-tRNA synthetic enzyme of the in vivo translation system that is used for second organism is right.In one embodiment, comparison and selection or screening comprise in vivo complementary calibrating.The concentration of selection or screening agent can change.
The organism of Miao Shuing comprises multiple organism and multiple combination herein.In one embodiment, organism is the prokaryotic organism body according to circumstances, its including (but not limited to) Methanococcus jannaschii, hot autotrophic methane bacteria, have a liking for the ancient green-ball bacterium of salt bacillus, intestinal bacteria, flicker, strong red-hot coccus, pick Yue Shi hot-bulb bacterium, the hot bacterium of quick gas, thermus thermophilus or its analogue.Perhaps, organism is a most eukaryotes, it is including (but not limited to) plant (including (but not limited to) complicated plant, such as monocotyledons or dicotyledons), algae, protobiont, fungi (including (but not limited to) yeast etc.), animal (including (but not limited to) Mammals, insect, arthropods etc.) or its analogue.
A. the expression in non-eukaryote and eukaryote
The non-eukaryote of the non-natural amino acid polypeptides that the technology that discloses in this part can be applicable to describe herein and the expression in the eukaryote.
High level expression for the polynucleotide that obtains to be cloned, usually the polynucleotide subclone of required polypeptide of will encoding instructs the strong promoter transcribe to containing, transcribes/expression vector of translation termination in, and if for the nucleic acid of coded protein, then subclone is used for the ribosome bind site of translation initiation usually.Be fit to the bacterium promotor and describe (for example) in philtrums such as people such as Sambrook and Ausubel.
The bacterial expression system that is used for express polypeptide can be used for (comprising, but be not limited to) (people such as Palva, Gene 22:229-235 (1983) in intestinal bacteria, genus bacillus (Bacillussp.), Pseudomonas fluorescens, Pseudomonas aeruginosa, pseudomonas putida and the Salmonellas (Salmonella); People such as Mosbach, Nature 302:543-545 (1983)).The test kit of these expression systems can be buied on the market.The eukaryotic expression system that is used for mammalian cell, yeast and insect cell can be buied on the market.Use therein under the situation of quadrature tRNA and aminoacyl tRNA synthetase (elsewhere description in this article) express polypeptide, the host cell that is used to express is to use the ability of quadrature component to select according to it.Exemplary host cell comprises gram-positive microorganism (Gram-positive bacteria) (including (but not limited to) bacillus brevis (B.brevis) or subtilis (B.subtilis) or streptomycete (Streptomyce)) and Gram-negative bacteria (Gram-negative bacteria) (intestinal bacteria or Pseudomonas fluorescens, Pseudomonas aeruginosa, pseudomonas putida), and yeast and other eukaryotic cells.Comprise that the right cell of O-tRNA/O-RS can be according to described use the herein.
Eukaryotic host cell or non-eukaryotic host cell provide the ability of the polypeptide that synthesizes the alpha-non-natural amino acid that comprises big dosage as described herein.On the one hand, composition is according to circumstances including (but not limited to) at least 10 micrograms, at least 50 micrograms, at least 75 micrograms, at least 100 micrograms, at least 200 micrograms, at least 250 micrograms, at least 500 micrograms, at least 1 milligram, at least 10 milligrams, at least 100 milligrams, at least 1 gram or the above polypeptide that comprises alpha-non-natural amino acid of 1 gram, maybe the amount that can obtain by polypeptide production method in vivo (producing and the details of purifying provides in this article about recombinant protein).On the other hand, polypeptide is according to circumstances with in (comprising, but be not limited to) cell lysates, damping fluid, in medicine damping fluid or other liquid suspensions (including (but not limited to) about 1nl to the volume between about 100L) (comprise, but be not limited to) whenever rise to 10 microgram polypeptide less, whenever rise to few 50 microgram polypeptide, whenever rise to few 75 microgram polypeptide, whenever rise to few 100 microgram polypeptide, whenever rise to few 200 microgram polypeptide, whenever rise to few 250 microgram polypeptide, whenever rise to few 500 microgram polypeptide, whenever rise to few 1 milligram of polypeptide or whenever rise to the concentration of lacking 10 milligrams of polypeptide or every liter of polypeptide more than 10 milligrams and be present in the composition.In comprising the eukaryotic cell of at least a alpha-non-natural amino acid, produce a large amount of (including (but not limited to) greater than the common possible amount that obtains by additive method (including (but not limited to) in vitro translation)) protein and be the feature of method, technology and the composition of description herein.
Eukaryotic host cell or non-eukaryotic host cell provide the proteinic ability of the alpha-non-natural amino acid that biosynthesizing comprises big dosage as described herein.For instance, the polypeptide that comprises alpha-non-natural amino acid can be in cell extract, cell lysates, substratum, (comprise, but be not limited to) at least 10 micrograms per litre in damping fluid and/or its analogue, at least 50 micrograms per litre, at least 75 micrograms per litre, at least 100 micrograms per litre, at least 200 micrograms per litre, at least 250 micrograms per litre or at least 500 micrograms per litre, at least 1 mg/litre, at least 2 mg/litre, at least 3 mg/litre, at least 4 mg/litre, at least 5 mg/litre, at least 6 mg/litre, at least 7 mg/litre, at least 8 mg/litre, at least 9 mg/litre, at least 10 mg/litre, at least 20,30,40,50,60,70,80,90,100,200,300,400,500,600,700,800,900 mg/litre, 1 grams per liter, 5 grams per liters, the concentration of the polypeptide that 10 grams per liters or 10 grams per liters are above produces.
1. Expression system, cultivation and separation
In the expression system of the non-natural amino acid polypeptides that the technology that discloses in this part can be applicable to describe herein, cultivation and the separation.Non-natural amino acid polypeptides can any number suitable expression system (including (but not limited to) yeast, insect cell, mammalian cell and bacterium) express.Being described in herein of exemplary expression system provides.
YeastAs used herein, term " yeast " comprises any one in each primary yeast of gene that can express the coding non-natural amino acid polypeptides.These yeast are including (but not limited to) ascosporogenous yeast (Endomycetale (Endomycetales), basidiomycetes yeast and the yeast that belongs to Deuteromycotina (Fungi imperfecti) (gemma guiding principle (Blastomycetes)) group.Ascosporogenous yeast is divided into two sections, Spermophthoraceae (Spermophthoraceae) and Saccharomycetaceae (Saccharomycetaceae).The latter comprises four subfamilies, Schizosaccharomycoideae (Schizosaccharomycoideae) (for example, Schizosaccharomyces (genus Schizosaccharomyces)), Nadsonioideae (Nadsonioideae), fat yeast subfamily (Lipomycoideae) and yeast subfamily (Saccharomycoideae) (for example, Pichia (genus Pichia), genus kluyveromyces (genus Kluyveromyces) and Saccharomycodes (genus Saccharomyces)).The basidiomycetes yeast comprises Leucosporidium (genus Leucosporidium), Rhodosporidium (genus Rhodosporidium), lock Sporobolomyces (genus Sporidiobolus), basidiomycetes (genus Filobasidium) and lock load bacterium (genus Filobasidiella).The yeast that belongs to Deuteromycotina (gemma guiding principle) group is divided into two sections, Sporobolomycetaceae (Sporobolomycetaceae) (for example, Sporobolomyces (genusSporobolomyces) and Bullera (genus Bullera)) and Cryptococcaceae (Cryptococcaceae) (for example, mycocandida (genus Candida)).
In certain embodiments, the method for describing in this article, use Pichia in technology and the composition, genus kluyveromyces, Saccharomycodes, Schizosaccharomyces, Hansenula (Hansenula), species in torulopsis (Tondopsis) and the mycocandida are (including (but not limited to) pichia pastoris (P.pastoris), P.guillerimondii, yeast saccharomyces cerevisiae (S.cerevisiae), cereuisiae fermentum (S.carlsbergensis), saccharomyces diastaticus (S.diastaticus), Douglas yeast (S.douglasii), S.khiyveri, S.norbensis, ellipsoideus yeast (S.oviformis), Kluyveromyces lactis (K.lactis), Kluyveromyces fragilis (K.fragilis), white candiyeast (C.albicans), maltose candiyeast (C.maltosa) and multiple-shaped nuohan inferior yeast (H.polymorpha)).
The suitable yeast that selection is used to express non-natural amino acid polypeptides is in those skilled in the art's skill.When selecting the yeast host that is used to express, suitable host can have those hosts of (for example) good secretion capacity, low proteolytic activity and overall activity including (but not limited to) displaying.Yeast usually can be available from multiple source, it is including (but not limited to) (the Berkeley of University of California, CA) the yeast heredity reserve center of biophysics and medical physics system, (Yeast Genetic Stock Center, Department of Biophysics and Medical Physics, University of California (Berkeley, CA)) and American type culture collection (American TypeCulture Collection) (" ATCC ") (Manassas, VA).
Term " yeast host " or " yeast host cell " comprise the yeast that can be used as or be used as the acceptor of recombinant vectors or other transfer DNAs.Described term comprises the offspring of the original yeast host cell of accepting recombinant vectors or other transfer DNAs.Should be appreciated that because accidental or have a mind to sudden change, the offspring of single parental cell form or genome or with the total DNA of original parent complementary in full accord.The offspring who fully is similar to the parental cell of desiring the parent that characterized by relevant nature (such as the nucleotide sequence that has the coding non-natural amino acid polypeptides) is contained among the offspring that definition means thus.
Develop expression and conversion carrier (comprising extrachromosomal replication or integrative vector) and be used for being converted into many yeast hosts.For instance, develop expression vector and be used for yeast saccharomyces cerevisiae (people such as Sikorski, GENETICS (1998) 112:19; People such as Ito, J.BACTERIOL. (1983) 153:163; People such as Hinnen, PROC.NATL.ACAD.SCI.USA (1978) 75:1929); White candiyeast (people such as Kurtz, M OL.C ELL.B IOL. (1986) 6:142); Maltose candiyeast (people such as Kunze, J.BASIC MICROBIOL. (1985) 25:141); Multiple-shaped nuohan inferior yeast (people such as Gleeson, J.GEN.MICROBIOL. (1986) 132:3459; People such as Roggenkamp, M OL.GEN.GENET. (1986) 202:302); Kluyveromyces fragilis (people such as Das, J.BACTERIOL. (1984) 158:1165); Kluyveromyces lactis (people such as De Louvencourt, J.BACTERIOL. (1983) 154:737; People such as Van den Berg, BIO/TECHNOLOGY (1990) 8:135); P.guillerimondii (people such as Kunze, J.BASIC MICROBIOL. (1985) 25:141); Pichia pastoris (United States Patent (USP) the 5th, 324, No. 639; The 4th, 929, No. 555; And the 4th, 837, No. 148; People such as Cregg, MOL.CELL.BIOL. (1985) 5:3376); Schizosaccharomyces pombe (Schizosaccharomycespombe) (Beach and Nurse, NATURE (1981) 300:706); And separate fat Ye Shi yeast (Y.lipolytica) (people such as Davidow, CURR.GENET. (1985) 10:380 (1985); People such as Gaillardin, CURR.G ENET. (1985) 10:49); Aspergillus nidulans (A.nidulans) (people such as Ballance, BIOCHEM.BIOPHYS.RES.COMMUN. (1983) 112:284-89; People such as Tilburn, G ENE(1983) 26:205-221; And people such as Yelton, PROC NATL.ACAD.SCI.USA (1984) 81:1470-74); Aspergillus niger (A.nigcr) (Kelly and Hynes, EMBO J. (1985) 4:475-479); T.reesia (EP 0 244 234); And filamentous fungus, such as, neurospora (Neurospora), mould (Penicillium), curved neck mould (Tolypocladium) (WO 91/00357), each is all to be incorporated herein by reference.
The control sequence of yeast vector is including (but not limited to) from such as alcoholdehydrogenase (ADH) (EP 0 284 044); Enolase; Glucokinase; The G-6-P isomerase; Glyceraldehyde-3-phosphate dehydrogenase (GAP or GAPDH); Hexokinase; Phosphofructokinase; The 3-phoshoglyceric acid mutase; And the promoter region of the gene of pyruvate kinase (PyK) (EP 0 329 203).The yeast PH05 gene of coding acid phosphatase also can provide suitable promoter sequence (people such as Miyanohara, PROC.NATL.A CAD.SCI.USA (1983) 80:1).Other suitable promoter sequences that are used for yeast host can comprise the glycerol 3-phosphate acid kinase (people such as Hitzeman, J.BIOL.CHEM. (1980) 255 (4): 12073-12080); With other glycolytic ferments, such as pyruvic carboxylase, triosephosphate isomerase, and glucose phosphate isomerase (people such as Holland, BIOCHEMISTRY (1978) 17 (23): 4900-4907; People such as Hess, J.ADV.ENZYME REG. (1969) 7:149-167) promotor.Induced Yeast promoter with other advantages of transcribing of being controlled by growth conditions can comprise alcoholdehydrogenase 2; Different cell pigment C; Acid phosphatase; Metallothionein(MT); Glyceraldehyde-3-phosphate dehydrogenase; The degrading enzyme relevant with nitrogen metabolism; And the promoter region of the enzyme of responsible maltose and galactose utilization.The suitable carrier and the promotor that are used for yeast expression are further described among the EP 0,073 657.
The yeast enhanser also can use with Yeast promoter.In addition, synthetic promoter also can serve as Yeast promoter.For instance, the upstream activation sequences (UAS) of Yeast promoter can be connected with the transcription activating zone of another Yeast promoter, produce synthetic hybrid promoter.The example of these hybrid promoters comprises the ADH that is connected with GAP transcription activating zone and regulates sequence.Referring to United States Patent (USP) the 4th, 880, No. 734 and the 4th, 876, No. 197, it is all to be incorporated herein by reference.Other examples of hybrid promoter comprise by the promotor of forming with the adjusting sequence of ADH2, GAL4, GAL10 or the PHO5 gene of the combination of the transcription activating of glycolytic ferment gene (such as GAP or PyK) zone.Referring to EP 0 164 556.In addition, Yeast promoter can comprise the natural promotor that exists in the non-yeast source with ability that combining yeast RNA polymerase and initiation transcribe.
Other controlling elementss of part that can constitute Yeast expression carrier are including (for example) terminator (people such as Holland, J.BlOL.C from GAPDH or enolase gene HEM. (1981) 256:1385).In addition, the replication orgin from 2 μ plasmid starting points is applicable to yeast.The suitable selection gene that is used for yeast is the trpl gene that exists in the yeast plasmid.Referring to people such as Tschumper, GENE (1980) 10:157; People such as Kingsman, GENE (1979) 7:141.The trpl gene is provided for lacking the selective marker of the zymic mutants which had of the ability of growing in tryptophane.Similarly, Leu2 defective yeast bacterial strain (ATCC 20,622 or 38,626) is by the known plasmid complementation with Leu2 gene.
Foreign DNA is introduced method in the yeast host including (but not limited to) the complete yeast host cell that transforms spheroplast or handle through alkali metal cation.For instance, zymic transforms can be according to people such as Hsiao, P ROC.N ATL.A CAD.S CI.USA people such as (1979) 76:3829 and Van Solingen, the method for describing among J.BACT. (1977) 130:946 is carried out.Yet, also can be according to S with the additive method (such as merging) that DNA introduces in the cell by nuclear injection, electroporation or protoplastis AMBROOKDeng the people, M OLECULARC LONING: A L AB.M ANUAL(2001) common described use the in.Yeast host cell can use the known standard technique of those skilled in the art to cultivate subsequently.
The proteinic additive method of expressing heterologous No. the 6th, 361,969, No. 20020055169, United States Patent (USP) that be described in the U.S. Patent Publication case in yeast host cell; The 6th, 312, No. 923; The 6th, 183, No. 985; The 6th, 083, No. 723; The 6th, 017, No. 731; The 5th, 674, No. 706; The 5th, 629, No. 203; The 5th, 602, No. 034; With the 5th, 089, No. 398; U.S. review patent RE37, No. 343 and RE35, No. 749; PCT publication application case WO 99/07862; WO 98/37208; With WO 98/26080; European patent application EP 0 946 736; EP 0 732 403; EP 0 480480; WO 90/10277; EP 0 460 071; EP 0 340 986; EP 0 329 203; EP 0 324 274; And among the EP0 164 556.Also referring to people such as Gellissen, ANTONIE VAN LEEUWENHOEK (1992) 62 (1-2): 79-93; People such as Romanos, YEAST (1992) 8 (6): 423-488; Goeddel, M ETHODSINENZYMOLOGY (1990) 185:3-7, its each is all to be incorporated herein by reference.
During the amplification stage of using standard feed batch fermentation method, the yeast host bacterial strain can be grown in mash-back.Because the carbon of specific yeast host utilizes approach or expresses the difference of master mode, fermentation process can be regulated.Only for instance, the fermentation of yeast belong yeast host may need single glucose charging, compound nitrogen source (for example, caseic hydrolysate), with additional multivitamin, and for optimum growh and expression, methylotrophy yeast pichia pastoris may need glycerine, methyl alcohol and trace quantity mineral charging, but only needs simple ammonium (nitrogen) salt.Referring to No. the 5th, 324,639, (for example) United States Patent (USP); People such as Eniott, J.PROTEIN CHEM. (1990) 9:95; And people such as Fieschko, BIOTECH.BlOENG. (1987) 29:1113, its each is all to be incorporated herein by reference.
Yet these fermentation process can have some common trait that does not rely on employed yeast host bacterial strain.For instance, limiting growth nutrient substance (being generally carbon) can add to during the amplification stage in the mash-back to allow maximum growth.In addition, fermentation process uses usually through designing the fermention medium with carbon, nitrogen, basic salt, phosphorus and other the less important nutrient substances (VITAMIN, trace mineral and salt etc.) that contains q.s.The case description of fermention medium that is applicable to pichia spp is in United States Patent (USP) the 5th, 324, and No. 639 and the 5th, 231, in No. 178, its each is all to be incorporated herein by reference.
Infect the insect cell of baculovirusTerm " insect host " or " insect host cell " refer to the insect that can be used as or be used as the acceptor of recombinant vectors or other transfer DNAs.Described term comprises the offspring of the protentomon host cell of transfection.Should be appreciated that because accidental or have a mind to sudden change, the offspring of single parental cell form or genome or with the total DNA of original parent complementary on needn't be in full accord.The offspring who fully is similar to the parental cell of desiring the parent that characterized by relevant nature (such as the nucleotide sequence that has the coding non-natural amino acid polypeptides) is contained among the offspring that definition means thus.
The those skilled in the art knows the selection of the suitable insect cell that is used for express polypeptide.Some insect species fully are described in the affiliated field and can buy on the market, and it is including (but not limited to) Aedes aegypti (Aedes aegypti), silkworm (Bombyx mori), fruit bat (Drosophila melanogaster), fall army worm (Spodoptera frugiperda) and cabbage looper (Trichoplusia ni).When selecting the insect host that is used to express, suitable host can be including (but not limited to) showing those hosts that especially have good secretion capacity, low proteolytic activity and overall activity.Insect usually can be available from multiple source, it is including (but not limited to) (the Berkeley of University of California, CA) insect heredity reserve center (the Insect Genetic Stock Center of biophysics and medical physics system, Department ofBiophysics and Medical Physics, University of California (Berkeley, CA)), and American type culture collection (American Type Culture Collection) (" ATCC ") (Manassas, VA).
Usually, the component that infects the insect expression system of baculovirus comprises transfer vector, is generally bacterial plasmid, and it contains the genomic fragment of baculovirus and is used to insert the convenient restriction site of the heterologous gene of institute's desire expression; Have with transfer vector in the wild-type baculovirus (this allows that the heterologous gene homologous recombination enters in the baculovirus genome) of baculovirus specific fragment homologous sequence; And suitable insect host cell and growth medium.At carrier construction, transfectional cell, select bacterial plaque, in culture, make cell growth with and similar procedure in material, method and the technology used in affiliated field for known and can obtain to describe the handbook of these technology.
After inserting heterologous gene in the transfer vector, carrier and wild-type virus genome are transfected in the insect host cell, carrier and viral genome are recombinated therein.The recombinant virus of packing is through expression and discriminating and purification of Recombinant spot.Be used for the material of baculovirus/insect cell expression system and method can kit form available from (for example) Invitrogen Corp. (Carlsbad, CA).Illustrative technique is described in SUMMERS AND SMITH, and among the TEXAS AGRICULTURALEXPERIMENT STATION BULLETIN No. 1555 (1987), it is to be incorporated herein by reference.Also referring to RICHARDSON, 39 METHODS IN MOLECULAR BIOLOGY:BACULOVIRUS EXPRESSION PROTOCOLS (1995); People such as AUSUBEL, CURRENTPROTOCOLS IN MOLECULAR B IOLOGY16.9-16.11 (1994); KING and POSSEE, THEBACULOVIRUS SYSTEM:A LABORATORY GUIDE (1992); And people such as O ' REILLY, BACULOVIRUS EXPRESSION VECTORS:A LABORATORY MANUAL (1992).
Use baculovirus/insect cell expression system produce various heterologous proteins be described in below with reference in the document and these technology can be suitable for preparing the non-natural amino acid polypeptides of describing herein.Referring to No. the 6th, 368,825, (for example) United States Patent (USP); The 6th, 342, No. 216; The 6th, 338, No. 846; The 6th, 261, No. 805; The 6th, 245,528,6,225, No. 060; The 6th, 183, No. 987; The 6th, 168, No. 932; The 6th, 126, No. 944; The 6th, 096, No. 304; The 6th, 013, No. 433; The 5th, 965, No. 393; The 5th, 939, No. 285; The 5th, 891, No. 676; The 5th, 871, No. 986; The 5th, 861, No. 279; The 5th, 858, No. 368; The 5th, 843, No. 733; The 5th, 762, No. 939; The 5th, 753, No. 220; The 5th, 605, No. 827; The 5th, 583, No. 023; The 5th, 571, No. 709; The 5th, 516, No. 657; The 5th, 290, No. 686; WO 02/06305; WO01/90390; WO 01/27301; WO 01/05956; WO 00/55345; WO 00/20032 WO 99/51721; WO 99/45130; WO 99/31257; WO 99/10515; WO 99/09193; WO 97/26332; WO 96/29400; WO 96/25496; WO 96/06161; WO 95/20672; WO 93/03173; WO 92/16619; WO 92/03628; WO 92/01801; WO 90/14428; WO 90/10078; WO 90/02566; WO 90/02186; WO 90/01556; WO 89/01038; WO 89/01037; WO 88/07082, and its each is all to be incorporated herein by reference.
Be applicable to that the carrier in baculovirus/insect cell expression system expresses and transfer vector including (but not limited to) the insect that derives from baculovirus autographa california (Autographacalifornica) nucleopolyhedrosis virus (AcNPV), it is the virus expression carrier that does not rely on helper virus.The virus expression carrier that derives from this system uses strong virus polyhedrosis gene promotor to drive expression of heterologous genes usually.Usually referring to people such as Reilly, BACULOVIRUSEXPRESSION VECTORS:A LABORATORY MANUAL (1992).
Before foreign gene being inserted in the shaft-like viral genome, transposition was constructed in the body (transfer vector) in the middle of the said components that will comprise promotor, leader (in case of necessity), the encoding sequence of being paid close attention to and transcription termination sequence usually was assembled into.Middle transposition is constructed body and is maintained usually in the replicon, such as can be in host (such as bacterium) extra-chromosomal element (for example, plasmid) of stable maintenance.Replicon should have dubbing system, thereby it is maintained be used for the suitable host that clones and increase.More particularly, plasmid can contain polyhedrin polyadenylation signal (people such as Miller, ANN.R EV(1988) 42:177) and protokaryon penbritin (ampicillin) resistance (amp) gene and be used for selecting and the replication orgin of breeding .MICROBIOL. intestinal bacteria.
A kind of transfer vector commonly used that is used for foreign gene is introduced AcNPV is pAc373.Known many other carriers of those skilled in the art have also designed including (for example) pVL985, and it changes into ATT with the polyhedrin initiator codon by ATG, and its 32 base pair places in the ATT downstream introduce the BamHI cloning site.Referring to Luckow and Summers, VIROLOGY 170:31-39 (1989).Other commercially available carriers are including (for example) PBlueBac4.5/V5-His; PBlueBacHis2; PMelBac; PBlueBac4.5 (Invitrogen Corp., Carlsbad, CA).
After inserting heterologous gene, transfer vector and wild-type baculovirus genome cotransfection are gone among the insect cell host.The illustrative method that allogeneic dna sequence DNA is introduced in the required site in the baculovirus is described in SUMMERS and SMITH, TEXAS AGRICULTURAL EXPERIMENT No. the 1555th, BULLETIN of STATION (1987); People such as Smith, MOL.CELL.BIOL. (1983) 3:2156; Luckow and Summers are among VIROLOGY (1989) 170:31-39.For instance, can be by in the gene of homology double cross reorganization insertion such as polyhedron gene; Also can insert through through engineering approaches and operate in the restriction enzyme sites that enters in the required baculovirus gene.Referring to people such as Miller, BlOESSAYS (1989) 4:91.
Transfection can be used TROTTER and W OOD, 39 METHODS IN MOLECULAR BIOLOGY (1995); Mann and King, J.G EN.V IROL. the method for describing among (1989) 70:3501 realizes by electroporation.Perhaps, can use liposome with recombinant expression vector and baculovirus transfection insect cell.Referring to people such as (for example) Liebman, BlOTECHNlQUES (1999) 26 (1): 36; People such as Graves, BIOCHEMISTRY (1998) 37:6050; People such as Nomura, J.BIOL.CHEM. (1998) 273 (22): 13570; People such as Schmidt, PROTEINEXPRESSION AND PURIFICATION (1998) 12:323; People such as Siffert, NATURE GENETICS (1998) 18:45; T ILKINSDeng the people, CELL BIOLOGY:A LABORATORY HANDBOOK 145-154 (1998); People such as Cai, PROTEIN EXPRESSION AND PURIFICATION (1997) 10:263; People such as Dolphin, NATURE GENETICS (1997) 17:491; People such as Kost, GENE (1997) 190:139; People such as Jakobsson, J.B IOL.CHEM. (1996) 271:22203; People such as Rowles, J.BIOL CHEM. (1996) 271 (37): 22376; People such as Reversey, J.B IOL.CHEM. (1996) 271 (39): 23607-10; People such as Stanley, J.B IOL.CHEM. (1995) 270:4121; People such as Sisk, J.VIROL. (1994) 68 (2): 766; And people such as Peng, BIOTECHNIQUES (1993) 14.2:274.Commercially available liposome including (for example)
Figure A20058004432801611
With
Figure A20058004432801612
(Invitrogen, Corp., Carlsbad, CA).In addition, can use calcium phosphate transfection.Referring to T ROTTERAnd W OOD, 39METHODS IN MOLECULAR BIOLOGY (1995); Kitts, NAR (1990) 18 (19): 5667; And Mann and King, J.GEN.VIROL. (1989) 70:3501.
Rhabdovirus expression vector contains bacilliform virus promoter usually.Bacilliform virus promoter is any dna sequence dna that can be transcribed into mRNA in conjunction with the downstream (3 ') of shaft-like viral rna polymerase and start code sequence (for example, structure gene).Promotor should have near the transcription initiation region that is usually located at 5 of encoding sequence ' end.This transcription initiation region comprises RNA polymerase binding site and transcription initiation site usually.Bacilliform virus promoter also can have second territory that is known as enhanser, and its (if existence) is usually away from structure gene.In addition, expression can be modulability or composition.
The structure gene that later stage in infecting circulation transcribes in a large number provides the promoter sequence that especially is suitable for.Example comprises gene (people such as FRIESEN, the The Regulation of Baculovirus GeneExpression in THE MOLECULAR BIOLOGY OF BACULOVIRUSES (1986) that derives from the viral polyhedrin of coding; EP 0 127 839 and 0 155 476) and the sequence of coding p10 proteic gene people such as (, J.GEN.VlROL. (1988) 69:765) Vlak.
Wrap up the rhabdovirus expression vector that newly forms in the infectious recombinant baculovirus and the spot of growing subsequently can be by such as people such as Miller, BIOESSAYS (1989) 4:91; Those technology of describing among SUMMERS and the SMITH, TEXAS AGRICULTURALEXPERIMENT STATION BULLETINNO.1555 (1987) are come purifying.
Developed the recombination rhabdovirus expression vector that is used for infecting into some insect cells.For instance, developed the recombinant baculovirus that is particularly useful in following each insect: Aedes aegypti (ATCC CCL-125), silkworm (ATCC CRL-8910), fruit bat (ATCC number 1963), fall army worm and cabbage looper.Referring to WO 89/046,699; Wright, NATURE (1986) 321:718; People such as Carbonell, J.VlROL. (1985) 56:153; People such as Smith, MOL.CELL.BIOL. (1983) 3:2156.Usually referring to people such as Fraser, IN VITRO CELL.DEV.BIOL. (1989) 25:225.More particularly, the clone that is used for the rhabdovirus expression vector system is usually including (but not limited to) Sf9 (fall army worm) (ATCC CRL-1711), Sf21 (fall army worm) (Invitrogen Corp., catalog number (Cat.No.) 11497-013 (Carlsbad, CA)), Tri-368 (cabbage looper) and High-Five TMBTI-TN-5B1-4 (cabbage looper).
Be used for directly expressing with the cell and the substratum of amalgamation and expression heterologous polypeptide and can buying on the market in baculovirus/expression.
Bacterium bacterial expression technology is what know in affiliated field.Variety carrier can be used in the host bacterium.Carrier can be the low or high multi-copy vector of single copy.Carrier can be used for the clone and/or expresses.Because about the abundant document of carrier, the commercial applicability of many carriers, and even the handbook of carrier and its estriction map and feature is described, do not need extensive argumentation in this article.As everyone knows, carrier generally includes the mark of allowing selection, and these marks can provide cytotoxic agent resistance, prototrophy or immunity.Usually, have a plurality of marks, it provides different characteristics.
The bacterium promotor is can (3 ") are transcribed into any dna sequence dna of mRNA in conjunction with the downstream of bacteria RNA polysaccharase and start code sequence (for example, structure gene).Promotor should have near the transcription initiation region that is usually located at 5 of encoding sequence ' end.This transcription initiation region comprises RNA polymerase binding site and transcription initiation site usually.The bacterium promotor also can have second territory of the operator gene of being known as, and it can be overlapping at the place and the adjacent R NA polymerase binding site point of the synthetic beginning of RNA.Operator gene allows and negative regulate (can induce) and transcribe, because gene repression albumen can and and then suppress transcribing of specific gene in conjunction with operator gene.The constructive expression can be taken place under the situation that does not have negative regulatory element (such as operator gene).In addition, just regulating and to pass through gene activation protein binding sequence (if exist, it is usually near (5 ') of RNA polymerase binding sequence so) and obtain.The proteic example of gene activation is meta-bolites activated protein (CAP), and it helps transcribe [people such as Raibaud, ANNU.REV.GENET. (1984) 18:173] of lac operon in the initial intestinal bacteria.Therefore the modulated expression can be positivity or negative, and then strengthens or weaken and transcribe.
The sequence of encoding metabolic pathway enzyme provides the promoter sequence that especially is suitable for.Example comprises the promoter sequence that derives from such as the carbohydrate metabolism enzyme of semi-lactosi, lactose (lac) [people such as Chang, NATURE (1977) 198:1056] and maltose.Other examples comprise the promoter sequence that derives from biosynthetic enzyme, such as tryptophane (trp) (people such as Goeddel, NUC.ACIDS RES. (1980) 8:4057; People such as Yelverton, NUCL.ACIDS RES. (1981) 9:731; United States Patent (USP) the 4th, 738, No. 921; IFNPub. the 036 No. 776 and the 121 No. 775), its each is all to be incorporated herein by reference.People such as beta-galactosidase enzymes (bla) promoter systems [Weissmann (1981) " The cloning ofinterferon and other mistakes. " In Interferon 3 (I.Gresser volume)], phage PL[Shimatake, NATURE (1981) 292:128] and T5[United States Patent (USP) the 4th, 689, No. 406], its each is all to be incorporated herein by reference, and promoter systems also provides suitable promoter sequence.The preferred method of containing herein utilizes strong promoter (such as the T7 promotor) to produce with high level to bring out polypeptide.The example of these carriers is including (but not limited to) the pET29 series from Novagen, and the pPOP carrier of describing among the WO99/05297 (it is all to be incorporated herein by reference).These expression systems produce high-caliber polypeptide and do not jeopardize host cell vitality or growth parameter(s) in the host.
In addition, also serve as the bacterium promotor at the non-existent synthetic promoter of occurring in nature.For instance, the transcription-activating sequence of a bacterium or phage promoter can be connected with the operon sequence of another bacterium or phage promoter, produce synthetic hybrid promoter [United States Patent (USP) the 4th, 551, No. 433].For instance, the tac promotor is the heterozygosis trp-lac promotor that is made of trp promotor and the lac operon sequence that regulated by the lac repressor [people such as Amann, GENE (1983) 25:167; People such as de Boer, P ROC.NATL.ACAD.SCI. (1983) 80:21].In addition, the bacterium promotor can comprise the natural promotor that exists that has in conjunction with the non-bacterial origin of bacteria RNA polysaccharase and initial ability of transcribing.Non-bacterial origin natural exist promotor also can with the coupling of consistency RNA polymerase to produce the high level expression of some genes in the prokaryotic organism.Phage t7 RNA polymerase/promoter systems is example [people such as Studier, the J.M of coupling promoter systems OL.B IOL. (1986) 189:113; People such as Tabor, Proc Natl.Acad.Sci. (1985) 82:1074].In addition, hybrid promoter also can comprise phage promoter and intestinal bacteria operator region (No. the 267 851, IFNPub.).
Except that the function on subsequence, effectively ribosome bind site also is applicable to the expression of foreign gene in prokaryotic organism.In intestinal bacteria, ribosome bind site is known as Shine-Dalgarno (SD) sequence and the length that comprises initiator codon (ATG) and be positioned at upstream from start codon 3-11 Nucleotide place is the sequence [people such as Shine, NATURE (1975) 254:34] of 3-9 Nucleotide.Think that the SD sequence promotes mRNA and ribosomal combination the [people such as Steitz by making base pairing between SD sequence and 3 of the intestinal bacteria 16S rRNA ' end, " Genetic signals andnucleotide sequences in messenger RNA ", In Biological Regulation and Development:GeneExpression (R.F.Goldberger compiles, 1979)].For expression have the eukaryotic gene of weak ribosome bind site and prokaryotic gene [people such as Sambrook. " Expression of cloned genes in Escherichia coli ", MolecularCloning:A Laboratory Manual, 1989].
Term " host bacterium " or " bacterial host cell " refer to the bacterium that can be used as or be used as the acceptor of recombinant vectors or other transfer DNAs.Described term comprises the offspring of the primitive bacteria host cell of transfection.Should be appreciated that because accidental or have a mind to sudden change, the offspring of single parental cell form or genome or with the total DNA of original parent complementary on needn't be in full accord.The offspring who fully is similar to the mother cell of desiring the parent that characterized by relevant nature (such as the nucleotide sequence that has the required polypeptide of coding) is contained among the offspring that definition means thus.
The those skilled in the art knows the selection of the suitable host bacteria that is used to express required polypeptide.When the host bacterium of selecting to be used to express, suitable host can including (but not limited to) show especially have in the following feature at least one and preferably have those hosts at least in the following feature: good inclusion body forms ability, low proteolytic activity, good secretion capacity, good soluble proteins and produces ability and overall activity.Host bacterium usually can be available from multiple source, it is including (but not limited to) (the Berkeley of University of California, CA) bacterium heredity reserve center (the Bacterial Genetic Stock Center of biophysics and medical physics system, Department of Biophysics and MedicalPhysics, University of California (Berkeley, CA)); And American type culture collection (American Type Culture Collection) (" ATCC ") (Manassas, VA).The bacterium (for example BL21) that derives from the bacterium (for example W3110) of K bacterial strain or derive from the B bacterial strain is used in the fermentation of industry/medicine usually.Because its growth parameter(s) is extremely known and it is active to be, so these bacterial strains are especially suitable.In addition, these bacterial strains are avirulence, and for the reason of safety and environment, it is commercially important.Among one embodiment of the method for describing in this article and containing, escherichia coli host is including (but not limited to) the bacterial strain of BL21, DH10B or derivatives thereof.Among another embodiment of the method for describing in this article and containing, escherichia coli host is a Deproteinization enzyme bacterial strain, and it is including (but not limited to) OMP-and LON-.In another embodiment, host bacterium is the species of pseudomonas, such as Pseudomonas fluorescens, Pseudomonas aeruginosa and pseudomonas putida.The example of pseudomonas expression strain is Pseudomonas fluorescens biotype I, bacterial strain MB101 (Dow Chemical).
In case set up the recombinant host cell strain (that is, and express construct body introduced in the host cell and will have suitable expression construct the host cell of body and separate), just be suitable for preparing cultivation recombinant host cell strain under the condition of polypeptide.The cultural method of recombinant host cell strain should depend on that the expression that is utilized constructs the character and the host cell itself of body.The method of knowing in the field under the recombinant host bacterial strain uses is usually cultivated.Recombinant host cell is containing absorbable carbon, nitrogen and inorganic salt source usually, and contains other protein of knowing in VITAMIN, amino acid, somatomedin and the affiliated field according to circumstances and cultivate in the liquid nutrient medium of supplement and cultivate.The liquid nutrient medium that is used to cultivate host cell can contain the microbiotic that prevents unwanted microorganism growth or anti-mycotic agent and/or according to circumstances including (but not limited to) the antibiotic compound that is used to select contain the host cell of expression vector.
Recombinant host cell form is in batches or continuously cultivated, wherein with form collecting cell (under the situation that required therein polypeptide accumulates in cell) in batches or continuously or collect culture supernatants.For in prokaryotic host cell, preparing preferred batch culture and cell harvesting.
In one embodiment, the non-natural amino acid polypeptides of describing herein is to be to express in the recombination system purifying afterwards.Polypeptide can be by the purifying from host cell or substratum of known several different methods in the affiliated field.Usually, the multiple polypeptides solubility that in bacterial host cell, prepares relatively poor or soluble (with the form of inclusion body).In one embodiment, be the deliquescent purpose of the polypeptide that increases the reorganization preparation, utilize known those methods in the method that discloses and the affiliated field herein, can be easy in polypeptide, carry out aminoacid replacement through selecting.Under the situation of insoluble polypeptide, described polypeptide can be by centrifugal or filter and to collect from the host cell lysate and can further then carry out homogenizing of cell.Under the situation of the polypeptide of poorly soluble, can add compound including (but not limited to) polymine (PEI) to bring out the precipitation of the solvable polypeptide of part.Sedimentary polypeptide can be collected easily by centrifugal or filtration subsequently.Can use several different methods that the those skilled in the art knows that recombinant host cell is broken or homogenize to discharge inclusion body in cell.Host cell is broken or homogenize to use and know technology and carry out, and described technology homogenizes (dounce homogenization) including (but not limited to) enzymatic cytoclasis, ultrasonic degradation, Du Ensi or high pressure discharges broken.Among one embodiment of the method for describing in this article and containing, use the broken e. coli host cell of high pressure release tech to discharge the inclusion body of polypeptide.Found by only utilizing e. coli host cell can increase the productive rate of the insoluble polypeptide that is the inclusion body form by a passage of homogenizer.When handling the inclusion body of polypeptide, because such as the factor of dissolving, mechanical shearing or proteolysis, minimize and repeat the time of homogenizing so that the productive rate of maximization inclusion body and free of losses is favourable.
Under can using subsequently in the field any one in known numerous suitable solvating agents make insoluble or sedimentary polypeptide dissolving.For instance, with urea or guanidine hydrochloride dissolution polypeptide.The volume of dissolving polypeptide should minimize so that can use the batch weight that can conveniently handle to prepare in enormous quantities.Recombinant host can volume be that this factor can be important in the large-scale commercial applications device of batch growth of last kilolitre therein.In addition, when in the large-scale commercial applications device, making polypeptide,, if possible, should avoid damaging machine and container especially for human medicinal use, or the harsh chemicals of polypeptide product itself.Showed in the method for describing in this article and containing that available gentleer denaturing agent urea replaces than causticity denaturing agent guanidine hydrochloride dissolution polypeptide inclusion body.The use of urea significantly reduces the risk that used stainless steel equipment in the manufacturing of polypeptide and purge process is damaged, simultaneously effective dissolving polypeptide inclusion body.
Under the situation of soluble polypeptide, peptide can be secreted in periplasmic space or the substratum.In addition, soluble peptide can be present in the tenuigenin of host cell.Soluble peptide can be concentrated before the purification step carrying out.Can use standard technique (including (but not limited to) those technology of describing) from (for example) cell lysates or substratum, to concentrate soluble peptide herein.In addition, can use standard technique (including (but not limited to) those technology of describing) broken host cell and from the tenuigenin of host cell or periplasmic space, discharge soluble peptide herein.
When polypeptide is prepared as fusion rotein, preferably remove fusion sequence.Removing of fusion sequence can be realized by the method including (but not limited to) enzymatic lysis or chemical cracking, wherein preferred enzymatic lysis.The method that can use the those skilled in the art to know realizes that the enzymatic of fusion sequence removes.The selection that is used to remove the enzyme of fusion sequence is determined by the characteristic that merges, and reaction conditions is specified by the selection of enzyme.Can use including (but not limited to) the reagent of cyanogen bromide, TEV proteolytic enzyme and other reagent and realize chemical cracking.The cracked polypeptide is according to circumstances by well-known process purifying from the cracked fusion sequence.These methods will be determined by the characteristic and the character of fusion sequence and polypeptide.Purification process can be including (but not limited to) size exclusion chromatography, hydrophobic interaction chromatograph, ion-exchange chromatography or dialysis or its any combination.
Polypeptide is also purified according to circumstances to remove DNA from protein soln.DNA can be removed by known any appropriate methodology in the affiliated field, and it is including (but not limited to) precipitation or ion-exchange chromatography.In one embodiment, by removing DNA with nucleic acid precipitation agent (such as (but being not limited to) protamine sulfate) precipitation.Can use standard well-known process (including (but not limited to) centrifugal or filtration) that polypeptide is separated with sedimentary DNA.Using polypeptide to treat under the human environment, removing of host's nucleic acid molecule is an important factor, and the method for describing herein is reduced to pharmaceutically acceptable degree with host cell DNA.
The method of small-scale or large scale fermentation also can be used in the protein expression, and it is including (but not limited to) mash-back, concussion flask, fluidized bed bio reactor, hollow-fiber bioreactor, rolling bottle culture systems and steel basin bioreactor system.In these methods each can batch-type, stream adds formula or the continuous mode method is carried out.
Method standard under the human form of the non-natural amino acid polypeptides of Miao Shuing can be used usually herein in the field reclaims.For instance, substratum or cell lysates can be through centrifugal or filter to remove cell debris.Supernatant liquor can be through concentrating or being diluted to volume required or diafiltration goes into to be fit to be used to be further purified with the adjusting preparation in the damping fluid.Being further purified including (but not limited to) the deacylated tRNA amine that makes polypeptide variants of the non-natural amino acid polypeptides of Miao Shuing separated with corresponding complete form with clipped form herein.
Below in the exemplary program any one can be used for the non-natural amino acid polypeptides that purifying is described herein: affinity chromatography; Negatively charged ion or cation-exchange chromatography (using (comprise, but be not limited to) DEAE SEPHAROSE); The silica chromatogram; Reversed-phase HPLC; Gel-filtration (using (comprise, but be not limited to) SEPHADEX G-75); Hydrophobic interaction chromatograph; Size exclusion chromatography; Immobilized metal ion afinity chromatography; Ultrafiltration/diafiltration; Ethanol sedimentation; Ammonium sulfate precipitation; Chromatofocusing; Displcement chromatography; Electrophoretic procedures (including (but not limited to) the isoelectrofocusing of preparation type), differential solvability (including (but not limited to) ammonium sulfate precipitation), SDS-PAGE, extraction or its any combination.
Known according to the those skilled in the art and use standard program, herein the polypeptide of containing in the method and composition of Miao Shuing (including (but not limited to) the polypeptide that comprises alpha-non-natural amino acid, comprise the polypeptide of alpha-non-natural amino acid antibody, comprise alpha-non-natural amino acid polypeptide in conjunction with the thing of arranging in pairs or groups) but partial purification or in fact purifying be homogeneity.Therefore, the polypeptide of Miao Shuing can be reclaimed and purifying by in numerous methods of knowing in the affiliated field any one herein, these methods including (but not limited to) ammonium sulfate or ethanol sedimentation, acid or alkaline extraction, tubing string chromatogram, affine tubing string chromatogram, negatively charged ion or cation-exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatograph, hydroxyapatite chromatography, lectin chromatogram, gel electrophoresis with and any combination.When the maturation protein that preparation appropriately folds, can use albumen folding step more in case of necessity.In the highly purified final purification step of needs, can use high performance liquid chromatography (HPLC), affinity chromatography or other appropriate methodologies.In one embodiment, will be at the antibody of the alpha-non-natural amino acid polypeptide of alpha-non-natural amino acid (or comprise) preparation purified reagent based on the purifying of affinity as the polypeptide that comprises (comprise, but be not limited to) one or more alpha-non-natural amino acids.On demand partial purification or be purified to homogeneity after, polypeptide can be used for multiple effectiveness according to circumstances, it is including (but not limited to) as calibrating component, therapeutical agent, preventive, diagnostic reagent, research reagent and/or as the immunogen that is used for Antibody Preparation.
Except that other reference that indicate herein, know multiple purifying/protein folding method in the affiliated field, it is including (but not limited to) those methods that propose in the following document: R.Scopes, Protein Purification, Springer-Verlag, N.Y. (1982); Deutscher, Methods in Enzymologv the 182nd volume: Guide to Protein Purification,Academic Press, Inc.N.Y. (1990); Sandana (1997) Bioseparation of Proteins.Academic Press, Inc.; People such as Bollag. (1996) Protein Methods.The 2nd edition Wiley-Liss, NY; Walker (1996) The Protein Protocols HandbookHumana Press, NJ; Harris and Angal (1990) Protein Purification Applications:A Practical ApproachIRL Press at Oxford, Oxford, England; Harris and Angal Protein Purification Methods:A Practical ApproachIRL Press atOxford, Oxford, England; Scopes (1993) Protein Purification:Principles and Practice the 3rd editionSpringer Verlag, NY; Janson and Ryden (1998) Protein Purification:Principles.High Resolution Methods and Applications. the 2nd editionWiley-VCH, NY; And Walker (1998) Protein Protocols on CD-ROMHumana Press, NJ; And the reference of wherein quoting.
Preparation comprises that an advantage of the polypeptide of at least one alpha-non-natural amino acid is that common polypeptide can be folding with its native conformation in eukaryotic host cell or non-eukaryotic host cell.Yet in this article among some embodiment of the method and composition of Miao Shuing, after synthetic, expression and/or purifying, polypeptide can have the conformation of the required conformation that is different from related polypeptide.In this article in the one side of the method and composition of Miao Shuing, make expressed protein denaturation and renaturation subsequently according to circumstances.This optional sex change and renaturation are known method realizations in the field under utilizing, wherein these methods are including (but not limited to) chaperone (chaperonin) is added in the polypeptide of being paid close attention to, and polypeptide is dissolved in the chaotropic agent (including (but not limited to) Guanidinium hydrochloride), and utilize protein disulphideisomerase.
In general, sometimes need sex change and the expressed polypeptide of reduction and make these polypeptide be folded into preferred conformation more subsequently.For instance, described folding again can the realization by guanidine, urea, DTT, DTE and/or chaperone are added in the translation product of being paid close attention to.The those skilled in the art know reduction, sex change and recombinant protein matter method (referring to, above reference, and people such as Debinski. (1993) J.Biol.Chem..268:14065-14070; Kreitman and Pastan (1993) Bioconjug.Chem..4:581-585; And Buchner, wait the people, (1992) Anal.Biochem..205:263-270).People such as Debinski (for example) describe the sex change and the reduction of inclusion body protein among guanidine-DTE.Protein can contain in (comprise, but be not limited to) Sleep-promoting factor B and the arginic potential buffer solution of L-more folding.Folding again reagent can flow or otherwise move contacting with one or more polypeptide or other expression products, or vice versa.
Prepare at protokaryon under the situation of non-natural amino acid polypeptides, so the polypeptide of preparation may false folding and is therefore lacked biological activity or have the biological activity of reduction.Proteinic biological activity can be recovered by " folding again ".In one embodiment, by use (for example) one or more chaotropic agent (including (but not limited to) urea and/or guanidine) and can reduce disulfide linkage reductive agent (including (but not limited to) dithiothreitol (DTT), DTT or 2 mercapto ethanol, 2-ME) dissolving (wherein polypeptide is also for insoluble), separate folding and the reducing polypeptide chain makes the polypeptide of false folding folding again.Under the chaotropic agent of intermediate concentration, add oxygenant (including (but not limited to) oxygen, Gelucystine or cystamine) subsequently, it is allowed and forms disulfide linkage again.Under the polypeptide of separating folding or false folding can use in the field known standard method folding again, such as United States Patent (USP) the 4th, 511, No. 502, the 4th, 511, No. 503 and the 4th, those methods of describing in 512, No. 922, each of these documents is all to be incorporated herein by reference.Polypeptide also can be folded to form heterodimer or heteromultimeric altogether with other protein.After folding again or folding altogether, according to circumstances polypeptide is further purified.
The purifying of non-natural amino acid polypeptides also can use multiple technologies to realize, it is including (but not limited to) those technology of describing herein, for example hydrophobic interaction chromatograph, size exclusion chromatography, ion-exchange chromatography, RPLC, affinity chromatography with and similar techniques or its any combination.Other purifying also can comprise drying or precipitate purified proteinic step.
After purifying, non-natural amino acid polypeptides can be exchanged in the different damping fluids and/or concentrate by in the known several different methods in the affiliated field (including (but not limited to) diafiltration and dialysis) any one.The hGH experience that provides with single protein purification form is assembled and precipitation.In certain embodiments, purified non-natural amino acid polypeptides can be at least 90% pure (as measured by RPLC (RP-HPLC) or sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)).In some other embodiment, it is at least 95% pure that purified non-natural amino acid polypeptides can be, or at least 98% is pure, or at least 99% or high purity more.No matter the definite numerical value of the purity of non-natural amino acid polypeptides, non-natural amino acid polypeptides is all enough pure to be used as medical product or to be used for further processing, and it is including (but not limited to) engaging with water-soluble polymers (such as PEG).
In certain embodiments, the non-natural amino acid polypeptides molecule does not exist other activeconstituentss or protein (except vehicle, supporting agent, and stablizer, serum albumin with and analogue) situation under useful as therapeutics, and in certain embodiments, the non-natural amino acid polypeptides molecule can be compound with another kind of polypeptide or polymkeric substance.
2. The purifying of non-natural amino acid polypeptides
General purification processThe general purifying of the non-natural amino acid polypeptides that the technology that discloses in this part can be applicable to describe herein.
But pair cell lysate extract, substratum, inclusion body, the periplasmic space of host cell, the tenuigenin of host cell or comprise other materials of required polypeptide or by including (but not limited to) affinity chromatography, ion-exchange chromatography, hydrophobic interaction chromatograph, gel filtration chromatography, high performance liquid chromatography (" HPLC "), reversed-phase HPLC (" RP-HPLC "), expanded bed adsorption or its any combination and/or any separating step of multiple and carry out in the multiple separating step any one with any polypeptide mixture that any suitable order produces.
Equipment and other necessary materials of being used to carry out the technology of describing herein can buied on the market.Pump, run tank, monitor, register and total system are available from (for example) Applied Biosystems (Foster City, CA), Bio-Rad Laboratories, Inc. (Hercules, CA) and Amersham Biosciences, Inc. (Piscataway, NJ).Chromatographic material including (but not limited to) exchange group material, medium and damping fluid also can be available from these companies.
Balance in the tubing string chromatographic process that the professional equipment of use such as pump can more promptly be realized describing herein and other steps are such as washing and elution.Commercially available pump including (but not limited to)
Figure A20058004432801691
Pump P-50, peristaltic pump P-1, pump P-901 and pump P-903 (Amersham Biosciences, Piscataway, NJ).
The example of run tank comprise RediFrac run tank, FRAC-100 and FRAC-200 run tank and
Figure A20058004432801692
Run tank (Amersham Biosciences, Piscataway, NJ).Also available mixing tank is to form pH value and linear concentration gradient.Commercially available mixing tank comprise gradient mixer GM-1 and tandem mixing tank (Amersham Biosciences, Piscataway, NJ).
Can use any commercially available monitor to monitor chromatographic process.These monitors can be used for collecting the information as UV, fluorescence, pH value and conductivity.The example of detector comprise monitor UV-1,
Figure A20058004432801693
S II, monitor UV-MII, monitor UV-900, monitor UPC-900, monitor pH/C-900 and conductivity monitor (AmershamBiosciences, Piscataway, NJ).In fact, comprise various from Amersham Biosciences
Figure A20058004432801694
(Piscataway, total system NJ) can buied on the market in system.
In this article among the embodiment of the method and composition of Miao Shuing, for example, polypeptide can reduce and sex change by at first making the purified polypeptide sex change of gained in urea, then be diluted in to be in the TRIS damping fluid that contains reductive agent (such as DTT) that is fit to the pH value.In another embodiment, polypeptide is sex change in the urea in the concentration range of about 2M between about 9M, then is diluted in the TRIS damping fluid under about 5.0 pH values to about 8.0 the scope.Can cultivate the folding again mixture of this embodiment subsequently.In one embodiment, folding again mixture was at room temperature cultivated 4 to 24 hours.The polypeptide mixture of reduction and sex change subsequently can be through further isolated or purified.
Such as herein statement, the pH value that can at first regulate first polypeptide mixture is carried out any later separation step subsequently.In addition, can use under in the field known technology first polypeptide mixture or its any subsequent mixtures are concentrated.In addition, comprise that the exchange of skills that the elution damping fluid of first polypeptide mixture or its any subsequent mixtures can use the those skilled in the art to know is the damping fluid that is suitable for next separating step.
Ion-exchange chromatographyThe chromatography of ions of the non-natural amino acid polypeptides that the technology that discloses in this part can be applicable to describe herein.
In one embodiment, and optional other steps of conduct, can carry out ion-exchange chromatography to first polypeptide mixture.Usually referring to ION EXCHANGE CHROMATOGRAPHY:PRINCIPLES AND METHODS (catalog number (Cat.No.) 18-1114-21, Amersham Biosciences (Piscataway, NJ)).Commercially available ion-exchange tubing string comprises
Figure A20058004432801701
And
Figure A20058004432801702
Tubing string (Amersham Biosciences, Piscataway, NJ).These tubing strings utilize strong anion exchanger, such as Q
Figure A20058004432801703
Fast Flow, Q
Figure A20058004432801704
HighPerformance, and Q
Figure A20058004432801705
XL; Strong cation exchanger is such as SP
Figure A20058004432801706
HighPerformance, SP
Figure A20058004432801707
Fast Flow, and SP
Figure A20058004432801708
XL; Weak anion exchanger is such as DEAE
Figure A20058004432801709
Fast Flow; And weak cation exchanger, such as CM
Figure A200580044328017011
Fast Flow (Amersham Biosciences, Piscataway, NJ).Can carry out negatively charged ion or cationic exchange tubing string chromatogram to the polypeptide in any stage of being in purge process to separate purified in fact polypeptide.Can use any suitable cationic exchange matrix to carry out the cation-exchange chromatography step.Cationic exchange matrix is including (but not limited to) fibrous, porous, atresia, microgranular, bead or cross-linked cationic exchange group material.These cationic exchange substrate materials are including (but not limited to) the mixture of Mierocrystalline cellulose, agarose, dextran, polyacrylic ester, polyethylene, polystyrene, silica, polyethers or any aforementioned substances.After polypeptide was adsorbed on the cationite matrix, purified in fact polypeptide can be able to elution by matrix is contacted so that polypeptide is cemented out with the damping fluid with enough high pH value or ionic strength from matrix.The suitable damping fluid of high pH value elution that is used for purified in fact polypeptide is including (but not limited to) Citrate trianion, phosphoric acid salt, formate, acetate, HEPES, and concentration at least about 5mM at least about the MES damping fluid in the scope of 100mM.
Reverse-phase chromatographyThe reverse-phase chromatography of the non-natural amino acid polypeptides that the technology that discloses in this part can be applicable to describe herein.
Can carry out RP-HPLC with protein purification according to the known suitable scheme of those skilled in the art.Referring to people such as (for example) Pearson, ANAL BIOCHEM. (1982) 124:217-230 (1982); People such as Rivier, J.CHROM. (1983) 268:112-119; People such as Kunitani, J.CHROM. (1986) 359:391-402.Can carry out RP-HPLC to separate purified in fact polypeptide to polypeptide.In this respect, can use and have multiple length (including (but not limited to) at least about C 3Extremely at least about C 30, at least about C 3Extremely at least about C 20, or at least about C 3Extremely at least about C 18) the silica deutero-resin with alkyl functional base of resin.Perhaps, can use polymer resin.For instance, can use TosoHaas Amberchrome CG1000sd resin, it is a styrenic polymer resins.Also can use cyano group or polymer resin with multiple alkyl chain length.In addition, can be such as alcoholic acid solvent wash RP-HPLC tubing string.The suitable elution damping fluid that contains ion pair reagent and organic modifiers (such as methyl alcohol, Virahol, tetrahydrofuran (THF), acetonitrile or ethanol) can be used for polypeptide elution from the RP-HPLC tubing string is gone out.The most frequently used ion pair reagent is including (but not limited to) acetate, formic acid, perchloric acid, phosphoric acid, trifluoroacetic acid, hyptafluorobutyric acid, triethylamine, tetramethyl-ammonium, TBuA, acetate triethyl ammonium.Can use one or more gradient or isocratic condition to carry out elution, wherein gradient condition preferably reduces disengaging time and reduces peak width.Another kind method relates to uses two kinds of gradients with different solvents concentration range.The example of the suitable elution damping fluid of Shi Yonging can be including (but not limited to) ammonium acetate and acetonitrile solution in this article.
The hydrophobic interaction chromatograph purification techniqueThe hydrophobic interaction chromatograph purifying of the non-natural amino acid polypeptides that the technology that discloses in this part can be applicable to describe herein.
Can carry out hydrophobic interaction chromatograph (HIC) to polypeptide.Usually referring to HYDROPHOBIC INTERACTIONCHROMATOGRAPHY HANDBOOK:PRINCIPLES AND METHODS (catalog number (Cat.No.) 18-1020-90, Amersham Biosciences (Piscataway, NJ)), it is to be incorporated herein by reference.Being fit to HIC matrix can be including (but not limited to) the matrix that replaces through alkyl or aryl, such as the matrix that replaces through butyl, hexyl, octyl group or phenyl, described matrix comprises agarose, Sepharose, sepharose, Mierocrystalline cellulose, silica, dextran, polystyrene, polymethacrylate matrix, and the resin of mixed form, it is including (but not limited to) polyvinylamine resin or the polymethacrylate matrix that replaces through butyl or phenyl.The commercially available source of hydrophobic interaction tubing string stratographic including (but not limited to) And
Figure A20058004432801712
Tubing string (Amersham Biosciences, Piscataway, NJ).In brief, before loading, the HIC tubing string can use the known standard buffer solution of one of ordinary skill in the art (such as acetate/sodium chloride solution or contain the HEPES of ammonium sulfate) to come balance.Ammonium sulfate can be used as the damping fluid that is used to load the HIC tubing string.After loading polypeptide, can use standard buffer solution and condition washing tubing string removing unwanted material subsequently, but polypeptide is retained on the HIC tubing string.Available about 3 standard buffer solutions to about 10 tubing string volumes (wherein, such as the HEPES damping fluid that contains EDTA and the ammonium sulfate concentrations lower than level pad, or acetate/sodium-chlor damping fluid) the elution polypeptide.Use the linear salt gradient that reduces gradually of (for example) potassiumphosphate gradient also to can be used for the elution peptide molecule.Can (for example) concentrate eluant subsequently by filtering (such as diafiltration or ultrafiltration).Can utilize diafiltration to remove the salt that is used for the elution polypeptide.
Other purification techniquesOther purification techniques of the non-natural amino acid polypeptides that the technology that discloses in this part can be applicable to describe herein.
Can use (for example) gel-filtration (GELFILTRATION:PRINCIPLES AND METHODS (catalog number (Cat.No.) 18-1022-18 to first polypeptide mixture or its any subsequent mixtures, Amersham Biosciences, Piscataway, NJ), it is all to be incorporated herein by reference), hydroxylapatite chromatography (is fit to matrix including (but not limited to) HA-Ultrogel, High Resolution (Calbiochem), CHT pottery hydroxyapatite (BioRad), Bio-Gel HTP hydroxyapatite (BioRad)), HPLC, expanded bed adsorption, ultrafiltration, diafiltration, lyophilization with and another separating step of similar approach with remove any excess salt and be suitable for next separating step or even the damping fluid of allocating final medicament production replace damping fluid.Can use various technology (it is including (but not limited to) those technology of describing) to monitor the productive rate of the polypeptide that comprises purified in fact polypeptide under each step of describing herein herein.These technology also can be used for assessing the productive rate of last separating step purified in fact polypeptide afterwards.For instance, any one that can use some anti-phase high pressure liquid chromatography tubing strings with multiple alkyl chain length is (such as cyano group RP-HPLC, C 18RP-HPLC) and cationic exchange HPLC and gel-filtration HPLC monitor the productive rate of polypeptide.
Can use standard technique (such as SDS-PAGE) or measure purity by using west ink dot method (Western blot) and ELISA calibrating to measure polypeptide.For instance, polyclonal antibody can be at reclaiming isolating protein generation from negative control yeast fermentation and cationic exchange.These antibody also can be used for surveying the existence of polluting host cell proteins matter.
In certain embodiments, the polypeptide productive rate behind each purification step can be polypeptide in the initial substance of each purification step at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.9% or at least about 99.99%.
RP-HPLC material Vydac C4 (Vydac) is made up of silica gel particle, and its surface has C 4-alkyl chain.Polypeptide be difference separating of protein impurities according to the intensity of hydrophobic interaction.Carry out elution with the acetonitrile gradient in rare trifluoroacetic acid.Use stainless-steel tubing pillar (be filled with 2.8 and rise to 3.2 liters Vydac C4 silica gel) to be prepared type HPLC.By adding trifluoroacetic acid with hydroxyapatite Ultrogel elutant acidifying and be loaded on the Vydac C4 tubing string.For washing and elution, be used in the acetonitrile gradient in rare trifluoroacetic acid.With fraction collection and neutralize with phosphate buffered saline buffer immediately.Collect in the polypeptide cut in the IPC limit.
DEAE Sepharose (Pharmacia) material is made up of lip-deep diethylamino ethyl (DEAE) group that is covalently bound to the sepharose bead.Polypeptide mediates with combining by ionic interaction of DEAE group.Acetonitrile and trifluoroacetic acid do not have the ground of delay and pass through tubing string.After washing out, remove trace impurity at these materials by acetate buffer washing tubing string with low pH value.Subsequently with neutral phosphonic phthalate buffer washing tubing string and with the damping fluid elution polypeptide of ionic strength with increase.With DEAE Sepharose fast flow filling tubing string.The adjustable pipe column volume is loaded in the scope of every milliliter of gel 3-10mg polypeptide to guarantee polypeptide.With water and level pad (sodium phosphate/potassiumphosphate) washing tubing string.Load compiling cut and washing tubing string of HPLC elutant with level pad.With lavation buffer solution (sodium acetate buffer) washing tubing string, then wash subsequently with level pad.Subsequently, with elution damping fluid (sodium-chlor, sodium phosphate/potassiumphosphate) polypeptide elution from tubing string is gone out and according to main elution overview with single fraction collection.The elutant of DEAE Sepharose tubing string is adjusted to the appointment conductivity.The sterile filtration of gained drug substance is gone in the Teflon bottle and under-70 ℃ to store.
Can use the assessment of several different methods and program to comprise the productive rate and the purity of the polypeptide of one or more alpha-non-natural amino acids, it dyes SDS-PAGE, coomassie brilliant blue staining SDS-PAGE (coomassie stained SDS-PAGE), mass spectrum (including (but not limited to) MALDI-TOF) and the known additive method that is used for profiling protein matter of those skilled in the art including (but not limited to) Bradford calibrating, SDS-PAGE, silver.
Additive method is including (but not limited to) removing endotoxic step.Intracellular toxin is for being positioned at the lipopolysaccharides (LPS) on the Gram-negative host cell adventitia of (such as, intestinal bacteria).The method that reduces endotoxin content is including (but not limited to) the purification technique that uses silica carrier, glass powder or hydroxyapatite, reverse-phase chromatography, affinity chromatography, size exclusion chromatography, anion-exchange chromatography, hydrophobic interaction chromatograph, the combination of these methods, with and similar approach.May need to modify or additive method from the polypeptide of being paid close attention to, to remove such as moving proteic pollutent altogether.The method of the known measurement endotoxin content of one of ordinary skill in the art and its are including (but not limited to) king crab amoebocyte lysate (LimulusAmebocyte Lysate, LAL) calibrating.
Additive method and program comprise (but being not limited to) changes stain method, substance assistant laser desorpted/ionization-mass spectrum (MALDI-MS), liquid chromatography/mass spectrometry, isoelectrofocusing, analysis mode anionresin, chromatofocusing and circular dichroism spectrum with protein staining method coupled SDS-PAGE, immunity.
In certain embodiments, the any minor or formula I-XVIII, the XXX-XXXIV (A and B) of specific compound and the amino acid of XXXX-XXXXIII that comprise in the category that is in formula I-XVIII, XXX-XXXIV (A and B) and XXXX-XXXXIII can be incorporated in the polypeptide by biosynthesizing, and then produce non-natural amino acid polypeptides.In other embodiments, these amino acid are that specific site place in polypeptide incorporates into.In other embodiments, these amino acid are to use translation system to incorporate in the polypeptide.In other embodiments, these translation systems comprise: (i) polynucleotide of coded polypeptide, wherein polynucleotide comprises corresponding to the selection codon of incorporating above-mentioned amino acid whose predetermined site into, and (ii) comprises amino acid whose tRNA, and wherein tRNA has specificity to selecting codon.In other embodiment of these translation systems, the mRNA of polynucleotide in translation system, producing.In other embodiment of these translation systems, translation system comprises plasmid or comprises the phage of polynucleotide.In other embodiment of these translation systems, translation system comprises the genomic dna that comprises polynucleotide.In other embodiment of these translation systems, the polynucleotide stable integration is gone in the genomic dna.In other embodiment of these translation systems, translation system comprises that wherein said selection codon is to be selected from by amber codon, ocher codon, opal codon, unique codon, rare codon, non-natural codon, five base codons and the molecular group of four base passwords to selecting codon to have specific tRNA.In other embodiment of these translation systems, tRNA is for suppressing tRNA.In other embodiment of these translation systems, translation system comprises by the tRNA of aminoacylization to the above-mentioned amino acid.In other embodiment of these translation systems, translation system comprises that tRNA is had specific amino acyl synthetase.In other embodiment of these translation systems, translation system comprises quadrature tRNA and quadrature aminoacyl tRNA synthetase.In other embodiment of these translation systems, polypeptide is to be synthesized by rrna, and in other embodiments, translation system is the in vivo translation system that comprises the cell that is selected from the group that is made up of bacterial cell, archeobacteria cell and eukaryotic cell.In other embodiments, cell be Bacillus coli cells, yeast cell, from cell, mammalian cell, vegetable cell or the insect cell of the species of pseudomonas.In other embodiment of these translation systems, translation system is the in vitro translation system that comprises from bacterial cell, archeobacteria cell or eukaryotic cell extract.In other embodiments, cell extract is from Bacillus coli cells, from cell, yeast cell, mammalian cell, vegetable cell or the insect cell of the species of pseudomonas.In other embodiments, at least a portion polypeptide is by solid phase or solution phase peptide synthetic method or its combination is next synthesizes, and in other embodiments, further comprises polypeptide chain is received on another polypeptide.In other embodiments, comprise and be in formula I-XVIII, the any minor in the category of XXX-XXXIV (A and B) and XXXX-XXXXIII or the formula I-XVIII of specific compound, the amino acid of XXX-XXXIV (A and B) and XXXX-XXXXIII can be incorporated in the polypeptide by biosynthesizing, wherein polypeptide for the protein of the treatment protein homology that is selected from the group that forms by following each thing: α-1 antitrypsin, angiostatin, AHF, antibody, lipophorin, apoprotein, atrionatriuretic factor, atrial natriuretic polypeptins, atrial natriuretic peptide, the C-X-C chemokine, T39765, NAP-2, ENA-78, gro-a, gro-b, gro-c, IP-10, GCP-2, NAP-4, SDF-1, PF4, MIG, calcitonin, the c-kit ligand, cytokine, the CC chemokine, MCP-1, MCP-2, monocyte chemoattractant protein-3, monocyte inflammatory protein-1 α, monocyte inflammatory protein-i β, RANTES, 1309, R83915, R91733, HCC1, T58847, D31065, T64262, CD40, the CD40 ligand, the c-kit ligand, collagen protein, G CFS (CSF), complement factor 5a, complement inhibitor, complement receptor 1, cytokine, epithelium neutrophilic granulocyte activation peptide-78, MIP-16, MCP-1, Urogastron (EGF), epithelium neutrophilic granulocyte activation peptide, erythropoietin (EPO), exfoliative toxin,or exfoliatin, factors IX, factor VII, Factor IX, factor X, fibroblast growth factor (FGF), Fibrinogen, fibronectin, four-helix bundle albumen, G-CSF, glp-1, GM-CSF, glucocerebrosidase, gonad-stimulating hormone, somatomedin, growth factor receptors, grf, Shh, hemochrome, pHGF (hGF), r-hirudin, human growth hormone (hGH), the human serum albumin, ICAM-1, the ICAM-1 acceptor, LFA-1, the LFA-1 acceptor, Regular Insulin, insulin-like growth factor (IGF), IGF-I, IGF-II, Interferon, rabbit (IFN), IFN-α, IFN-β, IFN-γ, interleukin-(IL), IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, keratinocyte growth factor (KGF), lactoferrin, leukaemia inhibitory factor, luciferase, neural plain, neutrophil inhibitory factor (nif) (NIF), oncostatin M, bone morphogenic protein, oncoprotein, paracitonin, Rat parathyroid hormone 1-34, PD-ECSF, PDGF, peptide hormone, PTN, albumin A, Protein G, pth, pyrogenic exotoxin A, pyrogenic exotoxin B, pyrogenic exotoxin C, pyy, Relaxin, feritin, SCF, the atom synthetic proteins, soluble complement acceptor I, solubility I-CAM 1, soluble interleukin receptor, soluble TNF acceptor, somatomedin, Somat, tethelin, streptokinase, superantigen, staphyloentero-toxin, SEA, SEB, SEC1, SEC2, SEC3, SED, SEE, steroid hormone receptor, superoxide-dismutase, the toxic shock syndrome toxin, thymosin, the histiotype plasminogen activation factor, tumor growth factor (TGF), tumour necrosis factor, tumor necrosis factor alpha, tumor necrosis factor, Tumor Necrosis Factor Receptors (TNFR), VLA-4 albumen, VCAM-1 albumen, vascular endothelial growth factor (VEGF), urokinase, mos, ras, raf, met, p53, tat, fos, myc, jun, myb, rel, estrogen receptor, PgR, the testosterone acceptor, aldosterone receptor, ldl receptor and Kendall compound.
B. in vivo posttranslational modification
By produce the polypeptide of being paid close attention to at least one alpha-non-natural amino acid in eukaryotic cell, these polypeptide are modified after may comprising eukaryotic translation.In certain embodiments, protein comprises at least one alpha-non-natural amino acid and at least a posttranslational modification of being carried out by eukaryotic cell in vivo, and wherein posttranslational modification is not to be undertaken by prokaryotic cell prokaryocyte.For instance, posttranslational modification including (but not limited to) acetylize, acidylate, lipid-modified, palmitoylation, palmitate addition, phosphorylation, glycolipid key modify, glycosylation, with and similar mechanism.On the one hand, posttranslational modification comprises by GlcNAc-l-asparagine binding oligosaccharides (including (but not limited to) (GlcNAc-Man) 2-Man-GlcNAc-GlcNAc)) be connected on the l-asparagine.Referring to table 1, it lists some examples (can have other residues, it is not showed yet) of the N-binding oligosaccharides of eukaryotic protein.On the other hand, posttranslational modification comprises by GalNAc-Serine or GalNAc-Threonine binding, or GlcNAc-Serine or GlcNAc-Threonine binding are connected to oligosaccharides (including (but not limited to) Gal-GalNAc, Gal-GlcNAc etc.) on Serine or the Threonine.
Table 1: the example of the oligosaccharides that connects by the GlcNAc binding
Figure A20058004432801751
In another aspect, posttranslational modification comprises precursor (including (but not limited to) the calcitonin precursor, calcitonin gene related peptide precursor, Pre Pro PTH, preproinsulin, proinsulin, pre-pro-opiomelanocortin, before POMC with and analogue) proteolysis processing, be assembled into multi-subunit protein matter or macromole molectron, translate another site to the cell (including (but not limited to) organoid, such as endoplasmic reticulum, golgi body (golgiapparatus), nuclear, lysosome, peroxysome, plastosome, chloroplast(id), vacuole etc., or pass through Secretory Pathway).In certain embodiments, protein comprises secretion or positioning sequence, epitope label, FLAG label, polyhistidyl label, GST fusion or its analogue.
An advantage of alpha-non-natural amino acid is that its existence can be used for adding other chemical parts of other molecules.These modifications can in vivo or in vitro be carried out in eukaryotic cell or non-eukaryotic cell.Therefore, in certain embodiments, posttranslational modification is to be undertaken by alpha-non-natural amino acid.For instance, posttranslational modification can be undertaken by nucleophilic-electrophilic reaction.The great majority translation that is used for proteinic selective modification at present relates to arranging in pairs or groups with electrophilic reaction at nucleophilic and forms covalent linkage between the thing, and it is including (but not limited to) the reaction of α-halogen ketone and Histidine or cysteine side chain.Selectivity under these situations is to be determined by the number of nucleophilic residues in the protein and accessibility.Describe in this article or use in the polypeptide of the method preparation of describing herein, can use other to have more optionally reaction, it is including (but not limited to) the in vitro and in vivo reaction of non-natural ketone group amino acid and hydrazides or amino oxygen based compound.Referring to people such as (for example) Cornish, (1996) Am.Chem.Soc.118:8150-8151; People such as Mahal, (1997) Science,276:1125-1128; People such as Wang, (2001) Science292:498-500; People such as Chin, (2002) Am.Chem.Soc.124:9026-9027; People such as Chin, (2002) Proc.Natl.Acad.Sci..99:11020-11024; People such as Wang, (2003) Proc.Natl.Acad.Set100:56-61; People such as Zhang, (2003) Biochemistry.42:6735-6746; And people such as Chin, (2003) Science300:964-967.This allows with the almost any protein of many reagent (it comprises fluorophore, linking agent, sugar derivatives and cytotoxicity molecule) selected marker.Be No. the 10/686th, 944, the U.S. patent application case of " Glycoprotein synthesis " referring to the title of on January 16th, 2003 application also, it is to be incorporated herein by reference.Posttranslational modification (including (but not limited to) by azido-amino acid) also can connect (Staudinger ligation) (including (but not limited to) with triaryl phosphine reagent) by Staudinger and carries out.Referring to people such as (for example) Kiick, (2002) Incorporation of azides into recombinant proteins for chemoselectivemodification by the Staudinger ligtation, PNAS99 (1): 19-24.
IX. be used to prepare the alternate systems of non-natural amino acid polypeptides
Used some strategies in non-recombinant hosts cell, mutagenesis host cell or cell free system, alpha-non-natural amino acid is introduced in the protein.The alternate systems that discloses in this part can be applicable to prepare the non-natural amino acid polypeptides of describing herein.For instance, make amino acid (such as Lys, Cys and Tyr) derivatize make Methionin change N into reactive side chain 2-ethanoyl-Methionin.Chemosynthesis also provides the simple method of incorporating alpha-non-natural amino acid into.Along with the enzymatic of peptide fragment connects the latest developments that are connected with native chemical, might make bigger protein.Referring to (for example) P.E.Dawson and S.B.H.Kent, Annu.Rev.Biochem, 69:923 (2000).The chemistry peptide connects to be connected with native chemical and is described in United States Patent (USP) the 6th, 184, among No. 344, No. the 2004/0138412nd, U.S. Patent Publication case, No. the 2003/0208046th, U.S. Patent Publication case, WO 02/098902 and the WO 03/042235, it is all to be incorporated herein by reference.Wherein the inhibition tRNA through required alpha-non-natural amino acid chemical acylation being added to the general in vitro biosynthetic means that can support in the biosynthetic in vitro extract of protein has been used for incorporating the almost multiple proteins of any size into surpassing 100 kinds of alpha-non-natural amino acid locus specificity ground.Referring to (for example) V.W.Cornish, D.Mendel and P.G.Schultz, Angew.Chem.Int.Ed.Engl.1995,34:621-633 (1995); C.J.Noren, S.J.Anthony-Cahill, M.C.Griffith, P.G.Schultz, A general method for site-specificincorporation of unnatural amino acids into proteins, Science244 182-188 (1989); And J.D.Bain, C.G.Glabe, T.A.Dix, A.R.Chamberlin, E.S.Diala, Biosynthetic site-specificincorporation of a unnatural amino acid into a polypeptide, J.Am.Chem.Soc.1118013-8014 (1989).Multiple functional group has been introduced in the protein with research protein stability, protein folding, enzyme mechanism and signal transduction.
Developed and be known as the hybridization of in vivo method that selection pressure incorporates into exploitation wild-type synthetic enzyme.Referring to (for example) N.Budisa, C.Minks, S.Alefelder, W.Wenger, F.M.Dong, L.Moroder and R.Huber, FASEB J..13:41-51 (1999).Make and wherein supply the cut auxotrophic strain of specific natural amino acid whose associated metabolic approach to cell and grow in the minimum medium of the natural amino acid that contains limited concentration, transcribing of target gene is suppressed simultaneously.When the fixed growth stage begins, natural amino acid is exhausted and replace with the alpha-non-natural amino acid analogue.Induce the feasible protein accumulation that contains the non-natural analogue of expression of recombinant proteins.For instance, use this strategy, adjacent fluorophenylalanine, a fluorophenylalanine and P-fluoropnenylalanine are incorporated in the protein, and in UV spectrum, show two feature acromions that can be easy to differentiate.Referring to (for example) C.Minks, R.Huber, L.Moroder and N.Budisa, Anal.Biochem., 284:29-34 (2000); Used the fluoroform methyllanthionine to replace methionine(Met) in the phage T4 N,O-Diacetylmuramidase to pass through 19F NMR studies the interaction of itself and oligochitosan ligand, referring to (for example) H.Duewel, and E.Daub, V.Robinson and J.F.Honek, Biochemistry,36:3404-3416 (1997); And incorporate trifluoro leucine in place leucine into, it makes the thermostability of leucine zipper protein and chemical stability increase.Referring to (for example) Y.Tang, G.Ghirlanda, W.A.Petka, T.Nakajima, W.F.DeGrado and D.A.Tirrell, Angew. Chem.Int. Ed.Engl..40 (8): 1494-1496 (2001).In addition, selenium methionine(Met) and tellurium methionine(Met) are incorporated into be beneficial to resolve in the various recombinant proteins in the X-ray crystallography mutually.Referring to (for example) W.A.Hendrickson, J.R.Horton and D.M.Lemaster, EMBO J., 9 (5): 1665-1672 (1990); J.O.Boles, K.Lewinski, M.Kunkle, J.D.Odom, B.Dunlap, L.Lebioda and M.Hatada, Nat.Struct.Biol, 1:283-284 (1994); N.Budisa, B.Steipe, P.Demange, C.Eckerskorn, J.Kellermann and R.Huber, Eur.J.Biochem., 230:788-796 (1995); And N.Budisa, W.Karnbrock, S.Steinbacher, A.Humm, L.Prade, T.Neuefeind, L.Moroder and R.Huber, J.Mol.Biol.270:616-623 (1997).Methionine(Met) analogue with alkene or alkynes functional group is also incorporated into effectively, and it is allowed by chemical means and carries out proteinic other modifications.Referring to (for example) J.C.M.vanHest and D.A.Tirrell, FEBS Lett..428:68-70 (1998); J.C.M.van Hest, K.L.Kiick and D.A.Tirrell, J.Am.Chem.Soc.122:1282-1288 (2000); And K.L.Kiick and D.A.Tirrell, Tetrahedron, 56:9487-9493 (2000); United States Patent (USP) the 6th, 586, No. 207; U.S. Patent Publication case 2002/0042097, it is all to be incorporated herein by reference.
The success of this method depends on by aminoacyl-tRNA synthetase and discerns the alpha-non-natural amino acid analogue that it needs highly selective to guarantee the fidelity of protein translation usually.An approach that enlarges the category of this method is to loosen the substrate specificity of aminoacyl-tRNA synthetase, and it is realized under a limited number of situation.Only for instance, in intestinal bacteria phenylalanyl-tRNA synthetic enzyme (PheRS), replace Ala by Gly 294Increase the size of substrate binding pocket, and make tRNAPhe by fenclonine (p-Cl-Phe) acidylate.Referring to, M.Ibba, P.Kast and H.Hennecke, Biochemistry.33:7107-7112 (1994).The coli strain that contains this mutant PheRS is allowed and is incorporated fenclonine into or bromophenyl alanine is replaced phenylalanine.Referring to (for example) M.Ibba and H.Hennecke, FEBS Lett..364:272-275 (1995); And N.Sharma, R.Furter, P.Kast and D.A.Tirrell, FEBS Lett..467:37-40 (2000).Similarly, show near the point mutation Phe130Ser at the amino acid binding site place of intestinal bacteria tyrosyl-tRNA synthetic enzyme and allow that azatyrosine more effectively incorporates into than tyrosine.Referring to, F.Hamano-Takaku, T.Iwama, S.Saito-Yano, K.Takaku, Y.Monden, M.Kitabatake, D.Soil and S.Nishimura, L Biol.Chem..275 (51): 40324-40328 (2000).
Another strategy of in vivo alpha-non-natural amino acid being incorporated in the protein has the machine-processed synthetic enzyme of check and correction for modifying.The amino acid that is similar to the homology natural amino acid on the structure can not be distinguished and therefore be activated to these synthetic enzyme.This mistake obtains correcting in indivedual sites, and it gets on acidylate to keep the fidelity of protein translation with mispairing amino acid from tRNA.If the check and correction loss of activity of synthetic enzyme, so wrong activatory analog can be avoided editting function and be merged in.Confirm this method with valyl-tRNA synthetic enzyme (ValRS) recently.Referring to, V.Doring, H.D.Mootz, L.A.Nangle, T.L.Hendrickson, V.de Crecy-Lagard, P.Schimmel and P.Marliere, Science.292:501-504 (2001).ValRS can use Cys, Thr or the wrong aminoacyl tRNAVal of aminobutyric acid salt (Abu); Subsequently by these non-homogeneous amino acid of edit field hydrolysis.After the random mutagenesis escherichia coli chromosome, be chosen in the mutant coli strain that has sudden change in the editor site of ValRS.This editor's defective type ValRS mistake is loaded tRNAVal with Cys.Because Abu spatially similar Cys (in Abu Cys-SH group warp-CH 3Displacement), so when this mutant coli strain was grown in the presence of Abu, mutant ValRS also incorporated Abu in the protein into.Mass spectroscopy is illustrated in the Xie Ansuan of each Xie Ansuan position about 24% of natural protein and is replaced by Abu.
Solid phase synthesis and semisynthesis are also allowed synthetic many protein that contain new amino acid.For instance, referring to following discloses case and the reference wherein quoted, described open case is as follows: Crick, F.J.C., Barrett, L.Brenner, S.Watts-Tobin, R.General nature of the genetic code for proteins. Nature, 192 (4809): 1227-1232 (1961); Hofmann, K., Bohn, H.Studies on polypeptides.XXXVI.The effect ofpyrazole-imidazole replacements on the S-protein activating potency of an S-peptidefragment, J, Am Chem, 88 (24): 5914-5919 (1966); Kaiser, E.T.Synthetic approaches tobiologically active peptides and proteins including enyzmes, Acc Chem Res.22 (2): 47-54 (1989); Nakatsuka, T., Sasaki, T., Kaiser, E.T.Peptide segment coupling catalyzed by thesemisynthetic enzyme thiosubtilisin, J Am Chem Soc, 109,3808-3810 (1987); Schnolzer, M., Kent, S B H.Constructing proteins by dovetailing unprotected synthetic peptides:backbone-engineered HIV protease, Science, 256,221-225 (1992); Chaiken, I.M.Semisynthetic peptides and proteins, CRC Crit Rev Biochemu255-301 (1981); Offord, R.E.Protein engineering by chemical means? Protein Eng.,1 (3): 151-157 (1987); And Jackson, D.Y., Burnier, J., Quan, C, Stanley, M., Tom, J., Wells, J.A.A Designed Peptide Ligase forTotal Synthesis of Ribonuclease A with Unnatural Catalytic Residues Science,266,243-247 (1994).
Chemically modified has been used for the multiple non-natural side chain that comprises cofactor, spin labeling and oligonucleotide being introduced protein in vitro.Referring to (for example) Corey, D.R., Schultz, P.G.Generation of a hybridsequence-specific single-stranded deoxyribonuclease, Science, 238,1401-1403 (1987); Kaiser, E.T., Lawrence D.S., Rokita, S.E.The chemical modification of enzymatic specificity, Ann. Rev Biochem, 54,565-595 (1985); Kaiser, E.T., Lawrence, D.S.Chemical mutation ofenyzme active sites, Science,226,505-511 (1984); Neet, K.E., Nanci A, Koshland, D.E.Properties of thiol-subtilisin, J Biol.Chem, 243 (24): 6392-6401 (1968); Polgar, L.B., M.L.Anew enzyme containing a synthetically formed active site.Thiol-subtilisin. J.Am Chem Soc, 88 (13): 3153-3154 (1966); And Pollack, S.J., Nakayama, G.Schultz, P.G.Introduction ofnucleophiles and spectroscopic probes into antibody combining sites, Science, 1 (242): 1038-1040 (1988).
Perhaps, use has been used for some biophysics probes are incorporated in vitro synthetic protein through the biosynthetic means of the aminoacyl-tRNA of chemically modified.Referring to following discloses case and the reference wherein quoted: Brunner, J.NewPhotolabeling and crosslinking methods, Annu.Rev Biochem, 483-514 (1993); And Krieg, U.C., Walter, P., Hohnson, A.E.Photocrosslinking of the signal sequence of nascentpreprolactin of the 54-kilodalton polypeptide of the signal recognition particle Proc.Natl. Acad.Sci.83,8604-8608 (1986).
In the past, proved and can in vitro through the inhibition tRNA of chemical aminoacylization alpha-non-natural amino acid locus specificity ground have been incorporated in the protein by in the reaction of the protein synthesis through stylizing, adding with the gene that contains required amber nonsense mutation.Use these methods, can use that the bacterial strain as the nutritive deficiency type replaces numerous common 20 seed amino acids with the imporosity homologue concerning specific amino acids, for example, with fluorophenylalanine substituted benzene L-Ala.Referring to (for example) Noren, C.J., Anthony-Cahill, Griffith, M.C., Schultz, P.G.A general method for site-specificincorporation of unnatural amino acids into proteins, Science.244:182-188 (1989); People such as M.W.Nowak, Science268:439-42 (1995); Bain, J.D., Glabe, C.G., Dix, T.A., Chamberlin, A.R., Diala, E.S.Biosynthetic site-specific Incoiporation of a non-natural amino acid into apolypeptide, J.Am Chem Soc.111:8013-8014 (1989); People such as N.Budisa, FASEB J.13:41-51 (1999); Ellman, J.A., Mendel, D., Anthony-Cahill, S., Noren, C.J., Schultz, P.G.Biosyntheticmethod for introducing unnatural ammo acids site-specifically into proteins. Methods in Enz.,202,301-336 (1992); And Mendel, D., Cornish, V.W. and Schultz, P.G.Site-DirectedMutagenesis with an Expanded Genetic Code, Annu Rev Biophys.Biomol Strnct.24,435-62 (1995).
For instance, preparation identification terminator codon UAG and through the inhibition tRNA of alpha-non-natural amino acid chemistry aminoacylization.Use conventional rite-directed mutagenesis be formed in the protein gene the site of paying close attention to introduce terminator codon TAG.Referring to (for example) Sayers, J.R., Schmidt, W.Eckstein, F.5 ', 3 ' Exonuclease in phosphorothioate-basedoligonucleotide-directed mutagenesis, Nucleic Acids Res.16 (3): 791-802 (1988).In vitro transcribe when acidylate being suppressed tRNA and mutant gene/when making up in the translation system, alpha-non-natural amino acid is responded the UAG codon and is incorporated into and obtain containing the sort of amino acid whose protein in specified location.Use [ 3H]-experiment of Phe and use the experiment confirm of alpha hydroxy acid only amino acid needed incorporate into by UAG codon appointed positions place and this amino acid not the site of any other in protein incorporate into.Referring to people such as (for example) Noren, the same; People such as Kobayashi, (2003) Nature Structural Biology 10 (6): 425-432; And Ellman, J.A., Mendel, D., Schultz, P.G.Site-specific incorporation of novel backbone structures into proteins, Science, 255,197-200 (1992).
The microinjection technology also has been used for incorporating alpha-non-natural amino acid into protein.Referring to (for example) M.W.Nowak, P.C.Kearney, J.R.Sampson, M.E.Saks, C.G.Labarca, S.K.Silverman, W.G.Zhong, J.Thorson, J.N.Abelson, N.Davidson, P.G.Schultz, D.A.Dougherty and H.A.Lester Science,268:439-442 (1995); And D.A.Dougherty, Curr.Opin.Chem.Biol, 4:645 (2000).In xenopus leavis oocytes, be injected in vitro two kinds of RNA materials making jointly: have at the amino acid position place that is paid close attention to the UAG terminator codon the coding target protein mRNA and suppress tRNA through the amber of required alpha-non-natural amino acid aminoacylization.The translating mechanism of ovocyte is inserting alpha-non-natural amino acid by UAG appointed positions place subsequently.This method is allowed the in vivo structure-functional study of integral membrane proteins, and described membranin is not subjected in vitro expression system effect usually.Example is including (but not limited to) fluorescence amino acid being incorporated in tachykinin neurokinin-2 acceptor to come measuring distance by FRET (fluorescence resonance energy transfer), referring to (for example) G.Turcatti, K.Nemeth, M.D.Edgerton, U.Meseth, F.Talabot, M.Peitsch, J.Knowles, H.Vogel and A.Chollet, I Biol.Chem., 271 (33): 19991-19998 (1996); Incorporate biotinylation amino acid into to differentiate the surperficial exposed residue in the ionic channel, referring to (for example) J.P.Gallivan, H.A.Lester and D.A.Dougherty, Chem.Biol., 4 (10): 739-749 (1997); Use cage to cover the tyrosine analogue with the conformational change in the real-time monitoring ion passage, referring to (for example) J.C.Miller, S.K.Silverman, P.M.England, D.A.Dougherty and H.A.Lester, Neuron, 20:619-624 (1998); And, use the α hydroxy-amino-acid that changes the ionic channel skeleton to survey its door control mechanism.Referring to (for example) P.M.England, Y.Zhang, D.A.Dougherty and H.A.Lester, Cell, 96:89-98 (1999); And T.Lu, A.Y.Ting, J.Mainland, L.Y.Jan, P.G.Schultz and J.Yang, Nat.Neurosci., 4 (3): 239-246 (2001).
The ability in the protein in vivo alpha-non-natural amino acid directly incorporated into provide the high yield of mutein, technology simple and easy, in cell or the advantage that may in live organism, study the possibility of mutein and in therapeutic treatment, use these muteins.The alpha-non-natural amino acid that will have all size, acidity, nucleophilicity, hydrophobicity and other character is incorporated into ability in the protein can greatly expand us rationally and the ability of the structure of systematicness ground operon protein, with the organism of surveying protein function and producing new protein or have novel character.
Incorporate in the process of P-fluoropnenylalanine attempting locus specificity ground, the yeast amber is suppressed tRNAPheCUA/ phenyl alanyl-tRNA synthetic enzyme to being used for p-F-Phe resistance, Phe auxotroph coli strain.Referring to (for example) R.Furter, Protein Sci.,7:419-426 (1998).
Also might use acellular (in vitro) translation system to obtain the expression of required polynucleotide.In these systems, it can comprise mRNA as template (in vitro translation) or as the DNA (combination is in vitro transcribed and translated) of template, is in vitro syntheticly instructed by rrna.Use sizable effort and developed the cell-free protein expression system.Referring to (for example) Kim, D.-M. and J.R.Swartz, Biotechnology and Bio engineering, 74 (4): 309-316 (2001); Kim, D.-M. and J.R.Swartz, Biotechnology Letters, 22,1537-1542, (2000); Kim, D.-M. and J.R.Swartz, Biotechnology Progress, 16,385-390, (2000); Kim, D.-M. and J.R.Swartz, Biotechnology and Bioengineering, 66 (3): 180-188, (1999); And Patnaik, R. and J.R.Swartz, Biotechniques 24 (5): 862-868, (1998); United States Patent (USP) the 6th, 337, No. 191; No. the 2002/0081660th, U.S. Patent Publication case; WO 00/55353; WO 90/05785, and it is all to be incorporated herein by reference.The another kind of method of polypeptide expression that can be applicable to comprise alpha-non-natural amino acid is including (but not limited to) mRNA-peptide integration technology.Referring to (for example) R.Roberts and J.Szostak, Proc.Natl Acad.Sci. (USA) 9412297-12302 (1997); People such as A.FranKel, Chemistry ﹠amp; Biology 10,1043-1050 (2003).In this method, the mRNA template that is connected on the tetracycline is translated as peptide on rrna.If modify one or more tRNA molecule, alpha-non-natural amino acid also can be incorporated in the peptide so.After reading last mRNA codon, tetracycline is caught the C-end of peptide.Have interesting property if find gained mRNA-peptide joiner in vitro in the calibrating, its identity can be easy to disclose according to the mRNA sequence so.In this way, but screening comprises the storehouse of the polypeptide of one or more alpha-non-natural amino acids has required character with discriminating polypeptide.Recently, reported in vitro rrna translation and allowed the synthetic peptide that replaces through alpha-non-natural amino acid with purified components.Referring to people such as (for example) A.Forster, Proc.Natl Acad.Sci. (USA) 100 (11): 6353-6357 (2003).
X. the posttranslational modification of the alpha-non-natural amino acid component of polypeptide
For simplicity, the posttranslational modification of the alpha-non-natural amino acid component of polypeptide of describing in this part (XA to XJ) is general and/or describe with particular instance.Yet, general description or particular instance that the posttranslational modification of the alpha-non-natural amino acid component of the polypeptide of describing in this part should not only limit to provide in this part, but the posttranslational modification of the alpha-non-natural amino acid component of the polypeptide of describing in this part equally fully is applicable to and is in formula I-XVIII, all compounds in the category of XXX-XXXIV (A and B) and XXXX-XXXXIII, it comprises the specification sheets that is in herein, claim and the graphic middle formula I-XVIII that describes, any minor or specific compound in the category of XXX-XXXIV (A and B) and XXXX-XXXXIII.
Method, composition, technology and strategy have been developed to incorporate alpha-non-natural amino acid on proteinic in vivo translate duration locus specificity ground.Have and the natural alpha-non-natural amino acid that has the orthogonal side chain chemistry of amino acid whose those side chains by incorporating into, this technology makes the locus specificity derivatize of recombinant protein become possibility.Therefore, the major advantage of the method for describing herein, composition, technology and strategy is that nowadays the protein of derivatize can be prepared as definite homology product.Yet, the method of Miao Shuing, composition, reaction mixture, technology and strategy are not limited to the non-natural amino acid polypeptides that formed by protein translation technology in vivo herein, but comprise the non-natural amino acid polypeptides that forms by any technology, described technology comprise (only for instance) marking protein connection, chemosynthesis, based on the technology of ribozyme (referring to (for example) herein title be the part of " expression in the alternate systems ").
Incorporate alpha-non-natural amino acid into chemistry that ability wide spread in the recombinant protein can realize being used to translate the back derivatize, wherein this derivatize occurs in vivo or in vitro.More particularly, the protein derivedization of formation oxime key provides some advantages on the alpha-non-natural amino acid part of polypeptide.First, natural exist amino acid do not form usually the oxime key and therefore through design with the reagent that forms the oxime key can with the alpha-non-natural amino acid component locus specificity reaction (supposing that certainly alpha-non-natural amino acid and corresponding reagent have designed with formation oxime key) of polypeptide, therefore the mixture with the derived protein that uses prior art processes to produce is opposite, and the proteinic ability of site selectivity derivatize provides single homogeneous product.The second, the oxime adducts is stable under biotic condition, and its hint is effective candidate that treatment is used by oxime exchange deutero-protein.The 3rd, the stability of gained oxime key can be handled according to the character (that is, functional group and/or structure) of oxime key alpha-non-natural amino acid formed thereon.Therefore, as shown in example 16, the pH value stabilization of the oxime key of alpha-non-natural amino acid can be changed to far away more than 1 week from being less than 1 hour.Therefore, in certain embodiments, the oxime key of non-natural amino acid polypeptides has and is less than 1 hour half life of decomposition, in other embodiments for being less than 1 day, in other embodiments for being less than 2 days, in other embodiments for being less than for 1 week and being in other embodiments more than 1 week.In other embodiments, the gained oxime is stable at least two weeks under appropriate acidic conditions, and in other embodiments, the gained oxime was stablized 5 days under appropriate acidic conditions at least.In other embodiments, non-natural amino acid polypeptides was being stablized 1 day under the pH value between about 2 and about 8 at least; In other embodiments, under about 2 to about 6 pH value, stablized at least 1 day; In other embodiments, under about 2 to about 4 pH value, stablized at least 1 day.In other embodiments, use strategy, method, composition and the technology described herein, the those skilled in the art should be able to synthesize that to have a skilled manpower of being adjusted to required (for example, be used for the treatment of purposes (such as continuing release), or diagnostic uses, or industrial use or military use) the oxime key of non-natural amino acid polypeptides of half life of decomposition.
Above-mentioned non-natural amino acid polypeptides is applicable to and (comprises, but be not limited to) research of novel therapeutic agents, diagnostic reagent, katalaze enzyme, industrial enzyme, conjugated protein (including (but not limited to) antibody and antibody fragment) and (comprise, but be not limited to) protein structure and function.Referring to (for example) Dougherty, (2000) Unnatural Amino Acids as Probes ofProtein Structure and Function, Current Opinion in Chemical Biology, 4:645-652.Other purposes of above-mentioned non-natural amino acid polypeptides comprise (only for instance) purposes, cosmetic use, plant biology purposes, environmental application, energy generation purposes and/or military use based on calibrating.Yet above-mentioned non-natural amino acid polypeptides can experience further modification to incorporate new or modified functional group into, and it comprises the therapeutic efficiency of handling polypeptide; The security overview of improvement polypeptide; Pharmacokinetics, pharmacology and/or the drug effect of regulating polypeptide (for example, increase water-soluble, biological usability; Increase serum half-life; Increase the treatment transformation period; Regulate immunogenicity; Regulate biological activity; Or prolong cycling time); Provide other functional groups to polypeptide; In polypeptide, incorporate label, mark or detectable signal into; The separating property of facilitation polypeptide; And any combination of aforementioned modification.
Be the method for the separating property of facilitation polypeptide in certain embodiments, it comprises that utilization comprises the homology biosynthesizing non-natural amino acid polypeptides of at least one alpha-non-natural amino acid, and described alpha-non-natural amino acid is to be selected from by containing the oxime alpha-non-natural amino acid, contain the carbonyl alpha-non-natural amino acid and containing the group that the azanol alpha-non-natural amino acid is formed.In other embodiments, these alpha-non-natural amino acids are incorporated into as described herein in the polypeptide by biosynthesizing.In other or alternate embodiment, these non-natural amino acid polypeptides comprise at least a amino acid whose alpha-non-natural amino acid that is selected from formula I-XVIII, XXX-XXXIV (A and B) or XXXX-XXXXIII.
The method of Miao Shuing, composition, strategy and technology are not limited to particular type, kind or the family of polypeptide herein.In fact, almost any polypeptide can comprise the alpha-non-natural amino acid that at least one is described herein.Only for instance, polypeptide can with the treatment protein homology that is selected from the group that forms by following each thing: α-1 antitrypsin, angiostatin, AHF, antibody, lipophorin, apoprotein, atrionatriuretic factor, atrial natriuretic polypeptins, atrial natriuretic peptide, the C-X-C chemokine, T39765, NAP-2, ENA-78, gro-a, gro-b, gro-c, IP-10, GCP-2, NAP-4, SDF-1, PF4, MIG, calcitonin, the c-kit ligand, cytokine, the CC chemokine, MCP-1, MCP-2, monocyte chemoattractant protein-3, monocyte inflammatory protein-1 α, monocyte inflammatory protein-i β, RANTES, 1309, R83915, R91733, HCC1, T58847, D31065, T64262, CD40, the CD40 ligand, the c-kit ligand, collagen protein, G CFS (CSF), complement factor 5a, complement inhibitor, complement receptor 1, cytokine, epithelium neutrophilic granulocyte activation peptide-78, MIP-16, MCP-1, Urogastron (EGF), epithelium neutrophilic granulocyte activation peptide, erythropoietin (EPO), exfoliative toxin,or exfoliatin, factors IX, factor VII, Factor IX, factor X, fibroblast growth factor (FGF), Fibrinogen, fibronectin, four-helix bundle albumen, G-CSF, glp-1, GM-CSF, glucocerebrosidase, gonad-stimulating hormone, somatomedin, growth factor receptors, grf, Shh, hemochrome, pHGF (hGF), r-hirudin, human growth hormone (hGH), the human serum albumin, ICAM-1, the ICAM-1 acceptor, LFA-1, the LFA-1 acceptor, Regular Insulin, insulin-like growth factor (IGF), IGF-I, IGF-II, Interferon, rabbit (IFN), IFN-α, IFN-β, IFN-γ, interleukin-(IL), IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, keratinocyte growth factor (KGF), lactoferrin, leukaemia inhibitory factor, luciferase, neural plain, neutrophil inhibitory factor (nif) (NIF), oncostatin M, bone morphogenic protein, oncoprotein, paracitonin, Rat parathyroid hormone 1-34, PD-ECSF, PDGF, peptide hormone, PTN, albumin A, Protein G, pth, pyrogenic exotoxin A, pyrogenic exotoxin B, pyrogenic exotoxin C, pyy, Relaxin, feritin, SCF, the atom synthetic proteins, soluble complement acceptor I, solubility I-CAM 1, soluble interleukin receptor, soluble TNF acceptor, somatomedin, Somat, tethelin, streptokinase, superantigen, staphyloentero-toxin, SEA, SEB, SEC1, SEC2, SEC3, SED, SEE, steroid hormone receptor, superoxide-dismutase, the toxic shock syndrome toxin, thymosin, the histiotype plasminogen activation factor, tumor growth factor (TGF), tumour necrosis factor, tumor necrosis factor alpha, tumor necrosis factor, Tumor Necrosis Factor Receptors (TNFR), VLA-4 albumen, VCAM-1 albumen, vascular endothelial growth factor (VEGF), urokinase, mos, ras, raf, met, p53, tat, fos, myc, jun, myb, rel, estrogen receptor, PgR, the testosterone acceptor, aldosterone receptor, ldl receptor and Kendall compound.Non-natural amino acid polypeptides also can with any polypeptide member homology of tethelin supergene family.
These modifications comprise is incorporated on the alpha-non-natural amino acid component of polypeptide other functional groups, and these functional groups are including (but not limited to) mark; Dyestuff; Polymkeric substance; Water-soluble polymers; The derivative of polyoxyethylene glycol; Photocrosslinking agent; Cytotoxic compound; Medicine; Affinity marker; The photoaffinity mark; Reactive compounds; Resin; Second protein or polypeptide or polypeptide analog; Antibody or antibody fragment; Metal chelator; Cofactor; Lipid acid; Carbohydrate; Polynucleotide; DNA; RNA; Antisense polynucleotide; Carbohydrate, water-soluble branch-shape polymer, cyclodextrin, biomaterial; Nanoparticle; Spin labeling; Fluorophore, metallic part; Radioactive segment; Novel functional group; With the covalently or non-covalently interactional group of other molecules; The light cage covers part; But the part that actinic radiation excites; Ligand; But photoisomerization part; Vitamin H; The vitamin H analogue; Be combined with the part of heavy atom; But the group of chemical cracking; But the group of photodestruciton; The side chain that prolongs; The sugar of carbon bond connection; Redox active agent; Amino thioic acid sulfoacid; The toxicity part; Through isotope-labeled part; The biophysics probe; The phosphorescence group; Chemiluminescent groups; The electron dense group; The magnetic group; Insert group; Chromophoric group; Energy transfer agent; Biologically active agent; Detectable label; Small molecules; Inhibition Yeast Nucleic Acid, radioactive nuleus thuja acid; Neutron capture agent; The derivative of vitamin H; Quantum dot; Nanometer is transmitted plain; Radioactivity is transmitted plain; Abzyme, activation composite activating agent, virus, adjuvant, aglycone, anaphylactogen, angiostatin, hormone antagonist, antioxidant, fit, guide RNA, Saponin/TSM, shuttle vectors, macromole, plan epitope, acceptor, reverse micelle with and any combination.
In addition, non-natural amino acid polypeptides can contain the part that can change other functional groups into, such as, only for instance, carbonyl, dicarbapentaborane or azanol.Figure 63 A explanation non-natural amino acid polypeptides changes the chemical transformation of the non-natural amino acid polypeptides that contains carbonyl or dicarbapentaborane into, and Figure 63 B explanation non-natural amino acid polypeptides changes the chemical transformation of the non-natural amino acid polypeptides that contains azanol into.The non-natural amino acid polypeptides that gained contains the non-natural amino acid polypeptides of carbonyl or dicarbapentaborane and contains azanol can be used for or incorporate into be used for preparing, method, composition, technology and the strategy of alpha-non-natural amino acid that purifying, sign and using described herein, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides any one in.Chemical part change into other functional groups (such as, only for instance, carbonyl, dicarbapentaborane or azanol) chemical transformation can use known technology of those skilled in the art and material to realize, such as (for example) in March, A DVANCEDO RGANICC HEMISTRYThe 5th edition, (Wiley 2001); And Carey and Sundberg, A DVANCEDO RGANICC HEMISTRYThe 4th edition, described in A volume and the B volume (Plenum 2000,2001), (all documents are all to incorporate into by reference).
Therefore, only for instance, any one the non-natural amino acid polypeptides that contains in the following amino acid can use the method and composition of describing further to modify herein:
Figure A20058004432801851
Wherein:
A is optionally, and when exist, be low-grade alkylidene, be substituted low-grade alkylidene, rudimentary cycloalkylidene, be substituted rudimentary cycloalkylidene, lower alkenylene, be substituted lower alkenylene, alkynylene, the assorted alkyl in rudimentary Asia, be substituted inferior mix alkyl, rudimentary inferior Heterocyclylalkyl, be substituted rudimentary inferior Heterocyclylalkyl, arylidene, be substituted arylidene, inferior heteroaryl, be substituted inferior heteroaryl, alkarylene, be substituted alkarylene, inferior aralkyl or be substituted inferior aralkyl;
B is optionally, and is the connexon that is selected from the group that is made up of following each group when exist: low-grade alkylidene, be substituted low-grade alkylidene, lower alkenylene, be substituted lower alkenylene, alkyl is mixed in rudimentary Asia, be substituted rudimentary Asia mix alkyl ,-O-,-O-(alkylidene group or be substituted alkylidene group)-,-S-,-S-(alkylidene group or be substituted alkylidene group)-,-S (O) k-(wherein k is 1,2 or 3) ,-S (O) k(alkylidene group or be substituted alkylidene group)-,-C (O)-,-NS (O) 2-,-OS (O) 2-,-C (O)-(alkylidene group or be substituted alkylidene group)-,-C (S)-,-C (S)-(alkylidene group or be substituted alkylidene group)-,-N (R ')-,-NR '-(alkylidene group or be substituted alkylidene group)-,-C (O) N (R ')-,-CON (R ')-(alkylidene group or be substituted alkylidene group)-,-CSN (R ')-,-CSN (R ')-(alkylidene group or be substituted alkylidene group)-,-N (R ') CO-(alkylidene group or be substituted alkylidene group)-,-N (R ') C (O) O-,-S (O) kN (R ')-,-N (R ') C (O) N (R ')-,-N (R ') C (S) N (R ')-,-N (R ') S (O) kN (R ')-,-N (R ')-N=,-C (R ')=N-,-C (R ')=N-N (R ')-,-C (R ')=N-N=,-C (R ') 2-N=N-and-C (R ') 2-N (R ')-N (R ')-, wherein R ' is H, alkyl independently of one another or is substituted alkyl;
J is Or
Figure A20058004432801862
R is H, alkyl, is substituted alkyl, cycloalkyl or is substituted cycloalkyl;
R " independently of one another for H, alkyl, " during group, two R " forms Heterocyclylalkyl according to circumstances to be substituted alkyl or protecting group, maybe when there being more than one R;
R 1Be H, amino protecting group, resin; And
R 2Be OH, ester protecting group, resin;
R 3With R 4Be H, halogen, low alkyl group or be substituted low alkyl group independently of one another, or R 3With R 4Or two R 3Group forms cycloalkyl or Heterocyclylalkyl according to circumstances;
Or-the A-B-J-R group common form comprise at least one carbonyl (comprising dicarbapentaborane), through protection carbonyl (comprising) or dicyclo or three cyclic rings alkyl or Heterocyclylalkyls through sheltering carbonyl (comprising) through sheltering dicarbapentaborane through the protection dicarbapentaborane;
Or-the J-R group common form comprise at least one carbonyl (comprising dicarbapentaborane), through protection carbonyl (comprising) or through sheltering monocycle or the double-ring cycloalkyl or the Heterocyclylalkyl of carbonyl (comprising) through sheltering dicarbapentaborane through the protection dicarbapentaborane;
Figure A20058004432801871
Wherein:
A is optionally, and when exist, be low-grade alkylidene, be substituted low-grade alkylidene, rudimentary cycloalkylidene, be substituted rudimentary cycloalkylidene, lower alkenylene, be substituted lower alkenylene, alkynylene, the assorted alkyl in rudimentary Asia, be substituted inferior mix alkyl, rudimentary inferior Heterocyclylalkyl, be substituted rudimentary inferior Heterocyclylalkyl, arylidene, be substituted arylidene, inferior heteroaryl, be substituted inferior heteroaryl, alkarylene, be substituted alkarylene, inferior aralkyl or be substituted inferior aralkyl;
B is optionally, and is the connexon that is selected from the group that is made up of following each group when exist: low-grade alkylidene, be substituted low-grade alkylidene, lower alkenylene, be substituted lower alkenylene, alkyl is mixed in rudimentary Asia, be substituted rudimentary Asia mix alkyl ,-O-,-O-(alkylidene group or be substituted alkylidene group)-,-S-,-S-(alkylidene group or be substituted alkylidene group)-,-S (O) k-(wherein k is 1,2 or 3) ,-S (O) k(alkylidene group or be substituted alkylidene group)-,-C (O)-,-NS (O) 2-,-OS (O) 2-,-C (O)-(alkylidene group or be substituted alkylidene group)-,-C (S)-,-C (S)-(alkylidene group or be substituted alkylidene group)-,-N (R ')-,-NR '-(alkylidene group or be substituted alkylidene group)-,-C (O) N (R ')-,-CON (R ')-(alkylidene group or be substituted alkylidene group)-,-CSN (R ')-,-CSN (R ')-(alkylidene group or be substituted alkylidene group)-,-N (R ') CO-(alkylidene group or be substituted alkylidene group)-,-N (R ') C (O) O-,-S (O) kN (R ')-,-N (R ') C (O) N (R ')-,-N (R ') C (S) N (R ')-,-N (R ') S (O) kN (R ')-,-N (R ')-N=,-C (R ')=N-,-C (R ')=N-N (R ')-,-C (R ')=N-N=,-C (R ') 2-N=N-and-C (R ') 2-N (R ')-N (R ')-, wherein R ' is H, alkyl independently of one another or is substituted alkyl;
R is H, alkyl, is substituted alkyl, cycloalkyl or is substituted cycloalkyl;
R 1Be H, amino protecting group, resin; And
R 2Be OH, ester protecting group, resin;
R 3With R 4Be H, halogen, low alkyl group or be substituted low alkyl group independently of one another, or R 3With R 4Or two R 3Group forms cycloalkyl or Heterocyclylalkyl according to circumstances;
R 5For H, alkyl, be substituted alkyl, thiazolinyl, be substituted thiazolinyl, alkynyl, be substituted alkynyl, alkoxyl group, be substituted alkoxyl group, alkyl alkoxy, be substituted alkyl alkoxy, polyoxyalkylene, be substituted polyoxyalkylene, aryl, be substituted aryl, heteroaryl, be substituted heteroaryl, alkaryl, be substituted alkaryl, aralkyl, be substituted aralkyl ,-(alkylidene group or be substituted alkylidene group)-ON (R ") 2,-(alkylidene group or be substituted alkylidene group)-C (O) SR " ,-(alkylidene group or be substituted alkylidene group)-S-S-(aryl or be substituted aryl) ,-C (O) R " ,-C (O) 2R " or-C (O) N (R ") 2, R wherein " independently of one another for hydrogen, alkyl, be substituted alkyl, thiazolinyl, be substituted thiazolinyl, alkoxyl group, be substituted alkoxyl group, aryl, be substituted aryl, heteroaryl, alkaryl, be substituted alkaryl, aralkyl, or be substituted aralkyl;
Or R 5Be L-X, wherein X is selected from the group that is made up of following each thing: mark; Dyestuff; Polymkeric substance; Water-soluble polymers; The derivative of polyoxyethylene glycol; Photocrosslinking agent; Cytotoxic compound; Medicine; Affinity marker; The photoaffinity mark; Reactive compounds; Resin; Second protein or polypeptide or polypeptide analog; Antibody or antibody fragment; Metal chelator; Cofactor; Lipid acid; Carbohydrate; Polynucleotide; DNA; RNA; Antisense polynucleotide; Carbohydrate, water-soluble branch-shape polymer, cyclodextrin, biomaterial; Nanoparticle; Spin labeling; Fluorophore, metallic part; Radioactive segment; Novel functional group; With the covalently or non-covalently interactional group of other molecules; The light cage covers part; But but the part that actinic radiation excites, ligand photoisomerization part; Vitamin H; The vitamin H analogue; Be combined with the part of heavy atom; But the group of chemical cracking; But the group of photodestruciton; The side chain that prolongs; The sugar of carbon bond connection; Redox active agent; Amino thioic acid sulfoacid; The toxicity part; Through isotope-labeled part; The biophysics probe; The phosphorescence group; Chemiluminescent groups; The electron dense group; The magnetic group; Insert group; Chromophoric group; Energy transfer agent; Biologically active agent; Detectable label; Small molecules; Inhibition Yeast Nucleic Acid, radioactive nuleus thuja acid; Neutron capture agent; The derivative of vitamin H; Quantum dot; Nanometer is transmitted plain; Radioactivity is transmitted plain; Abzyme, activation composite activating agent, virus, adjuvant, aglycone, anaphylactogen, angiostatin, hormone antagonist, antioxidant, fit, guide RNA, Saponin/TSM, shuttle vectors, macromole, plan epitope, acceptor, reverse micelle with and any combination; And L is optionally, and is the connexon that is selected from the group that is made up of following each group when existing: alkylidene group, be substituted alkylidene group, alkenylene, be substituted alkenylene ,-O-,-O-(alkylidene group or be substituted alkylidene group)-,-S-,-S-(alkylidene group or be substituted alkylidene group)-,-S (O) k-(wherein k is 1,2 or 3) ,-S (O) k(alkylidene group or be substituted alkylidene group)-,-C (O)-,-C (O)-(alkylidene group or be substituted alkylidene group)-,-C (S)-,-C (S)-(alkylidene group or be substituted alkylidene group)-,-N (R ')-,-NR '-(alkylidene group or be substituted alkylidene group)-,-C (O) N (R ')-,-CON (R ')-(alkylidene group or be substituted alkylidene group)-,-CSN (R ')-,-CSN (R ')-(alkylidene group or be substituted alkylidene group)-,-N (R ') CO-(alkylidene group or be substituted alkylidene group)-,-N (R ') C (O) O-,-(alkylidene group or be substituted alkylidene group)-O-N=CR '-,-(alkylidene group or be substituted alkylidene group)-C (O) NR '-(alkylidene group or be substituted alkylidene group)-,-(alkylidene group or be substituted alkylidene group)-S (O) k-(alkylidene group or be substituted alkylidene group)-S-,-(alkylidene group or be substituted alkylidene group)-S-S-,-S (O) kN (R ')-,-N (R ') C (O) N (R ')-,-N (R ') C (S) N (R ')-,-N (R ') S (O) kN (R ')-,-N (R ')-N=,-C (R ')=N-,-C (R ')=N-N (R ')-,-C (R ')=N-N=,-C (R ') 2-N=N-and-C (R ') 2-N (R ')-N (R ')-, wherein R ' is H, alkyl independently of one another or is substituted alkyl;
Figure A20058004432801891
Wherein:
A is optionally, and when exist, be low-grade alkylidene, be substituted low-grade alkylidene, rudimentary cycloalkylidene, be substituted rudimentary cycloalkylidene, lower alkenylene, be substituted lower alkenylene, alkynylene, the assorted alkyl in rudimentary Asia, be substituted inferior mix alkyl, rudimentary inferior Heterocyclylalkyl, be substituted rudimentary inferior Heterocyclylalkyl, arylidene, be substituted arylidene, inferior heteroaryl, be substituted inferior heteroaryl, alkarylene, be substituted alkarylene, inferior aralkyl or be substituted inferior aralkyl;
B is optionally, and is the connexon that is selected from the group that is made up of following each group when exist: low-grade alkylidene, be substituted low-grade alkylidene, lower alkenylene, be substituted lower alkenylene, alkyl is mixed in rudimentary Asia, be substituted rudimentary Asia mix alkyl ,-O-,-O-(alkylidene group or be substituted alkylidene group)-,-S-,-S-(alkylidene group or be substituted alkylidene group)-,-S (O) k-(wherein k is 1,2 or 3) ,-S (O) k(alkylidene group or be substituted alkylidene group)-,-C (O)-,-NS (O) 2-,-OS (O) 2-,-C (O)-(alkylidene group or be substituted alkylidene group)-,-C (S)-,-C (S)-(alkylidene group or be substituted alkylidene group)-,-N (R ')-,-NR '-(alkylidene group or be substituted alkylidene group)-,-C (O) N (R ')-,-CON (R ')-(alkylidene group or be substituted alkylidene group)-,-CSN (R ')-,-CSN (R ')-(alkylidene group or be substituted alkylidene group)-,-N (R ') CO-(alkylidene group or be substituted alkylidene group)-,-N (R ') C (O) O-,-S (O) kN (R ')-,-N (R ') C (O) N (R ')-,-N (R ') C (S) N (R ')-,-N (R ') S (O) kN (R ')-,-N (R ')-N=,-C (R ')=N-,-C (R ')=N-N (R ')-,-C (R ')=N-N=,-C (R ') 2-N=N-and-C (R ') 2-N (R ')-N (R ')-, wherein R ' is H, alkyl independently of one another or is substituted alkyl;
K is-NR 6R 7Or-N=CR 6R 7
R is H, alkyl, is substituted alkyl, cycloalkyl or is substituted cycloalkyl;
R 1Be H, amino protecting group, resin; And
R 2Be OH, ester protecting group, resin;
R 3With R 4Be H, halogen, low alkyl group or be substituted low alkyl group independently of one another, or R 3With R 4Or two R 3Group forms cycloalkyl or Heterocyclylalkyl according to circumstances;
R 6With R 7Be selected from the group that forms by following each group independently of one another: H, alkyl, be substituted alkyl, thiazolinyl, be substituted thiazolinyl, alkoxyl group, be substituted alkoxyl group, polyoxyalkylene, be substituted polyoxyalkylene, aryl, be substituted aryl, heteroaryl, be substituted heteroaryl, alkaryl, be substituted alkaryl, aralkyl and be substituted aralkyl ,-C (O) R " ,-C (O) 2R " ,-C (O) N (R ") 2, R wherein " independently of one another for hydrogen, alkyl, be substituted alkyl, thiazolinyl, be substituted thiazolinyl, alkoxyl group, be substituted alkoxyl group, aryl, be substituted aryl, heteroaryl, alkaryl, be substituted alkaryl, aralkyl or be substituted aralkyl; Or R 6Or R 7Be L-X, wherein
X is selected from the group that is made up of following each thing: mark; Dyestuff; Polymkeric substance; Water-soluble polymers; The derivative of polyoxyethylene glycol; Photocrosslinking agent; Cytotoxic compound; Medicine; Affinity marker; The photoaffinity mark; Reactive compounds; Resin; Second protein or polypeptide or polypeptide analog; Antibody or antibody fragment; Metal chelator; Cofactor; Lipid acid; Carbohydrate; Polynucleotide; DNA; RNA; Antisense polynucleotide; Carbohydrate, water-soluble branch-shape polymer, cyclodextrin, biomaterial; Nanoparticle; Spin labeling; Fluorophore, metallic part; Radioactive segment; Novel functional group; With the covalently or non-covalently interactional group of other molecules; The light cage covers part; But the part that actinic radiation excites; Ligand; But photoisomerization part; Vitamin H; The vitamin H analogue; Be combined with the part of heavy atom; But the group of chemical cracking; But the group of photodestruciton; The side chain that prolongs; The sugar of carbon bond connection; Redox active agent; Amino thioic acid sulfoacid; The toxicity part; Through isotope-labeled part; The biophysics probe; The phosphorescence group; Chemiluminescent groups; The electron dense group; The magnetic group; Insert group; Chromophoric group; Energy transfer agent; Biologically active agent; Detectable label; Small molecules; Inhibition Yeast Nucleic Acid, radioactive nuleus thuja acid; Neutron capture agent; The derivative of vitamin H; Quantum dot; Nanometer is transmitted plain; Radioactivity is transmitted plain; Abzyme, activation composite activating agent, virus, adjuvant, aglycone, anaphylactogen, angiostatin, hormone antagonist, antioxidant, fit, guide RNA, Saponin/TSM, shuttle vectors, macromole, plan epitope, acceptor, reverse micelle with and any combination; And L is optionally, and is the connexon that is selected from the group that is made up of following each group when existing: alkylidene group, be substituted alkylidene group, alkenylene, be substituted alkenylene ,-O-,-O-(alkylidene group or be substituted alkylidene group)-,-S-,-S-(alkylidene group or be substituted alkylidene group)-,-S (O) k-(wherein k is 1,2 or 3) ,-S (O) k(alkylidene group or be substituted alkylidene group)-,-C (O)-,-C (O)-(alkylidene group or be substituted alkylidene group)-,-C (S)-,-C (S)-(alkylidene group or be substituted alkylidene group)-,-N (R ')-,-NR '-(alkylidene group or be substituted alkylidene group)-,-C (O) N (R ')-,-CON (R ')-(alkylidene group or be substituted alkylidene group)-,-CSN (R ')-,-CSN (R ')-(alkylidene group or be substituted alkylidene group)-,-N (R ') CO-(alkylidene group or be substituted alkylidene group)-,-N (R ') C (O) O-,-S (O) kN (R ')-,-N (R ') C (O) N (R ')-,-N (R ') C (S) N (R ')-,-N (R ') S (O) kN (R ')-,-N (R ')-N=,-C (R ')=N-,-C (R ')=N-N (R ')-,-C (R ')=N-N=,-C (R ') 2-N=N-and-C (R ') 2-N (R ')-N (R ')-, wherein R ' is H, alkyl independently of one another or is substituted alkyl;
Figure A20058004432801901
Wherein:
A is optionally, and when exist, be low-grade alkylidene, be substituted low-grade alkylidene, rudimentary cycloalkylidene, be substituted rudimentary cycloalkylidene, lower alkenylene, be substituted lower alkenylene, alkynylene, the assorted alkyl in rudimentary Asia, be substituted inferior mix alkyl, rudimentary inferior Heterocyclylalkyl, be substituted rudimentary inferior Heterocyclylalkyl, arylidene, be substituted arylidene, inferior heteroaryl, be substituted inferior heteroaryl, alkarylene, be substituted alkarylene, inferior aralkyl or be substituted inferior aralkyl;
R is H, alkyl, is substituted alkyl, cycloalkyl or is substituted cycloalkyl;
R 1Be H, amino protecting group, resin, amino acid, polypeptide or polynucleotide;
R 2Be OH, ester protecting group, resin, amino acid, polypeptide or polynucleotide;
X 1Be C, S or S (O); And L is alkylidene group, is substituted alkylidene group, N (R ') (alkylidene group) or N (R ') (being substituted alkylidene group) that wherein R ' is H, alkyl independently of one another or is substituted alkyl; Or
Figure A20058004432801911
Wherein:
A is optionally, and when exist, be low-grade alkylidene, be substituted low-grade alkylidene, rudimentary cycloalkylidene, be substituted rudimentary cycloalkylidene, lower alkenylene, be substituted lower alkenylene, alkynylene, the assorted alkyl in rudimentary Asia, be substituted inferior mix alkyl, rudimentary inferior Heterocyclylalkyl, be substituted rudimentary inferior Heterocyclylalkyl, arylidene, be substituted arylidene, inferior heteroaryl, be substituted inferior heteroaryl, alkarylene, be substituted alkarylene, inferior aralkyl or be substituted inferior aralkyl;
M is-C (R 3)-,
Figure A20058004432801912
Figure A20058004432801913
Wherein (a) indication and A group bond and (b) indication and carbonyl bond out of the ordinary, R 3With R 4Be independently selected from H, halogen, alkyl, be substituted alkyl, cycloalkyl or be substituted cycloalkyl, or R 3With R 4Or two R 3Group or two R 4Group forms cycloalkyl or Heterocyclylalkyl according to circumstances;
R is H, halogen, alkyl, is substituted alkyl, cycloalkyl or is substituted cycloalkyl;
T 3Be a key, C (R) (R), O or S, and R is H, halogen, alkyl, is substituted alkyl, cycloalkyl or is substituted cycloalkyl;
R 1Be H, amino protecting group, resin, amino acid, polypeptide or polynucleotide; And
R 2Be OH, ester protecting group, resin, amino acid, polypeptide or polynucleotide.
The one side of the method and composition of Miao Shuing is the composition of the polypeptide that comprises at least a alpha-non-natural amino acid with at least one (including (but not limited to) at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or more than at least ten or ten) posttranslational modification herein.Alpha-non-natural amino acid through posttranslational modification can be identical or different, including (but not limited to) the different loci that can exist in the polypeptide that comprises different alpha-non-natural amino acids through posttranslational modification more than 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20 or 20 more than 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20 or 20.On the other hand, the composition specific amino acids that comprises at least one (but being less than all) of existing in the polypeptide wherein polypeptide that the alpha-non-natural amino acid through posttranslational modification replaces.For given polypeptide with more than one alpha-non-natural amino acid through posttranslational modification, alpha-non-natural amino acid through posttranslational modification can identical or differently (comprise, but be not limited to, polypeptide can comprise two or more dissimilar alpha-non-natural amino acids through posttranslational modification, maybe can comprise two identical alpha-non-natural amino acids through posttranslational modification).For given polypeptide with plural alpha-non-natural amino acid through posttranslational modification, can be identical, different or be the combination through the alpha-non-natural amino acid of posttranslational modification different of the alpha-non-natural amino acid through posttranslational modification of a plurality of identical type through the alpha-non-natural amino acid of posttranslational modification with at least one.
A. the method that is used for the posttranslational modification non-natural amino acid polypeptides: contain carbonyl alpha-non-natural amino acid and the reaction that contains the reagent of azanol
The natural amino acid whose side chain shortage height electrophilicity site that exists.Therefore, incorporating alpha-non-natural amino acid with the side chain that contains electrophilic reagent (comprise (only for instance) contain such as the carbonyl of ketone or aldehyde or the amino acid of dicarbapentaborane) into makes this side chain of locus specificity derivatize of the nucleophilic attack by carbonyl or dicarbapentaborane become possibility.The attack nucleophilic reagent is under the situation of azanol therein, can produce oxime deutero-protein.The method that is used for derivatize and/or further modifies can be used on before the derivatize step or after the derivatize step polypeptide of purifying carry out.In addition, be used for derivatize and/or the method for further modifying can be used on these modify before or after synthetic polymer, polysaccharide or the polynucleotide of purifying carry out.In addition, the derivatize step can take place to the slight alkalinity condition in appropriateness acidity, and described condition is including (for example) under the pH value between about 2-8, or the pH value between about 4-8 down.
Reaction according to the polypeptide that contains carbonyl or dicarbapentaborane and the molecule that replaces through azanol comes the method for derivatize polypeptide to have tangible advantage.The first, azanol in the pH of 2-8 value scope (and in other embodiments in the pH of 4-8 value scope) experiences condensation with the compound that contains carbonyl or dicarbapentaborane to produce the oxime adducts.It is under these conditions, natural that to have amino acid whose side chain be anergy.The second, this selective chemical makes locus specificity derivatize recombinant protein become possibility: nowadays deutero-protein can be prepared as definite homology product.The 3rd, the needed mild conditions of reaction of the azanol of realization description herein and the polypeptide of describing herein that contains carbonyl or dicarbapentaborane can reversibly destroy the tertiary structure (certain, unless wherein Fan Ying purpose is this tertiary structure of destruction) of polypeptide usually.At last, as if although the azanol group is amino by the intestinal bacteria metabolism, azanol is created in oxime adducts stable under the biotic condition with the condensation that contains the molecule of carbonyl or dicarbapentaborane.
Only for instance, following alpha-non-natural amino acid is to can be used for further modifying the amino acid whose type that contains carbonyl or dicarbapentaborane of the responding property of reagent that contains azanol of description herein of the non-natural amino acid polypeptides that contains carbonyl or dicarbapentaborane:
Figure A20058004432801931
Wherein:
A is optionally, and when exist, be low-grade alkylidene, be substituted low-grade alkylidene, rudimentary cycloalkylidene, be substituted rudimentary cycloalkylidene, lower alkenylene, be substituted lower alkenylene, alkynylene, the assorted alkyl in rudimentary Asia, be substituted inferior mix alkyl, rudimentary inferior Heterocyclylalkyl, be substituted rudimentary inferior Heterocyclylalkyl, arylidene, be substituted arylidene, inferior heteroaryl, be substituted inferior heteroaryl, alkarylene, be substituted alkarylene, inferior aralkyl or be substituted inferior aralkyl;
B is optionally, and is the connexon that is selected from the group that is made up of following each group when exist: low-grade alkylidene, be substituted low-grade alkylidene, lower alkenylene, be substituted lower alkenylene, alkyl is mixed in rudimentary Asia, be substituted rudimentary Asia mix alkyl ,-O-,-O-(alkylidene group or be substituted alkylidene group)-,-S-,-S-(alkylidene group or be substituted alkylidene group)-,-S (O) k-(wherein k is 1,2 or 3) ,-S (O) k(alkylidene group or be substituted alkylidene group)-,-C (O)-,-NS (O) 2-,-OS (O) 2-,-C (O)-(alkylidene group or be substituted alkylidene group)-,-C (S)-,-C (S)-(alkylidene group or be substituted alkylidene group)-,-N (R ')-,-NR '-(alkylidene group or be substituted alkylidene group)-,-C (O) N (R ')-,-CON (R ')-(alkylidene group or be substituted alkylidene group)-,-CSN (R ')-,-CSN (R ')-(alkylidene group or be substituted alkylidene group)-,-N (R ') CO-(alkylidene group or be substituted alkylidene group)-,-N (R ') C (O) O-,-S (O) kN (R ')-,-N (R ') C (O) N (R ')-,-N (R ') C (S) N (R ')-,-N (R ') S (O) kN (R ')-,-N (R ')-N=,-C (R ')=N-,-C (R ')=N-N (R ')-,-C (R ')=N-N=,-C (R ') 2-N=N-and-C (R ') 2-N (R ')-N (R ')-, wherein R ' is H, alkyl independently of one another or is substituted alkyl;
J is
Figure A20058004432801941
Or
Figure A20058004432801942
R is H, alkyl, is substituted alkyl, cycloalkyl or is substituted cycloalkyl;
R " independently of one another for H, alkyl, " during group, two R " forms Heterocyclylalkyl according to circumstances to be substituted alkyl or protecting group, maybe when there being more than one R;
R 1Be H, amino protecting group, resin; And
R 2Be OH, ester protecting group, resin;
R 3With R 4Be H, halogen, low alkyl group or be substituted low alkyl group independently of one another, or R 3With R 4Or two R 3Group forms cycloalkyl or Heterocyclylalkyl according to circumstances;
Or-the A-B-J-R group common form comprise at least one carbonyl (comprising dicarbapentaborane), through protection carbonyl (comprising) or dicyclo or three cyclic rings alkyl or Heterocyclylalkyls through sheltering carbonyl (comprising) through sheltering dicarbapentaborane through the protection dicarbapentaborane;
Or-the J-R group common form comprise at least one carbonyl (comprising dicarbapentaborane), through protection carbonyl (comprising) or through sheltering monocycle or the double-ring cycloalkyl or the Heterocyclylalkyl of carbonyl (comprising) through sheltering dicarbapentaborane through the protection dicarbapentaborane.
In certain embodiments, formula (I) compound has reactivity with azanol under appropriate acidic conditions in the aqueous solution.In certain embodiments, described acidic conditions is pH 2 to 8.
Only for instance, be aforementioned purpose, formula (I) compound comprises the compound with following structure:
Figure A20058004432801943
Wherein:
A is optionally, and when exist, be low-grade alkylidene, be substituted low-grade alkylidene, rudimentary cycloalkylidene, be substituted rudimentary cycloalkylidene, lower alkenylene, be substituted lower alkenylene, alkynylene, the assorted alkyl in rudimentary Asia, be substituted inferior mix alkyl, rudimentary inferior Heterocyclylalkyl, be substituted rudimentary inferior Heterocyclylalkyl, arylidene, be substituted arylidene, inferior heteroaryl, be substituted inferior heteroaryl, alkarylene, be substituted alkarylene, inferior aralkyl or be substituted inferior aralkyl;
R is H, alkyl, is substituted alkyl, cycloalkyl or is substituted cycloalkyl;
R 1Be H, amino protecting group, resin, amino acid, polypeptide or polynucleotide;
R 2Be OH, ester protecting group, resin, amino acid, polypeptide or polynucleotide;
X 1Be C, S or S (O); And L is a key, alkylidene group, is substituted alkylidene group, N (R ') (alkylidene group) or N (R ') (being substituted alkylidene group) that wherein R ' is H, alkyl independently of one another or is substituted alkyl.
Only in further example, be aforementioned purpose, formula (I) compound comprises the have formula compound of structure of (XXXX):
Wherein:
A is optionally, and when exist, be low-grade alkylidene, be substituted low-grade alkylidene, rudimentary cycloalkylidene, be substituted rudimentary cycloalkylidene, lower alkenylene, be substituted lower alkenylene, alkynylene, the assorted alkyl in rudimentary Asia, be substituted inferior mix alkyl, rudimentary inferior Heterocyclylalkyl, be substituted rudimentary inferior Heterocyclylalkyl, arylidene, be substituted arylidene, inferior heteroaryl, be substituted inferior heteroaryl, alkarylene, be substituted alkarylene, inferior aralkyl or be substituted inferior aralkyl;
M is-C (R 3)-,
Figure A20058004432801952
Figure A20058004432801953
Wherein (a) indication and A group bond and (b) indication and carbonyl bond out of the ordinary, R 3With R 4Be independently selected from H, halogen, alkyl, be substituted alkyl, cycloalkyl or be substituted cycloalkyl, or R 3With R 4Or two R 3Group or two R 4Group forms cycloalkyl or Heterocyclylalkyl according to circumstances;
R is H, halogen, alkyl, is substituted alkyl, cycloalkyl or is substituted cycloalkyl;
T 3Be a key, C (R) (R), O or S, and R is H, halogen, alkyl, is substituted alkyl, cycloalkyl or is substituted cycloalkyl;
R 1Be H, amino protecting group, resin, amino acid, polypeptide or polynucleotide; And
R 2Be OH, ester protecting group, resin, amino acid, polypeptide or polynucleotide.
Comprise the alpha-non-natural amino acid that these contain carbonyl or dicarbapentaborane polypeptide type in fact and unrestricted, thereby be positioned at azanol reagent on the polypeptide and can and do not produce the modified alpha-non-natural amino acid (certainly, unless if this destruction is the purpose of reaction) of gained of the tertiary structure of destroying polypeptide with carbonyl or dicarbapentaborane reaction as long as contain the alpha-non-natural amino acid of carbonyl or dicarbapentaborane.
Only for instance, the reagent that below contains azanol is for to responding property of describing herein of alpha-non-natural amino acid that contains carbonyl or dicarbapentaborane and the type that can be used for further modifying the reagent that contains azanol of the non-natural amino acid polypeptides that contains carbonyl or dicarbapentaborane:
Figure A20058004432801961
Wherein:
X independently of one another for H, alkyl, be substituted alkyl, thiazolinyl, be substituted thiazolinyl, alkynyl, be substituted alkynyl, alkoxyl group, be substituted alkoxyl group, alkyl alkoxy, be substituted alkyl alkoxy, polyoxyalkylene, be substituted polyoxyalkylene, aryl, be substituted aryl, heteroaryl, be substituted heteroaryl, alkaryl, be substituted alkaryl, aralkyl, be substituted aralkyl ,-(alkylidene group or be substituted alkylidene group)-ON (R ") 2,-(alkylidene group or be substituted alkylidene group)-C (O) SR " ,-(alkylidene group or be substituted alkylidene group)-S-S-(aryl or be substituted aryl) ,-C (O) R " ,-C (O) 2R " or-C (O) N (R ") 2, R wherein " independently of one another for hydrogen, alkyl, be substituted alkyl, thiazolinyl, be substituted thiazolinyl, alkoxyl group, be substituted alkoxyl group, aryl, be substituted aryl, heteroaryl, alkaryl, be substituted alkaryl, aralkyl or be substituted aralkyl;
Or X is selected from the group that is made up of following each thing independently of one another: mark; Dyestuff; Polymkeric substance; Water-soluble polymers; The derivative of polyoxyethylene glycol; Photocrosslinking agent; Cytotoxic compound; Medicine; Affinity marker; The photoaffinity mark; Reactive compounds; Resin; Second protein or polypeptide or polypeptide analog; Antibody or antibody fragment; Metal chelator; Cofactor; Lipid acid; Carbohydrate; Polynucleotide; DNA; RNA; Antisense polynucleotide; Carbohydrate, water-soluble branch-shape polymer, cyclodextrin, biomaterial; Nanoparticle; Spin labeling; Fluorophore, metallic part; Radioactive segment; Novel functional group; With the covalently or non-covalently interactional group of other molecules; The light cage covers part; But the part that actinic radiation excites; Ligand; But photoisomerization part; Vitamin H; The vitamin H analogue; Be combined with the part of heavy atom; But the group of chemical cracking; But the group of photodestruciton; The side chain that prolongs; The sugar of carbon bond connection; Redox active agent; Amino thioic acid sulfoacid; The toxicity part; Through isotope-labeled part; The biophysics probe; The phosphorescence group; Chemiluminescent groups; The electron dense group; The magnetic group; Insert group; Chromophoric group; Energy transfer agent; Biologically active agent; Detectable label; Small molecules; Inhibition Yeast Nucleic Acid, radioactive nuleus thuja acid; Neutron capture agent; The derivative of vitamin H; Quantum dot; Nanometer is transmitted plain; Radioactivity is transmitted plain; Abzyme, activation composite activating agent, virus, adjuvant, aglycone, anaphylactogen, angiostatin, hormone antagonist, antioxidant, fit, guide RNA, Saponin/TSM, shuttle vectors, macromole, plan epitope, acceptor, reverse micelle with and any combination;
L is selected from the group that is made up of following each group independently of one another: alkylidene group, be substituted alkylidene group, alkenylene, be substituted alkenylene ,-O-,-O-(alkylidene group or be substituted alkylidene group)-,-S-,-S-(alkylidene group or be substituted alkylidene group)-,-S (O) k-(wherein k is 1,2 or 3) ,-S (O) k(alkylidene group or be substituted alkylidene group)-,-C (O)-,-C (O)-(alkylidene group or be substituted alkylidene group)-,-C (S)-,-C (S)-(alkylidene group or be substituted alkylidene group)-,-N (R ')-,-NR '-(alkylidene group or be substituted alkylidene group)-,-C (O) N (R ')-,-CON (R ')-(alkylidene group or be substituted alkylidene group)-,-(alkylidene group or be substituted alkylidene group) NR ' C (O) O-(alkylidene group or be substituted alkylidene group)-,-O-CON (R ')-(alkylidene group or be substituted alkylidene group)-,-CSN (R ')-,-CSN (R ')-(alkylidene group or be substituted alkylidene group)-,-N (R ') CO-(alkylidene group or be substituted alkylidene group)-,-N (R ') C (O) O-,-N (R ') C (O) O-(alkylidene group or be substituted alkylidene group)-,-S (O) kN (R ')-,-N (R ') C (O) N (R ')-,-N (R ') C (O) N (R ')-(alkylidene group or be substituted alkylidene group)-,-N (R ') C (S) N (R ')-,-N (R ') S (O) kN (R ')-,-N (R ')-N=,-C (R ')=N-,-C (R ')=N-N (R ')-,-C (R ')=N-N=,-C (R ') 2-N=N-and-C (R ') 2-N (R ')-N (R ')-;
L 1For optionally, and when existing be-C (R ') p-NR '-C (O) O-(alkylidene group or be substituted alkylidene group)-, wherein p is 0,1 or 2; R ' is H, alkyl independently of one another or is substituted alkyl;
W is-N (R 8) 2, R wherein 8Be H or amino protecting group independently of one another; And n is 1 to 3;
Its restricted condition is L-L 1-W provides at least one azanol base that can react with the carbonyl (comprising dicarbapentaborane) on alpha-non-natural amino acid or " modified or not modified " non-natural amino acid polypeptides jointly.
In some embodiment of formula (XIX) compound, X be comprise alkyl, be substituted alkyl, thiazolinyl, be substituted thiazolinyl, alkynyl, be substituted alkynyl, alkoxyl group, be substituted alkoxyl group, alkyl alkoxy, be substituted alkyl alkoxy, polyoxyalkylene, be substituted polyoxyalkylene, aryl, be substituted aryl, heteroaryl, be substituted heteroaryl, alkaryl, be substituted alkaryl, aralkyl or be substituted the polymkeric substance of aralkyl.In some embodiment of formula (XIX) compound, X is the polymkeric substance that comprises polyoxyalkylene or be substituted polyoxyalkylene.In some embodiment of formula (XIX) compound, X is for comprising-[(alkylidene group or be substituted alkylidene group)-O-(hydrogen, alkyl or be substituted alkyl)] xThe polymkeric substance of (wherein x is 20-10,000).In some embodiment of formula (XIX) compound, X is the m-PEG with the molecular weight in the scope of 2 KDa to 40 KDa.In some embodiment of formula (XIX) compound, X is the biologically active agent that is selected from the group that is made up of peptide, protein, enzyme, antibody, medicine, dyestuff, lipid, nucleosides, oligonucleotide, cell, virus, liposome, particulate and micella.In some embodiment of formula (XIX) compound, X is the medicine that is selected from the group that is made up of microbiotic, mycocide, antiviral agent, antiphlogistic, antineoplastic agent, cardiovascular agents, antianxiety agent, hormone, somatomedin and steroid agent.In some embodiment of formula (XIX) compound, X is the enzyme that is selected from the group that is made up of horseradish peroxidase, alkaline phosphatase, beta-galactosidase enzymes and glucose oxidase.In some embodiment of formula (XIX) compound, X is the detectable label that is selected from the group that is grouped into by fluorescence part, phosphorescence part, chemiluminescent moiety, chelating moiety, electron dense part, magnetic part, insertion portion, radioactive segment, chromophoric group part and energy transfer portion.In some embodiment of formula (XIX) compound, L be selected from by-N (R ') CO-(alkylidene group or be substituted alkylidene group)-,-CON (R ')-(alkylidene group or be substituted alkylidene group)-,-N (R ') C (O) N (R ')-(alkylidene group or be substituted alkylidene group)-,-O-CON (R ')-(alkylidene group or be substituted alkylidene group)-,-O-(alkylidene group or be substituted alkylidene group)-,-C (O) N (R ')-and-group of N (R ') C (O) O-(alkylidene group or be substituted alkylidene group)-form.
In some embodiment of formula (XIX) compound, be the compound of structure with formula (XX):
X-L-O-NH 2
(XX).。
In some embodiment of formula (XX) compound, for being selected from the compound of the group that forms by following each thing:
Figure A20058004432801981
Figure A20058004432801982
And
Wherein in other embodiments, these m-PEG or PEG group have the molecular weight in the scope of 5 kDa to 30 kDa.
In some embodiment of formula (XIX) compound, be the compound of structure with formula (XXI):
Figure A20058004432801991
In some embodiment of formula (XXI) compound, for being selected from the compound of the group that forms by following each thing:
Figure A20058004432801992
Figure A20058004432801993
And
Figure A20058004432801994
In some embodiment of formula (XIX) compound, be the compound of structure with formula (XXII):
Figure A20058004432801995
In some embodiment of formula (XXII) compound, L for-(alkylidene group or be substituted alkylidene group)-N (R ') C (O) O-(alkylidene group or be substituted alkylidene group)-.In some embodiment of formula (XXII) compound, be the compound of structure with formula (XXIII):
Figure A20058004432802001
Wherein in other embodiment of formula (XXII) compound, these m-PEG groups have the molecular weight in the scope of 5kDa to 30kDa.
In some embodiment of formula (XIX) compound, be the compound of structure with formula (XXIV):
Figure A20058004432802002
In some embodiment of formula (XXIV) compound, L for-(alkylidene group or be substituted alkylidene group)-N (R ') C (O) O-(alkylidene group or be substituted alkylidene group)-or-N (R ') C (O) O-(alkylidene group or be substituted alkylidene group)-.In some embodiment of formula (XXIV) compound, be the compound of structure with formula (XXV):
Wherein in other embodiment of formula (XXIV) compound, these m-PEG groups have the molecular weight in the scope of 5kDa to 30kDa.
In some embodiment of formula (XIX) compound, be the compound of structure with formula (XXVI):
Figure A20058004432802011
R wherein 10Be H or amino protecting group independently of one another.
In some embodiment of formula (XXVI) compound, polyoxyalkylene is PEG.In other embodiment of formula (XXVI) compound, the PEG group has the molecular weight in the scope of 5kDa to 30kDa.Be compound in another embodiment of formula (XXVI) compound corresponding to following formula:
Figure A20058004432802012
Being used for that azanol is coupled to three illustrative embodiment that contain the method on the carbonyl alpha-non-natural amino acid on the polypeptide is and is shown in Fig. 7.In these illustrative embodiment, azanol deutero-reagent is added in the buffered soln (pH 2-8) of the non-natural amino acid polypeptides that contains carbonyl.A plurality of hours skies are at the most carried out in reaction at ambient temperature.For quickening to engage, add those additives such as oblatio among Fig. 8; These compounds are also referred to as promotor in this article.In certain embodiments, promotor or additive can base catalysiss.Come the purifying gained to contain the oxime non-natural amino acid polypeptides by HPLC, FPLC or size exclusion chromatography.
In one embodiment, a plurality of connexon chemistry can react with the non-natural amino acid polypeptides locus specificity ground through carbonyl or dicarbapentaborane replacement.In one embodiment, the connexon method utilization of describing herein contains azanol functional group's connexon (simple function, difunctionality or multi-functional) at least one connexon end.Azanol deutero-connexon produces with the proteinic condensation that replaces through ketone stablizes the oxime key.Difunctionality and/or multifunctional connexon are (for example, azanol with one or more other binding chemistry) (for example allows differing molecular, other protein, polymkeric substance or small molecules) locus specificity is connected on the non-natural amino acid polypeptides, and simple function connexon (replacing through azanol on all ends) promotes the locus specificity dimerization or the oligomerization of non-natural amino acid polypeptides.By this connexon strategy is combined with the in vivo translation technology of describing, make the proteinic three-dimensional structure that describes in detail through chemical refining become possibility herein.
Be the method for the polypeptide that is used for the amino acid that derivatize comprises formula I-XVIII, XXX-XXXIV (A and B) or XXXX-XXXXIII (it comprises any minor or specific compound in the category that is in formula I-XVIII, XXX-XXXIV (A and B) or XXXX-XXXXIII) in certain embodiments, wherein said method comprises makes the amino acid whose polypeptide that comprises at least one formula I-XVIII, XXX-XXXIV (A and B) or XXXX-XXXXIII contact with the reagent of formula (XIX).In certain embodiments, polypeptide is purified before or after the reagent with formula (XIX) contacts.Be in other embodiments comprise at least one corresponding to formula (XI) contain the amino acid whose gained of the oxime polypeptide of deriving, and in other embodiments, these polypeptide under appropriate acidic conditions in the aqueous solution stable at least 1 month of deriving.In other embodiments, these are derived polypeptide stable 2 weeks under appropriate acidic conditions at least.In other embodiments, these polypeptide of deriving were stablized 5 days under appropriate acidic conditions at least.In other embodiments, described condition is pH 2 to 8.In certain embodiments, keep the deriving tertiary structure of polypeptide.In other embodiments, these derivatizes of polypeptide further comprise the polypeptide chain of deriving are connected on another polypeptide.In other embodiments, these polypeptides derived and the treatment protein homology that is selected from the group that forms by following each thing: α-1 antitrypsin, angiostatin, AHF, antibody, lipophorin, apoprotein, atrionatriuretic factor, atrial natriuretic polypeptins, atrial natriuretic peptide, the C-X-C chemokine, T39765, NAP-2, ENA-78, gro-a, gro-b, gro-c, IP-10, GCP-2, NAP-4, SDF-1, PF4, MIG, calcitonin, the c-kit ligand, cytokine, the CC chemokine, MCP-1, MCP-2, monocyte chemoattractant protein-3, monocyte inflammatory protein-1 α, monocyte inflammatory protein-i β, RANTES, 1309, R83915, R91733, HCC1, T58847, D31065, T64262, CD40, the CD40 ligand, the c-kit ligand, collagen protein, G CFS (CSF), complement factor 5a, complement inhibitor, complement receptor 1, cytokine, epithelium neutrophilic granulocyte activation peptide-78, MIP-16, MCP-1, Urogastron (EGF), epithelium neutrophilic granulocyte activation peptide, erythropoietin (EPO), exfoliative toxin,or exfoliatin, factors IX, factor VII, Factor IX, factor X, fibroblast growth factor (FGF), Fibrinogen, fibronectin, four-helix bundle albumen, G-CSF, glp-1, GM-CSF, glucocerebrosidase, gonad-stimulating hormone, somatomedin, growth factor receptors, grf, Shh, hemochrome, pHGF (hGF), r-hirudin, human growth hormone (hGH), the human serum albumin, ICAM-1, the ICAM-1 acceptor, LFA-1, the LFA-1 acceptor, Regular Insulin, insulin-like growth factor (IGF), IGF-I, IGF-II, Interferon, rabbit (IFN), IFN-α, IFN-β, IFN-γ, interleukin-(IL), IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, keratinocyte growth factor (KGF), lactoferrin, leukaemia inhibitory factor, luciferase, neural plain, neutrophil inhibitory factor (nif) (NIF), oncostatin M, bone morphogenic protein, oncoprotein, paracitonin, Rat parathyroid hormone 1-34, PD-ECSF, PDGF, peptide hormone, PTN, albumin A, Protein G, pth, pyrogenic exotoxin A, pyrogenic exotoxin B, pyrogenic exotoxin C, pyy, Relaxin, feritin, SCF, the atom synthetic proteins, soluble complement acceptor I, solubility I-CAM 1, soluble interleukin receptor, soluble TNF acceptor, somatomedin, Somat, tethelin, streptokinase, superantigen, staphyloentero-toxin, SEA, SEB, SEC1, SEC2, SEC3, SED, SEE, steroid hormone receptor, superoxide-dismutase, the toxic shock syndrome toxin, thymosin, the histiotype plasminogen activation factor, tumor growth factor (TGF), tumour necrosis factor, tumor necrosis factor alpha, tumor necrosis factor, Tumor Necrosis Factor Receptors (TNFR), VLA-4 albumen, VCAM-1 albumen, vascular endothelial growth factor (VEGF), urokinase, mos, ras, raf, met, p53, tat, fos, myc, jun, myb, rel, estrogen receptor, PgR, the testosterone acceptor, aldosterone receptor, ldl receptor and Kendall compound.
For producing the method for polypeptide dimer, wherein said method comprises in certain embodiments:
(i) comprise amino acid whose first polypeptide of formula (I) with the reagent derivatize of formula (XXVI), and the gained derived protein of step (i) is contacted with amino acid whose second protein that comprises formula (I), and then form the dimer that comprises first polypeptide and second polypeptide.For producing the method for polypeptide dimer, wherein first polypeptide and second polypeptide comprise the amino acid corresponding to formula (II) in other embodiments.In certain embodiments, polypeptide is purified before or after the reagent with formula (XXVI) contacts.In other embodiments, the gained derived protein of step (i) comprises that at least one contains oxime amino acid corresponding to formula (XXVIII):
Figure A20058004432802031
B. the method that is used for the posttranslational modification non-natural amino acid polypeptides: make alpha-non-natural amino acid that contains oxime and the reagent react that contains carbonyl
Has tangible advantage based on containing the protein derived method of oxime protein with the permutoid reaction of the molecule that replaces through carbonyl or dicarbapentaborane.The first, research indication based on amino acid whose oxime adducts by with have more reactive balance experience oxime exchange that contains the compound of carbonyl or dicarbapentaborane than the compound that is used to produce original oxime.This permutoid reaction occurs in the pH value scope of 4-8: under the described conditions, naturally exist amino acid whose side chain not have a reactivity.Therefore, be used to prepare the general method that is suitable for the molecule that replaces through carbonyl or dicarbapentaborane that contains the oxime proteins react the proteinic approach of multiple locus specificity deutero-that obtains can be provided.At this in vivo under the situation of translation technology, be used for preparation and be generally used for the general method of the form that replaces through carbonyl or dicarbapentaborane of proteinic those molecules of derivatize (comprising (only for instance) hydrophilic polymer) for valuable and the approach that obtains multiple locus specificity deutero-non-natural amino acid polypeptides can be provided such as polyoxyethylene glycol.The second, this selective chemical makes the locus specificity derivatize of recombinant protein become possibility: nowadays derived protein can be determines the homology product.The 3rd, realize that the needed mild conditions of describing of permutoid reaction can reversibly destroy the tertiary structure of polypeptide (certain, unless wherein Fan Ying purpose is this tertiary structure of destruction) usually herein.Finally, permutoid reaction is created in new oxime adducts stable under the biotic condition.
Only for instance, following alpha-non-natural amino acid for can be used for producing new the responding property of describing of reagent that contains carbonyl or dicarbapentaborane that contains the oxime non-natural amino acid polypeptides herein contain the amino acid whose type of oxime:
Figure A20058004432802041
Wherein:
A is optionally, and when exist, be low-grade alkylidene, be substituted low-grade alkylidene, rudimentary cycloalkylidene, be substituted rudimentary cycloalkylidene, lower alkenylene, be substituted lower alkenylene, alkynylene, the assorted alkyl in rudimentary Asia, be substituted inferior mix alkyl, rudimentary inferior Heterocyclylalkyl, be substituted rudimentary inferior Heterocyclylalkyl, arylidene, be substituted arylidene, inferior heteroaryl, be substituted inferior heteroaryl, alkarylene, be substituted alkarylene, inferior aralkyl or be substituted inferior aralkyl;
B is optionally, and is the connexon that is selected from the group that is made up of following each group when exist: low-grade alkylidene, be substituted low-grade alkylidene, lower alkenylene, be substituted lower alkenylene, alkyl is mixed in rudimentary Asia, be substituted rudimentary Asia mix alkyl ,-O-,-O-(alkylidene group or be substituted alkylidene group)-,-S-,-S-(alkylidene group or be substituted alkylidene group)-,-S (O) k-(wherein k is 1,2 or 3) ,-S (O) k(alkylidene group or be substituted alkylidene group)-,-C (O)-,-NS (O) 2-,-OS (O) 2-,-C (O)-(alkylidene group or be substituted alkylidene group)-,-C (S)-,-C (S)-(alkylidene group or be substituted alkylidene group)-,-N (R ')-,-NR '-(alkylidene group or be substituted alkylidene group)-,-C (O) N (R ')-,-CON (R ')-(alkylidene group or be substituted alkylidene group)-,-CSN (R ')-,-CSN (R ')-(alkylidene group or be substituted alkylidene group)-,-N (R ') CO-(alkylidene group or be substituted alkylidene group)-,-N (R ') C (O) O-,-S (O) kN (R ')-,-N (R ') C (O) N (R ')-,-N (R ') C (S) N (R ')-,-N (R ') S (O) kN (R ')-,-N (R ')-N=,-C (R ')=N-,-C (R ')=N-N (R ')-,-C (R ')=N-N=,-C (R ') 2-N=N-and-C (R ') 2-N (R ')-N (R ')-, wherein R ' is H, alkyl independently of one another or is substituted alkyl;
R is H, alkyl, is substituted alkyl, cycloalkyl or is substituted cycloalkyl;
R 1Be H, amino protecting group, resin, amino acid, polypeptide or polynucleotide; And
R 2Be OH, ester protecting group, resin, amino acid, polypeptide or polynucleotide;
R 3With R 4Be H, halogen, low alkyl group or be substituted low alkyl group independently of one another, or R 3With R 4Or two R 3Group forms cycloalkyl or Heterocyclylalkyl according to circumstances;
R 5For H, alkyl, be substituted alkyl, thiazolinyl, be substituted thiazolinyl, alkynyl, be substituted alkynyl, alkoxyl group, be substituted alkoxyl group, alkyl alkoxy, be substituted alkyl alkoxy, polyoxyalkylene, be substituted polyoxyalkylene, aryl, be substituted aryl, heteroaryl, be substituted heteroaryl, alkaryl, be substituted alkaryl, aralkyl, be substituted aralkyl ,-(alkylidene group or be substituted alkylidene group)-ON (R ") 2,-(alkylidene group or be substituted alkylidene group)-C (O) SR " ,-(alkylidene group or be substituted alkylidene group)-S-S-(aryl or be substituted aryl) ,-C (O) R " ,-C (O) 2R " or-C (O) N (R ") 2, R wherein " independently of one another for hydrogen, alkyl, be substituted alkyl, thiazolinyl, be substituted thiazolinyl, alkoxyl group, be substituted alkoxyl group, aryl, be substituted aryl, heteroaryl, alkaryl, be substituted alkaryl, aralkyl or be substituted aralkyl;
Or R 5Be L-X, wherein X is selected from the group that is made up of following each thing: mark; Dyestuff; Polymkeric substance; Water-soluble polymers; The derivative of polyoxyethylene glycol; Photocrosslinking agent; Cytotoxic compound; Medicine; Affinity marker; The photoaffinity mark; Reactive compounds; Resin; Second protein or polypeptide or polypeptide analog; Antibody or antibody fragment; Metal chelator; Cofactor; Lipid acid; Carbohydrate; Polynucleotide; DNA; RNA; Antisense polynucleotide; Carbohydrate, water-soluble branch-shape polymer, cyclodextrin, biomaterial; Nanoparticle; Spin labeling; Fluorophore, metallic part; Radioactive segment; Novel functional group; With the covalently or non-covalently interactional group of other molecules; The light cage covers part; But the part that actinic radiation excites; Ligand; But photoisomerization part; Vitamin H; The vitamin H analogue; Be combined with the part of heavy atom; But the group of chemical cracking; But the group of photodestruciton; The side chain that prolongs; The sugar of carbon bond connection; Redox active agent; Amino thioic acid sulfoacid; The toxicity part; Through isotope-labeled part; The biophysics probe; The phosphorescence group; Chemiluminescent groups; The electron dense group; The magnetic group; Insert group; Chromophoric group; Energy transfer agent; Biologically active agent; Detectable label; Small molecules; Inhibition Yeast Nucleic Acid, radioactive nuleus thuja acid; Neutron capture agent; The derivative of vitamin H; Quantum dot; Nanometer is transmitted plain; Radioactivity is transmitted plain; Abzyme, activation composite activating agent, virus, adjuvant, aglycone, anaphylactogen, angiostatin, hormone antagonist, antioxidant, fit, guide RNA, Saponin/TSM, shuttle vectors, macromole, plan epitope, acceptor, reverse micelle with and any combination; And L is optionally, and is the connexon that is selected from the group that is made up of following each group when existing: alkylidene group, be substituted alkylidene group, alkenylene, be substituted alkenylene ,-O-,-O-(alkylidene group or be substituted alkylidene group)-,-S-,-S-(alkylidene group or be substituted alkylidene group)-,-S (O) k-(wherein k is 1,2 or 3) ,-S (O) k(alkylidene group or be substituted alkylidene group)-,-C (O)-,-C (O)-(alkylidene group or be substituted alkylidene group)-,-C (S)-,-C (S)-(alkylidene group or be substituted alkylidene group)-,-N (R ')-,-NR '-(alkylidene group or be substituted alkylidene group)-,-C (O) N (R ')-,-CON (R ')-(alkylidene group or be substituted alkylidene group)-,-CSN (R ')-,-CSN (R ')-(alkylidene group or be substituted alkylidene group)-,-N (R ') CO-(alkylidene group or be substituted alkylidene group)-,-N (R ') C (O) O-,-(alkylidene group or be substituted alkylidene group)-O-N=CR '-,-(alkylidene group or be substituted alkylidene group)-C (O) NR '-(alkylidene group or be substituted alkylidene group)-,-(alkylidene group or be substituted alkylidene group)-S (O) k-(alkylidene group or be substituted alkylidene group)-S-,-(alkylidene group or be substituted alkylidene group)-S-S-,-S (O) kN (R ')-,-N (R ') C (O) N (R ')-,-N (R ') C (S) N (R ')-,-N (R ') S (O) kN (R ')-,-N (R ')-N=,-C (R ')=N-,-C (R ')=N-N (R ')-,-C (R ')=N-N=,-C (R ') 2-N=N-and-C (R ') 2-N (R ')-N (R ')-, wherein R ' is H, alkyl independently of one another or is substituted alkyl.
Only in further example, following alpha-non-natural amino acid also for can be used for producing new the responding property of describing of reagent that contains carbonyl or dicarbapentaborane that contains the oxime non-natural amino acid polypeptides herein contain the amino acid whose type of oxime:
Figure A20058004432802061
Wherein:
A is optionally, and when exist, be low-grade alkylidene, be substituted low-grade alkylidene, rudimentary cycloalkylidene, be substituted rudimentary cycloalkylidene, lower alkenylene, be substituted lower alkenylene, alkynylene, the assorted alkyl in rudimentary Asia, be substituted inferior mix alkyl, rudimentary inferior Heterocyclylalkyl, be substituted rudimentary inferior Heterocyclylalkyl, arylidene, be substituted arylidene, inferior heteroaryl, be substituted inferior heteroaryl, alkarylene, be substituted alkarylene, inferior aralkyl or be substituted inferior aralkyl;
B is optionally, and is the connexon that is selected from the group that is made up of following each group when exist: low-grade alkylidene, be substituted low-grade alkylidene, lower alkenylene, be substituted lower alkenylene, alkyl is mixed in rudimentary Asia, be substituted rudimentary Asia mix alkyl ,-O-,-O-(alkylidene group or be substituted alkylidene group)-,-S-,-S-(alkylidene group or be substituted alkylidene group)-,-S (O) k-(wherein k is 1,2 or 3) ,-S (O) k(alkylidene group or be substituted alkylidene group)-,-C (O)-,-NS (O) 2-,-OS (O) 2-,-C (O)-(alkylidene group or be substituted alkylidene group)-,-C (S)-,-C (S)-(alkylidene group or be substituted alkylidene group)-,-N (R ')-,-NR '-(alkylidene group or be substituted alkylidene group)-,-C (O) N (R ')-,-CON (R ')-(alkylidene group or be substituted alkylidene group)-,-CSN (R ')-,-CSN (R ')-(alkylidene group or be substituted alkylidene group)-,-N (R ') CO-(alkylidene group or be substituted alkylidene group)-,-N (R ') C (O) O-,-S (O) kN (R ')-,-N (R ') C (O) N (R ')-,-N (R ') C (S) N (R ')-,-N (R ') S (O) kN (R ')-,-N (R ')-N=,-C (R ')=N-,-C (R ')=N-N (R ')-,-C (R ')=N-N=,-C (R ') 2-N=N-and-C (R ') 2-N (R ')-N (R ')-, wherein R ' is H, alkyl independently of one another or is substituted alkyl;
R 1Be H, amino protecting group, resin, amino acid, polypeptide or polynucleotide; And
R 2Be OH, ester protecting group, resin, amino acid, polypeptide or polynucleotide;
R 3With R 4Be H, halogen, low alkyl group or be substituted low alkyl group independently of one another, or R 3With R 4Or two R 3Group forms cycloalkyl or Heterocyclylalkyl according to circumstances;
R 6With R 7Be selected from the group that forms by following each group independently of one another: H, alkyl, be substituted alkyl, thiazolinyl, be substituted thiazolinyl, alkoxyl group, be substituted alkoxyl group, polyoxyalkylene, be substituted polyoxyalkylene, aryl, be substituted aryl, heteroaryl, be substituted heteroaryl, alkaryl, be substituted alkaryl, aralkyl and be substituted aralkyl ,-C (O) R " ,-C (O) 2R " ,-C (O) N (R ") 2, R wherein " independently of one another for hydrogen, alkyl, be substituted alkyl, thiazolinyl, be substituted thiazolinyl, alkoxyl group, be substituted alkoxyl group, aryl, be substituted aryl, heteroaryl, alkaryl, be substituted alkaryl, aralkyl or be substituted aralkyl; Or R 6Or R 7Be L-X, wherein
X is selected from the group that is made up of following each thing: mark; Dyestuff; Polymkeric substance; Water-soluble polymers; The derivative of polyoxyethylene glycol; Photocrosslinking agent; Cytotoxic compound; Medicine; Affinity marker; The photoaffinity mark; Reactive compounds; Resin; Second protein or polypeptide or polypeptide analog; Antibody or antibody fragment; Metal chelator; Cofactor; Lipid acid; Carbohydrate; Polynucleotide; DNA; RNA; Antisense polynucleotide; Carbohydrate, water-soluble branch-shape polymer, cyclodextrin, biomaterial; Nanoparticle; Spin labeling; Fluorophore, metallic part; Radioactive segment; Novel functional group; With the covalently or non-covalently interactional group of other molecules; The light cage covers part; But the part that actinic radiation excites; Ligand; But photoisomerization part; Vitamin H; The vitamin H analogue; Be combined with the part of heavy atom; But the group of chemical cracking; But the group of photodestruciton; The side chain that prolongs; The sugar of carbon bond connection; Redox active agent; Amino thioic acid sulfoacid; The toxicity part; Through isotope-labeled part; The biophysics probe; The phosphorescence group; Chemiluminescent groups; The electron dense group; The magnetic group; Insert group; Chromophoric group; Energy transfer agent; Biologically active agent; Detectable label; Small molecules; Inhibition Yeast Nucleic Acid, radioactive nuleus thuja acid; Neutron capture agent; The derivative of vitamin H; Quantum dot; Nanometer is transmitted plain; Radioactivity is transmitted plain; Abzyme, activation composite activating agent, virus, adjuvant, aglycone, anaphylactogen, angiostatin, hormone antagonist, antioxidant, fit, guide RNA, Saponin/TSM, shuttle vectors, macromole, plan epitope, acceptor, reverse micelle with and any combination; And L is optionally, and is the connexon that is selected from the group that is made up of following each group when existing: alkylidene group, be substituted alkylidene group, alkenylene, be substituted alkenylene ,-O-,-O-(alkylidene group or be substituted alkylidene group)-,-S-,-S-(alkylidene group or be substituted alkylidene group)-,-S (O) k-(wherein k is 1,2 or 3) ,-S (O) k(alkylidene group or be substituted alkylidene group)-,-C (O)-,-C (O)-(alkylidene group or be substituted alkylidene group)-,-C (S)-,-C (S)-(alkylidene group or be substituted alkylidene group)-,-N (R ')-,-NR '-(alkylidene group or be substituted alkylidene group)-,-C (O) N (R ')-,-CON (R ')-(alkylidene group or be substituted alkylidene group)-,-CSN (R ')-,-CSN (R ')-(alkylidene group or be substituted alkylidene group)-,-N (R ') CO-(alkylidene group or be substituted alkylidene group)-,-N (R ') C (O) O-,-S (O) kN (R ')-,-N (R ') C (O) N (R ')-,-N (R ') C (S) N (R ')-,-N (R ') S (O) kN (R ')-,-N (R ')-N=,-C (R ')=N-,-C (R ')=N-N (R ')-,-C (R ')=N-N=,-C (R ') 2-N=N-and-C (R ') 2-N (R ')-N (R ')-, wherein R ' is H, alkyl independently of one another or is substituted alkyl.
Only for instance, the reagent that below contains carbonyl or dicarbapentaborane is for having reactivity and can be used for realizing the type of permutoid reaction to form new oxime key and therefore to modify the reagent that contains carbonyl or dicarbapentaborane that contains the oxime non-natural amino acid polypeptides with the oxime alpha-non-natural amino acid of describing herein that contains:
Figure A20058004432802071
Wherein:
X independently of one another for H, alkyl, be substituted alkyl, thiazolinyl, be substituted thiazolinyl, alkynyl, be substituted alkynyl, alkoxyl group, be substituted alkoxyl group, alkyl alkoxy, be substituted alkyl alkoxy, polyoxyalkylene, be substituted polyoxyalkylene, aryl, be substituted aryl, heteroaryl, be substituted heteroaryl, alkaryl, be substituted alkaryl, aralkyl, be substituted aralkyl ,-(alkylidene group or be substituted alkylidene group)-ON (R ") 2,-(alkylidene group or be substituted alkylidene group)-C (O) SR " ,-(alkylidene group or be substituted alkylidene group)-S-S-(aryl or be substituted aryl) ,-C (O) R " ,-C (O) 2R " or-C (O) N (R ") 2, R wherein " independently of one another for hydrogen, alkyl, be substituted alkyl, thiazolinyl, be substituted thiazolinyl, alkoxyl group, be substituted alkoxyl group, aryl, be substituted aryl, heteroaryl, alkaryl, be substituted alkaryl, aralkyl, or be substituted aralkyl;
Or X is selected from the group that is made up of following each thing independently of one another: mark; Dyestuff; Polymkeric substance; Water-soluble polymers; The derivative of polyoxyethylene glycol; Photocrosslinking agent; Cytotoxic compound; Medicine; Affinity marker; The photoaffinity mark; Reactive compounds; Resin; Second protein or polypeptide or polypeptide analog; Antibody or antibody fragment; Metal chelator; Cofactor; Lipid acid; Carbohydrate; Polynucleotide; DNA; RNA; Antisense polynucleotide; Carbohydrate, water-soluble branch-shape polymer, cyclodextrin, biomaterial; Nanoparticle; Spin labeling; Fluorophore, metallic part; Radioactive segment; Novel functional group; With the covalently or non-covalently interactional group of other molecules; The light cage covers part; But the part that actinic radiation excites; Ligand; But photoisomerization part; Vitamin H; The vitamin H analogue; Be combined with the part of heavy atom; But the group of chemical cracking; But the group of photodestruciton; The side chain that prolongs; The sugar of carbon bond connection; Redox active agent; Amino thioic acid sulfoacid; The toxicity part; Through isotope-labeled part; The biophysics probe; The phosphorescence group; Chemiluminescent groups; The electron dense group; The magnetic group; Insert group; Chromophoric group; Energy transfer agent; Biologically active agent; Detectable label; Small molecules; Inhibition Yeast Nucleic Acid, radioactive nuleus thuja acid; Neutron capture agent; The derivative of vitamin H; Quantum dot; Nanometer is transmitted plain; Radioactivity is transmitted plain; Abzyme, activation composite activating agent, virus, adjuvant, aglycone, anaphylactogen, angiostatin, hormone antagonist, antioxidant, fit, guide RNA, Saponin/TSM, shuttle vectors, macromole, plan epitope, acceptor, reverse micelle with and any combination;
L is selected from the group that is made up of following each group independently of one another: alkylidene group, be substituted alkylidene group, alkenylene, be substituted alkenylene ,-O-,-O-(alkylidene group or be substituted alkylidene group)-,-S-,-S-(alkylidene group or be substituted alkylidene group)-,-S (O) k-(wherein k is 1,2 or 3) ,-S (O) k(alkylidene group or be substituted alkylidene group)-,-C (O)-,-C (O)-(alkylidene group or be substituted alkylidene group)-,-C (S)-,-C (S)-(alkylidene group or be substituted alkylidene group)-,-N (R ')-,-NR '-(alkylidene group or be substituted alkylidene group)-,-C (O) N (R ')-,-CON (R ')-(alkylidene group or be substituted alkylidene group)-,-(alkylidene group or be substituted alkylidene group) NR ' C (O) O-(alkylidene group or be substituted alkylidene group)-,-O-CON (R ')-(alkylidene group or be substituted alkylidene group)-,-CSN (R ')-,-CSN (R ')-(alkylidene group or be substituted alkylidene group)-,-N (R ') CO-(alkylidene group or be substituted alkylidene group)-,-N (R ') C (O) O-,-N (R ') C (O) O-(alkylidene group or be substituted alkylidene group)-,-S (O) kN (R ')-,-N (R ') C (O) N (R ')-,-N (R ') C (O) N (R ')-(alkylidene group or be substituted alkylidene group)-,-N (R ') C (S) N (R ')-,-N (R ') S (O) kN (R ')-,-N (R ')-N=,-C (R ')=N-,-C (R ')=N-N (R ')-,-C (R ')=N-N=,-C (R ') 2-N=N-and-C (R ') 2-N (R ')-N (R ')-;
L 1For optionally, and when existing be-C (R ') p-NR '-C (O) O-(alkylidene group or be substituted alkylidene group)-, wherein p is 0,1 or 2; R ' is H, alkyl independently of one another or is substituted alkyl;
W is-C (=O) R 9, R wherein 9Be H or OR '; And n is 1 to 3;
Or L-L wherein 1-W is common form comprise at least one carbonyl (comprising dicarbapentaborane), through protection carbonyl (comprising) or through sheltering monocycle or the double-ring cycloalkyl or the Heterocyclylalkyl of carbonyl (comprising) through sheltering dicarbapentaborane through the protection dicarbapentaborane;
Its restricted condition is L-L 1-W provide jointly at least one can experience and alpha-non-natural amino acid or " modified or not modified " non-natural amino acid polypeptides on the carbonyl (comprising dicarbapentaborane) of oximido generation oxime permutoid reaction.
Be used for realizing that two illustrative embodiment that contain oxime amino acid and contain the method for the oxime permutoid reaction between the reagent of carbonyl on the polypeptide are is shown in Fig. 9.In these illustrative embodiment, the reagent that contains carbonyl is added in the buffered soln (pH 2-8) that contains the oxime non-natural amino acid polypeptides.A plurality of hours skies are at the most carried out in reaction at ambient temperature.Come the modified oxime non-natural amino acid polypeptides that contains of purifying by HPLC, FPLC or size exclusion chromatography.
In one embodiment, but a plurality of connexon chemistry locus specificities ground and the non-natural amino acid polypeptides reaction that replaces through oxime.In one embodiment, the connexon method utilization of describing herein contains carbonyl or dicarbapentaborane functional group's connexon (simple function, difunctionality or multifunctional) at least one connexon end.Carbonyl or dicarbapentaborane deutero-connexon produce new stable oxime key with the condensation of the non-natural amino acid polypeptides that replaces through oxime.Difunctionality and/or multifunctional connexon are (for example, carbonyl or dicarbapentaborane with one or more other binding chemistry) (for example allow differing molecular, other protein, polymkeric substance or small molecules) locus specificity is connected on the non-natural amino acid polypeptides, and simple function connexon (replacing through carbonyl or dicarbapentaborane on all ends) promotes the locus specificity dimerization or the oligomerization of non-natural amino acid polypeptides.By this connexon strategy is combined with the in vivo translation technology of describing, make the proteinic three-dimensional structure that describes in detail through chemical refining become possibility herein.
C. the method that is used for the posttranslational modification non-natural amino acid polypeptides: make alpha-non-natural amino acid that contains azanol and the reagent react that contains carbonyl
Above-mentioned posttranslational modification technology and composition also can be used for and contain the reagent react of carbonyl or dicarbapentaborane to produce the modified alpha-non-natural amino acid that contains azanol that contains the oxime non-natural amino acid polypeptides.
Protein derived method based on the protein that contains azanol and the reaction of the molecule that replaces through carbonyl or dicarbapentaborane has tangible advantage.The first, azanol experiences condensation with the compound that contains carbonyl or dicarbapentaborane to produce the oxime adducts in the pH of 4-8 value scope.Under the described conditions, naturally exist amino acid whose side chain not have a reactivity.The second, this selective chemical makes the locus specificity derivatize of recombinant protein become possibility: nowadays derived protein can be prepared as determines the homology product.The 3rd, realize that the reagent that contains carbonyl or dicarbapentaborane of description herein and the needed mild conditions of describing of the reaction that contains the azanol polypeptide can reversibly destroy the tertiary structure of polypeptide (certain, unless wherein Fan Ying purpose is this tertiary structure of destruction) usually herein.At last, be created in oxime adducts stable under the biotic condition although as if azanol base amino acid, contain the reagent of carbonyl or dicarbapentaborane by the intestinal bacteria metabolism with the amino acid whose condensation that contains azanol.
Only for instance, following alpha-non-natural amino acid is for having reactive amino acid whose type of azanol that contains with the reagent that contains carbonyl or dicarbapentaborane of description herein that can be used for further modifying the non-natural amino acid polypeptides that contains azanol:
Figure A20058004432802101
Wherein:
A is optionally, and when exist, be low-grade alkylidene, be substituted low-grade alkylidene, rudimentary cycloalkylidene, be substituted rudimentary cycloalkylidene, lower alkenylene, be substituted lower alkenylene, alkynylene, the assorted alkyl in rudimentary Asia, be substituted inferior mix alkyl, rudimentary inferior Heterocyclylalkyl, be substituted rudimentary inferior Heterocyclylalkyl, arylidene, be substituted arylidene, inferior heteroaryl, be substituted inferior heteroaryl, alkarylene, be substituted alkarylene, inferior aralkyl or be substituted inferior aralkyl;
B is optionally, and is the connexon that is selected from the group that is made up of following each group when exist: low-grade alkylidene, be substituted low-grade alkylidene, lower alkenylene, be substituted lower alkenylene, alkyl is mixed in rudimentary Asia, be substituted rudimentary Asia mix alkyl ,-O-,-O-(alkylidene group or be substituted alkylidene group)-,-S-,-S-(alkylidene group or be substituted alkylidene group)-,-S (O) k-(wherein k is 1,2 or 3) ,-S (O) k(alkylidene group or be substituted alkylidene group)-,-C (O)-,-NS (O) 2-,-OS (O) 2-,-C (O)-(alkylidene group or be substituted alkylidene group)-,-C (S)-,-C (S)-(alkylidene group or be substituted alkylidene group)-,-N (R ')-,-NR '-(alkylidene group or be substituted alkylidene group)-,-C (O) N (R ')-,-CON (R ')-(alkylidene group or be substituted alkylidene group)-,-CSN (R ')-,-CSN (R ')-(alkylidene group or be substituted alkylidene group)-,-N (R ') CO-(alkylidene group or be substituted alkylidene group)-,-N (R ') C (O) O-,-S (O) kN (R ')-,-N (R ') C (O) N (R ')-,-N (R ') C (S) N (R ')-,-N (R ') S (O) kN (R ')-,-N (R ')-N=,-C (R ')=N-,-C (R ')=N-N (R ')-,-C (R ')=N-N=,-C (R ') 2-N=N-and-C (R ') 2-N (R ')-N (R ')-, wherein R ' is H, alkyl independently of one another or is substituted alkyl;
K is-NR 6R 7Or-N=CR 6R 7
R 1Be H, amino protecting group, resin, amino acid, polypeptide or polynucleotide; And
R 2Be OH, ester protecting group, resin, amino acid, polypeptide or polynucleotide;
R 3With R 4Be H, halogen, low alkyl group or be substituted low alkyl group independently of one another, or R 3With R 4Or two R 3Group forms cycloalkyl or Heterocyclylalkyl according to circumstances.
In some embodiment of formula (XIV) compound, K is NH 2
Comprise the alpha-non-natural amino acid that these contain azanol polypeptide type in fact and unrestricted, as long as containing the alpha-non-natural amino acid of azanol is positioned on the polypeptide so that contain the reagent of carbonyl or dicarbapentaborane and can and produce the modified alpha-non-natural amino acid (certainly, unless if this destruction is the purpose of reaction) of gained of the tertiary structure of destroying polypeptide with the azanol radical reaction.
Only for instance, the reagent that below contains carbonyl or dicarbapentaborane is for having reactivity with the alpha-non-natural amino acid of describing that contains azanol herein and can be used for further modifying the type of the reagent that contains carbonyl or dicarbapentaborane of the non-natural amino acid polypeptides that contains azanol:
Figure A20058004432802111
Wherein:
X independently of one another for H, alkyl, be substituted alkyl, thiazolinyl, be substituted thiazolinyl, alkynyl, be substituted alkynyl, alkoxyl group, be substituted alkoxyl group, alkyl alkoxy, be substituted alkyl alkoxy, polyoxyalkylene, be substituted polyoxyalkylene, aryl, be substituted aryl, heteroaryl, be substituted heteroaryl, alkaryl, be substituted alkaryl, aralkyl, be substituted aralkyl ,-(alkylidene group or be substituted alkylidene group)-ON (R ") 2,-(alkylidene group or be substituted alkylidene group)-C (O) SR " ,-(alkylidene group or be substituted alkylidene group)-S-S-(aryl or be substituted aryl) ,-C (O) R " ,-C (O) 2R " or-C (O) N (R ") 2, R wherein " independently of one another for hydrogen, alkyl, be substituted alkyl, thiazolinyl, be substituted thiazolinyl, alkoxyl group, be substituted alkoxyl group, aryl, be substituted aryl, heteroaryl, alkaryl, be substituted alkaryl, aralkyl, or be substituted aralkyl;
Or X is selected from the group that is made up of following each thing independently of one another: mark; Dyestuff; Polymkeric substance; Water-soluble polymers; The derivative of polyoxyethylene glycol; Photocrosslinking agent; Cytotoxic compound; Medicine; Affinity marker; The photoaffinity mark; Reactive compounds; Resin; Second protein or polypeptide or polypeptide analog; Antibody or antibody fragment; Metal chelator; Cofactor; Lipid acid; Carbohydrate; Polynucleotide; DNA; RNA; Antisense polynucleotide; Carbohydrate, water-soluble branch-shape polymer, cyclodextrin, biomaterial; Nanoparticle; Spin labeling; Fluorophore, metallic part; Radioactive segment; Novel functional group; With the covalently or non-covalently interactional group of other molecules; The light cage covers part; But the part that actinic radiation excites; Ligand; But photoisomerization part; Vitamin H; The vitamin H analogue; Be combined with the part of heavy atom; But the group of chemical cracking; But the group of photodestruciton; The side chain that prolongs; The sugar of carbon bond connection; Redox active agent; Amino thioic acid sulfoacid; The toxicity part; Through isotope-labeled part; The biophysics probe; The phosphorescence group; Chemiluminescent groups; The electron dense group; The magnetic group; Insert group; Chromophoric group; Energy transfer agent; Biologically active agent; Detectable label; Small molecules; Inhibition Yeast Nucleic Acid, radioactive nuleus thuja acid; Neutron capture agent; The derivative of vitamin H; Quantum dot; Nanometer is transmitted plain; Radioactivity is transmitted plain; Abzyme, activation composite activating agent, virus, adjuvant, aglycone, anaphylactogen, angiostatin, hormone antagonist, antioxidant, fit, guide RNA, Saponin/TSM, shuttle vectors, macromole, plan epitope, acceptor, reverse micelle with and any combination;
L is selected from the group that is made up of following each group independently of one another: alkylidene group, be substituted alkylidene group, alkenylene, be substituted alkenylene ,-O-,-O-(alkylidene group or be substituted alkylidene group)-,-S-,-S-(alkylidene group or be substituted alkylidene group)-,-S (O) k-(wherein k is 1,2 or 3) ,-S (O) k(alkylidene group or be substituted alkylidene group)-,-C (O)-,-C (O)-(alkylidene group or be substituted alkylidene group)-,-C (S)-,-C (S)-(alkylidene group or be substituted alkylidene group)-,-N (R ')-,-NR '-(alkylidene group or be substituted alkylidene group)-,-C (O) N (R ')-,-CON (R ')-(alkylidene group or be substituted alkylidene group)-,-(alkylidene group or be substituted alkylidene group) NR ' C (O) O-(alkylidene group or be substituted alkylidene group)-,-O-CON (R ')-(alkylidene group or be substituted alkylidene group)-,-CSN (R ')-,-CSN (R ')-(alkylidene group or be substituted alkylidene group)-,-N (R ') CO-(alkylidene group or be substituted alkylidene group)-,-N (R ') C (O) O-,-N (R ') C (O) O-(alkylidene group or be substituted alkylidene group)-,-S (O) kN (R ')-,-N (R ') C (O) N (R ')-,-N (R ') C (O) N (R ')-(alkylidene group or be substituted alkylidene group)-,-N (R ') C (S) N (R ')-,-N (R ') S (O) kN (R ')-,-N (R ')-N=,-C (R ')=N-,-C (R ')=N-N (R ')-,-C (R ')=N-N=,-C (R ') 2-N=N-and-C (R ') 2-N (R ')-N (R ')-;
L 1For optionally, and when existing be-C (R ') p-NR '-C (O) O-(alkylidene group or be substituted alkylidene group)-, wherein p is 0,1 or 2; R ' is H, alkyl independently of one another or is substituted alkyl;
W is-J-R that wherein J is
Figure A20058004432802121
R is H, alkyl, is substituted alkyl, cycloalkyl or is substituted cycloalkyl; R " be H, alkyl independently of one another, be substituted alkyl, or protecting group, " during group, two R " forms Heterocyclylalkyl according to circumstances maybe when there being more than one R; And n is 1 to 3;
Its restricted condition is L-L 1-W provide jointly at least one can with the carbonyl (comprising dicarbapentaborane) of azanol radical reaction on alpha-non-natural amino acid or " modified or not modified " non-natural amino acid polypeptides.
Be compound in some embodiment of formula (XIX) compound with structure of formula (XXI):
Figure A20058004432802131
The illustrative embodiment that the reagent that is used for containing carbonyl is coupled to the method on the alpha-non-natural amino acid that contains azanol on the polypeptide is and is shown in Figure 10.In this illustrative embodiment, will add to through carbonyl deutero-reagent in the buffered soln (pH 2-8) of the non-natural amino acid polypeptides that contains azanol.A plurality of hours skies are at the most carried out in reaction at ambient temperature.For quickening to engage, add those additives such as oblatio among Fig. 8.Come the purifying gained to contain the oxime non-natural amino acid polypeptides by HPLC, FPLC or size exclusion chromatography.
In one embodiment, but a plurality of connexon chemistry locus specificities ground and the non-natural amino acid polypeptides reaction that replaces through azanol.In one embodiment, the connexon method utilization of describing herein contains carbonyl or dicarbapentaborane functional group's connexon (simple function, difunctionality or multifunctional) at least one connexon end.Stablize the oxime key through carbonyl or dicarbapentaborane deutero-connexon with the proteinic condensation generation that replaces through azanol.Difunctionality and/or multifunctional connexon are (for example, carbonyl or dicarbapentaborane with one or more other binding chemistry) (for example allow differing molecular, other protein, polymkeric substance or small molecules) locus specificity is connected on the non-natural amino acid polypeptides, and simple function connexon (replacing through carbonyl or dicarbapentaborane on all ends) promotes the locus specificity dimerization or the oligomerization of non-natural amino acid polypeptides.By with this connexon strategy and the in vivo translation technology combination of describing, make the proteinic three-dimensional structure that describes in detail through chemical refining become possibility herein.
Be the method for the polypeptide that is used for the amino acid that derivatize comprises formula XIV-XVI (it comprises any minor or specific compound in the category that is in formula XIV-XVI) in certain embodiments, wherein said method comprises makes the amino acid whose polypeptide that comprises at least one formula XIV-XVI contact with the reagent of formula (XIX).In certain embodiments, polypeptide is purified before or after the reagent with formula (XIX) contacts.Be in other embodiments comprise at least one corresponding to formula (XXIX) contain the amino acid whose gained of the oxime polypeptide of deriving,
Figure A20058004432802132
Wherein:
A is optionally, and when existing, be low-grade alkylidene, be substituted low-grade alkylidene, lower alkenylene, be substituted lower alkenylene, arylidene, be substituted arylidene, inferior heteroaryl, be substituted inferior heteroaryl, alkarylene, be substituted alkarylene, inferior aralkyl or be substituted inferior aralkyl;
B is optionally, and is the connexon that is selected from the group that is made up of following each group when existing: low-grade alkylidene, be substituted low-grade alkylidene, lower alkenylene, be substituted lower alkenylene ,-O-,-O-(alkylidene group or be substituted alkylidene group)-,-S-,-S-(alkylidene group or be substituted alkylidene group)-,-S (O) k-(wherein k is 1,2 or 3) ,-S (O) k(alkylidene group or be substituted alkylidene group)-,-C (O)-,-NS (O) 2-,-OS (O) 2-,-C (O)-(alkylidene group or be substituted alkylidene group)-,-C (S)-,-C (S)-(alkylidene group or be substituted alkylidene group)-,-N (R ')-,-NR '-(alkylidene group or be substituted alkylidene group)-,-C (O) N (R ')-,-CON (R ')-(alkylidene group or be substituted alkylidene group)-,-CSN (R ')-,-CSN (R ')-(alkylidene group or be substituted alkylidene group)-,-N (R ') CO-(alkylidene group or be substituted alkylidene group)-,-N (R ') C (O) O-,-S (O) kN (R ')-,-N (R ') C (O) N (R ')-,-N (R ') C (S) N (R ')-,-N (R ') S (O) kN (R ')-,-N (R ')-N=,-C (R ')=N-,-C (R ')=N-N (R ')-,-C (R ')=N-N=,-C (R ') 2-N=N-and-C (R ') 2-N (R ')-N (R ')-, wherein R ' is H, alkyl independently of one another or is substituted alkyl;
L is the connexon that is independently selected from the group that is made up of following each group: alkylidene group, be substituted alkylidene group, alkenylene, be substituted alkenylene ,-O-,-O-(alkylidene group or be substituted alkylidene group)-,-S-,-S-(alkylidene group or be substituted alkylidene group)-,-S (O) k-(wherein k is 1,2 or 3) ,-S (O) k(alkylidene group or be substituted alkylidene group)-,-C (O)-,-C (O)-(alkylidene group or be substituted alkylidene group)-,-C (S)-,-C (S)-(alkylidene group or be substituted alkylidene group)-,-N (R ')-,-NR '-(alkylidene group or be substituted alkylidene group)-,-C (O) N (R ')-,-CON (R ')-(alkylidene group or be substituted alkylidene group)-,-(alkylidene group or be substituted alkylidene group) NR ' C (O) O-(alkylidene group or be substituted alkylidene group)-,-O-CON (R ')-(alkylidene group or be substituted alkylidene group)-,-CSN (R ')-,-CSN (R ')-(alkylidene group or be substituted alkylidene group)-,-N (R ') CO-(alkylidene group or be substituted alkylidene group)-,-N (R ') C (O) O-,-N (R ') C (O) O-(alkylidene group or be substituted alkylidene group)-,-S (O) kN (R ')-,-N (R ') C (O) N (R ')-,-N (R ') C (O) N (R ')-(alkylidene group or be substituted alkylidene group)-,-N (R ') C (S) N (R ')-,-N (R ') S (O) kN (R ')-,-N (R ')-N=,-C (R ')=N-,-C (R ')=N-N (R ')-,-C (R ')=N-N=,-C (R ') 2-N=N-and-C (R ') 2-N (R ')-N (R ')-, wherein R ' is H, alkyl independently of one another or is substituted alkyl;
R 1For optionally, and when existing H, amino protecting group, resin, amino acid, polypeptide or polynucleotide; And
R 2For optionally, and when existing OH, ester protecting group, resin, amino acid, polypeptide or polynucleotide;
R 3With R 4Be H, halogen, low alkyl group or be substituted low alkyl group independently of one another; And
X is detectable label, biologically active agent or polymkeric substance independently.
In other embodiments, these polypeptide of deriving were stablized 1 month in the aqueous solution under appropriate acidic conditions at least.In other embodiments, these are derived polypeptide stable 2 weeks under appropriate acidic conditions at least.In other embodiments, these polypeptide of deriving were stablized 5 days under appropriate acidic conditions at least.In other embodiments, described condition is pH2 to 8.In certain embodiments, keep the deriving tertiary structure of polypeptide.In other embodiments, these derivatizes of polypeptide further comprise the polypeptide chain of deriving are connected on another polypeptide.In other embodiments, these polypeptides derived and the treatment protein homology that is selected from the group that forms by following each thing: α-1 antitrypsin, angiostatin, AHF, antibody, lipophorin, apoprotein, atrionatriuretic factor, atrial natriuretic polypeptins, atrial natriuretic peptide, the C-X-C chemokine, T39765, NAP-2, ENA-78, gro-a, gro-b, gro-c, IP-10, GCP-2, NAP-4, SDF-1, PF4, MIG, calcitonin, the c-kit ligand, cytokine, the CC chemokine, MCP-1, MCP-2, monocyte chemoattractant protein-3, monocyte inflammatory protein-1 α, monocyte inflammatory protein-i β, RANTES, 1309, R83915, R91733, HCC1, T58847, D31065, T64262, CD40, the CD40 ligand, the c-kit ligand, collagen protein, G CFS (CSF), complement factor 5a, complement inhibitor, complement receptor 1, cytokine, epithelium neutrophilic granulocyte activation peptide-78, MIP-16, MCP-1, Urogastron (EGF), epithelium neutrophilic granulocyte activation peptide, erythropoietin (EPO), exfoliative toxin,or exfoliatin, factors IX, factor VII, Factor IX, factor X, fibroblast growth factor (FGF), Fibrinogen, fibronectin, four-helix bundle albumen, G-CSF, glp-1, GM-CSF, glucocerebrosidase, gonad-stimulating hormone, somatomedin, growth factor receptors, grf, Shh, hemochrome, pHGF (hGF), r-hirudin, human growth hormone (hGH), the human serum albumin, ICAM-1, the ICAM-1 acceptor, LFA-1, the LFA-1 acceptor, Regular Insulin, insulin-like growth factor (IGF), IGF-I, IGF-II, Interferon, rabbit (IFN), IFN-α, IFN-β, IFN-γ, interleukin-(IL), IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, keratinocyte growth factor (KGF), lactoferrin, leukaemia inhibitory factor, luciferase, neural plain, neutrophil inhibitory factor (nif) (NIF), oncostatin M, bone morphogenic protein, oncoprotein, paracitonin, Rat parathyroid hormone 1-34, PD-ECSF, PDGF, peptide hormone, PTN, albumin A, Protein G, pth, pyrogenic exotoxin A, pyrogenic exotoxin B, pyrogenic exotoxin C, pyy, Relaxin, feritin, SCF, the atom synthetic proteins, soluble complement acceptor I, solubility I-CAM1, soluble interleukin receptor, soluble TNF acceptor, somatomedin, Somat, tethelin, streptokinase, superantigen, staphyloentero-toxin, SEA, SEB, SEC1, SEC2, SEC3, SED, SEE, steroid hormone receptor, superoxide-dismutase, the toxic shock syndrome toxin, thymosin, the histiotype plasminogen activation factor, tumor growth factor (TGF), tumour necrosis factor, tumor necrosis factor alpha, tumor necrosis factor, Tumor Necrosis Factor Receptors (TNFR), VLA-4 albumen, VCAM-1 albumen, vascular endothelial growth factor (VEGF), urokinase, mos, ras, raf, met, p53, tat, fos, myc, jun, myb, rel, estrogen receptor, PgR, the testosterone acceptor, aldosterone receptor, ldl receptor and Kendall compound.
D. add functional group's example: be coupled to the high polymer on the non-natural amino acid polypeptides
Can use composition, method, technology and the strategy described to realize herein to the various modifications of the non-natural amino acid polypeptides of description herein.These modifications comprise is incorporated on the alpha-non-natural amino acid component of polypeptide other functional groups, and these functional groups are including (but not limited to) mark; Dyestuff; Polymkeric substance; Water-soluble polymers; The derivative of polyoxyethylene glycol; Photocrosslinking agent; Cytotoxic compound; Medicine; Affinity marker; The photoaffinity mark; Reactive compounds; Resin; Second protein or polypeptide or polypeptide analog; Antibody or antibody fragment; Metal chelator; Cofactor; Lipid acid; Carbohydrate; Polynucleotide; DNA; RNA; Antisense polynucleotide; Carbohydrate, water-soluble branch-shape polymer, cyclodextrin, biomaterial; Nanoparticle; Spin labeling; Fluorophore, metallic part; Radioactive segment; Novel functional group; With the covalently or non-covalently interactional group of other molecules; The light cage covers part; But the part that actinic radiation excites; Ligand; But photoisomerization part; Vitamin H; The vitamin H analogue; Be combined with the part of heavy atom; But the group of chemical cracking; But the group of photodestruciton; The side chain that prolongs; The sugar of carbon bond connection; Redox active agent; Amino thioic acid sulfoacid; The toxicity part; Through isotope-labeled part; The biophysics probe; The phosphorescence group; Chemiluminescent groups; The electron dense group; The magnetic group; Insert group; Chromophoric group; Energy transfer agent; Biologically active agent; Detectable label; Small molecules; Inhibition Yeast Nucleic Acid, radioactive nuleus thuja acid; Neutron capture agent; The derivative of vitamin H; Quantum dot; Nanometer is transmitted plain; Radioactivity is transmitted plain; Abzyme, activation composite activating agent, virus, adjuvant, aglycone, anaphylactogen, angiostatin, hormone antagonist, antioxidant, fit, guide RNA, Saponin/TSM, shuttle vectors, macromole, plan epitope, acceptor, reverse micelle with and any combination.Illustrative, limiting examples as the composition of describing herein, method, technology and strategy, below describe to concentrate on high polymer is added on the non-natural amino acid polypeptides, composition, method, technology and the strategy that should be appreciated that other description simultaneously also can be used for (suitably revising in case of necessity, and the those skilled in the art can use disclosure herein to carry out) add other functional groups, it is including (but not limited to) those functional groups listed above.
Multiple high polymer and other molecules can be coupled to herein on the non-natural amino acid polypeptides of describing regulating the biological property of non-natural amino acid polypeptides (or corresponding natural amino acid polypeptide), and/or provide new biological property to non-natural amino acid polypeptides (or corresponding natural amino acid polypeptide).These high polymers can pass through alpha-non-natural amino acid, or any sense substituent of alpha-non-natural amino acid, or any substituting group or the functional group that are added on the alpha-non-natural amino acid are coupled on the non-natural amino acid polypeptides.
Water-soluble polymers can be coupled on the alpha-non-natural amino acid in polypeptide (natural or synthetic), polynucleotide, polysaccharide or the synthetic polymer that is incorporated herein description.Water-soluble polymers can be by incorporating the alpha-non-natural amino acid in the polypeptide or any functional group or the substituting group of alpha-non-natural amino acid into, or be added into any functional group or substituting group coupling on the alpha-non-natural amino acid.In some cases, the non-natural amino acid polypeptides of describing herein comprises that one or more are coupled to alpha-non-natural amino acid on the water-soluble polymers and one or more bindings natural amino acid that exists to the water-soluble polymers.The covalently bound expression of hydrophilic polymer and bioactive molecules increases water-soluble (such as in physiological environment), biological usability, the increase serum half-life of bioactive molecules (comprise protein, peptide and especially hydrophobic molecule), a kind of method that increases the treatment transformation period, regulates immunogenicity, regulates biological activity or prolong cycling time.Other key characters of these hydrophilic polymers comprise biocompatibility, nontoxicity and non-immunogenicity.For the therepic use of final product preparation, it is pharmaceutically acceptable that preferred polymers should be.
The example of hydrophilic polymer including (but not limited to): poly alkyl ether with and alkoxy end-capped analogue (for example, polyoxyethylene glycol, polyoxyethylene glycol/polyoxy propylene glycol, with and methoxy or ethoxy end-blocking analogue, especially polyoxyethylene glycol, the latter also is known as polyoxyethylene glycol or PEG); Polyvinylpyrrolidone; The polyvinyl alkyl oxide; Ju oxazoline, Ju Wan oxazolin and poly-Qiang Wan oxazolin; Polyacrylamide, poly-alkyl acrylamide and poly-hydroxyalkyl acrylamide (for example, poly-hydroxypropyl Methacrylamide with and derivative); Poly-hydroxyalkyl acrylates; Polysialic acid with and analogue; The hydrophilic peptide sequence; Polysaccharide with and derivative, it comprises dextran and glucan derivative, for example, Sensor Chip CM 5, T 500, glycosaminoglycan; Mierocrystalline cellulose with and derivative, for example, carboxymethyl cellulose, hydroxy alkyl cellulose; Chitin with and derivative, for example, chitosan, succinyl chitosan, carboxymethyl chitin, cm-chitosan; Hyaluronic acid with and derivative; Starch; Alginate; Chondroitin sulfate; Albumin; Amylopectin and carboxymethyl amylopectin; Polyamino acid with and derivative, for example, polyglutamic acid, polylysine, poly aspartic acid, poly-asparagine; Copolymer-maleic anhydride, such as: Zelan 338, divinyl ethyl ether copolymer-maleic anhydride; Polyvinyl alcohol; Its multipolymer; Its terpolymer; Its mixture; And the derivative of aforementioned substances.Water-soluble polymers can be any structure form, and it is including (but not limited to) linear, forked or branch.In certain embodiments, have 2 especially suitable to the water-soluble polymers main chain of about 300 ends.Multifunctional polymer derivant is including (but not limited to) the linear polymer with two ends, wherein each functional group's bond terminal and can be identical or different.In certain embodiments, aqueous-based polymers comprises polyalkylene glycol moiety.The molecular weight of polymkeric substance can be in broad range, and it is including (but not limited to) between about 100Da and about 100,000Da or higher between scope.The molecular weight of polymkeric substance can be at about 100Da and about 100, and between the 000Da, it is including (but not limited to) 100,000Da, 95,000Da, 90,000Da, 85,000Da, 80,000Da, 75,000Da, 70,000Da, 65,000Da, 60,000Da, 55,000Da, 50,000Da, 45,000Da, 40,000Da, 35,000Da, 30,000Da, 25,000Da, 20,000Da, 15,000Da, 10,000Da, 9,000Da, 8,000Da, 7,000Da, 6,000Da, 5,000Da, 4,000Da, 3,000Da, 2,000Da, 1,000Da, 900Da, 800Da, 700Da, 600Da, 500Da, 400Da, 300Da, 200Da and 100Da.In certain embodiments, the molecular weight of polymkeric substance is in about 100Da and 50, between the 000Da.In certain embodiments, the molecular weight of polymkeric substance is in about 100Da and 40, between the 000Da.In certain embodiments, the molecular weight of polymkeric substance is about 1, and 000Da and 40 is between the 000Da.In certain embodiments, the molecular weight of polymkeric substance is about 5, and 000Da and 40 is between the 000Da.In certain embodiments, the molecular weight of polymkeric substance is about 10, and 000Da and 40 is between the 000Da.In certain embodiments, peg molecule is a branched polymers.The molecular weight of side chain PEG can be about 1,000Da and about 100, and between the 000Da, it is including (but not limited to) 100,000Da, 95,000Da, 90,000Da, 85,000Da, 80,000Da, 75,000Da, 70,000Da, 65,000Da, 60,000Da, 55,000Da, 50,000Da, 45,000Da, 40,000Da, 35,000Da, 30,000Da, 25,000Da, 20,000Da, 15,000Da, 10,000Da, 9,000Da, 8,000Da, 7,000Da, 6,000Da, 5,000Da, 4,000Da, 3,000Da, 2,000Da and 1,000Da.In certain embodiments, the molecular weight of side chain PEG is about 1, and 000Da and 50 is between the 000Da.In certain embodiments, the molecular weight of side chain PEG is about 1, and 000Da and 40 is between the 000Da.In certain embodiments, the molecular weight of side chain PEG is about 5, and 000Da and 40 is between the 000Da.In certain embodiments, the molecular weight of side chain PEG is about 5, and 000Da and 20 is between the 000Da.It will be understood by one of ordinary skill in the art that aforementioned list about water-soluble in fact main chain detailed absolutely not and only for illustrative, and estimate that all polymeric materials with above-mentioned quality are applicable in the method and composition of describing herein.
As mentioned above, an example of hydrophilic polymer is polyoxyethylene glycol (being abbreviated as PEG), and it has been widely used in the medicine, on the artificial implant, and wherein biocompatibility, nontoxicity and non-immunogenicity have importance other use.The polymkeric substance of Miao Shuing herein: polypeptide embodiment will use PEG as exemplary hydrophilic polymer, should be appreciated that wherein other hydrophilic polymers can be used for these embodiment similarly.
PEG is the water-soluble polymers of knowing, and it can be buied on the market or can prepare (Sandler and Karo, Polymer Synthesis by the ring-opening polymerization of ethylene glycol according to the method for knowing in the affiliated field, Academic Press, New York, the 3rd volume, 138-161 page or leaf).In PEG normally transparent, colourless, tasteless, the water soluble, to thermally-stabilised, to many chemical agent inertia, not hydrolysis or rotten, and nontoxic usually.Think that polyoxyethylene glycol is biocompatible, PEG can coexist with living tissue or organism in other words, and does not work the mischief.More particularly, PEG is essentially non-immunogenic, and PEG does not trend towards producing immune response in vivo in other words.When the molecule (such as biologically active agent) that has some functions that need in being connected to body was gone up, PEG trended towards sheltering medicament and can reduce or eliminate any immune response so that organism can tolerate the existence of medicament.The PEG joiner trends towards not producing the immune response of essence or causes and condenses or other unwanted effects.
Be extensive use of term " PEG " and contain any peg molecule (not considering size or the modification on the PEG end), and when binding is to non-natural amino acid polypeptides, can be expressed from the next:
XO-(CH 2CH 2O) n-CH 2CH 2-Y
Wherein n be 2 to 10,000 and X be H or end modified, it is including (but not limited to) C 1-4Alkyl, protecting group or terminal functional group.Term PEG is including (but not limited to) its any type of polyoxyethylene glycol, it comprises difunctionality PEG, multi-arm PEG, the PEG that derives, forked PEG, branch PEG (wherein each chain has about 1kDa to about 100kDa, about 1kDa about 50kDa or the about 1kDa molecular weight of about 20kDa extremely extremely), the PEG that dangles (PEG or the related polymer that promptly have one or more functional groups that overhang main polymer chain), or wherein has the PEG of degradable binding.In one embodiment, wherein n is that about 20 to about 2000 PEG is applicable in the method and composition of describing herein.In certain embodiments, aqueous-based polymers comprises polyalkylene glycol moiety.The molecular weight of PEG polymkeric substance can be in broad range, and it is including (but not limited to) between about 100Da and about 100,000Da or higher between scope.The molecular weight of PEG polymkeric substance can be at about 100Da and about 100, and between the 000Da, it is including (but not limited to) 100,000Da, 95,000Da, 90,000Da, 85,000Da, 80,000Da, 75,000Da, 70,000Da, 65,000Da, 60,000Da, 55,000Da, 50,000Da, 45,000Da, 40,000Da, 35,000Da, 30,000Da, 25,000Da, 20,000Da, 15,000Da, 10,000Da, 9,000Da, 8,000Da, 7,000Da, 6,000Da, 5,000Da, 4,000Da, 3,000Da, 2,000Da, 1,000Da, 900Da, 800Da, 700Da, 600Da, 500Da, 400Da, 300Da, 200Da and 100Da.In certain embodiments, the molecular weight of polymkeric substance is in about 100Da and 50, between the 000Da.In certain embodiments, the molecular weight of polymkeric substance is in about 100Da and 40, between the 000Da.In certain embodiments, the molecular weight of polymkeric substance is about 1, and 000Da and 40 is between the 000Da.In certain embodiments, the molecular weight of polymkeric substance is about 5, and 000Da and 40 is between the 000Da.In certain embodiments, the molecular weight of polymkeric substance is about 10, and 000Da and 40 is between the 000Da.In certain embodiments, peg molecule is a branched polymers.The molecular weight of side chain PEG can be about 1,000Da and about 100, and between the 000Da, it is including (but not limited to) 100,000Da, 95,000Da, 90,000Da, 85,000Da, 80,000Da, 75,000Da, 70,000Da, 65,000Da, 60,000Da, 55,000Da, 50,000Da, 45,000Da, 40,000Da, 35,000Da, 30,000Da, 25,000Da, 20,000Da, 15,000Da, 10,000Da, 9,000Da, 8,000Da, 7,000Da, 6,000Da, 5,000Da, 4,000Da, 3,000Da, 2,000Da and 1,000Da.In certain embodiments, the molecular weight of side chain PEG is about 1, and 000Da and 50 is between the 000Da.In certain embodiments, the molecular weight of side chain PEG is about 1, and 000Da and 40 is between the 000Da.In certain embodiments, the molecular weight of side chain PEG is about 5, and 000Da and 40 is between the 000Da.In certain embodiments, the molecular weight of side chain PEG is about 5, and 000Da and 20 is between the 000Da.The PEG molecule of broad range is described in (comprise, but be not limited to) Shearwater Polymers, and in Inc. catalogue, the Nektar Therapeutics catalogue, it is to be incorporated herein by reference.
In the document terminal functional group's particular instance including (but not limited to) carbonic acid N-succinimide ester (referring to (for example) United States Patent (USP) the 5th, 281, No. 698, the 5th, 468, No. 478), amine is (referring to people such as (for example) Buckmann, Makromol.Chem.182:1379 (1981); People such as Zalipsky, Eur.Polym.J.19:1177 (1983)), hydrazides is (referring to people such as (for example) Andresz, Makromol.Chem.179:301 (1978)), propionic acid succinimide ester and butyric acid succinimide ester are (referring to people such as (for example) Olson, at Poly (ethylene glycol) Chemistry ﹠amp; 170-181 page or leaf among the BiologicalApplications, Harris and Zalipsky compile, ACS, Washington, D.C., 1997; Also referring to United States Patent (USP) the 5th, 672, No. 662), the succsinic acid succinimide ester is (referring to people such as (for example) Abuchowski, people such as Cancer Biochem.Biophys.7:175 (1984) and Joppich, Makromol.Chem.180:1381 (1979)), the succinimido ester is (referring to (for example) United States Patent (USP) the 4th, 670, No. 417), the carbonic acid benzotriazole is (referring to (for example) United States Patent (USP) the 5th, 650, No. 234), glycidyl ether is (referring to people such as (for example) Pitha, Eur.J Biochem.94:11 (1979); People such as Elling, Biotech.Appl.Biochem.13:354 (1991)), oxygen base carbonylic imidazole is (referring to people such as (for example) Beauchamp, Anal.Biochem.131:25 (1983); People such as Tondelli, J.ControlledRelease 1:251 (1985)), p-nitrophenyl carbonate ester is (referring to people such as (for example) Veronese, Appl.Biochem.Biotech., 11:141 (1985); And people such as Sartore, Appl.Biochem.Biotech., 27:45 (1991)), aldehyde (referring to people such as (for example) Harris, J.Polym.Sci.Chem.Ed.22:341 (1984); United States Patent (USP) the 5th, 824, No. 784; United States Patent (USP) the 5th, 252, No. 714), maleimide is (referring to people such as (for example) Goodson, Bio/Technology8:343 (1990); People such as Romani are in Chemistry of Peptides and Proteins 2:29 (1984); And Kogan, Synthetic Comm.22:2417 (1992)), adjacent pyridyl disulfide is (referring to people such as (for example) Woghiren, Bioconj.Chem.4:314 (1993)), vinylcarbinol is (referring to people such as (for example) Sawhney, Macromolecules, 26:581 (1993)), vinyl sulphone is (referring to (for example) United States Patent (USP) the 5th, 900, No. 461).All above-mentioned reference and patent are all to be incorporated herein by reference.
In some cases, PEG at one end goes up with hydroxyl or methoxyl group and stops, that is, X is H or CH 3(" methoxyl group PEG ").Perhaps, PEG can stop by reactive group, and then forms the double functional copolymer.The type reaction group can comprise be generally used for 20 kinds of common amino acids in the functional group that exists (including (but not limited to) maleimide base group, activated carbonate (including (but not limited to) p-nitrophenyl ester), Acibenzolar is (including (but not limited to) N-hydroxy-succinamide, p-nitrophenyl ester) and aldehyde) and to 20 kinds of common amino acid inertia but with alpha-non-natural amino acid in the functional group of complementary functional group's specific reaction of existing (including (but not limited to) oximido, carbonyl or dicarbapentaborane and azanol base) those reactive groups.
The other end that should note the PEG that represented by Y in following formula should be connected on polypeptide (synthetic or natural), polynucleotide, polysaccharide or the synthetic polymer directly or indirectly by alpha-non-natural amino acid.When Y is the azanol base, contain so azanol PEG reagent can with polypeptide in contain carbonyl or dicarbapentaborane alpha-non-natural amino acid reaction to form by the PEG group of oxime key binding to the polypeptide.When Y is carbonyl or dicarbapentaborane, contain so carbonyl or dicarbapentaborane PEG reagent can with polypeptide in contain azanol alpha-non-natural amino acid reaction to form by the PEG group of oxime key binding to the polypeptide.When Y is carbonyl or dicarbapentaborane, the PEG reagent that contains carbonyl or dicarbapentaborane so also can with polypeptide in contain oxime the alpha-non-natural amino acid reaction to form by the PEG group of new oxime key binding to the polypeptide.The example of appropriate reaction condition, purification process and reagent spreads all over this specification sheets and subsidiary graphic being described.For instance, Fig. 7 oblatio non-natural amino acid polypeptides that contains carbonyl and PEG reagent react that contains azanol forms three examples of the non-natural amino acid polypeptides that contain oxime of binding to the PEG group.In addition, Fig. 9 oblatio non-natural amino acid polypeptides that contains oxime and PEG reagent react that contains carbonyl forms two examples of the new non-natural amino acid polypeptides that contain oxime of binding to the PEG group.And the non-natural amino acid polypeptides that Figure 10 oblatio contains azanol and the PEG reagent react that contains carbonyl form an example of the non-natural amino acid polypeptides that contain oxime of binding to the PEG group.
Only for instance and be not as to the type of the PEG reagent of the composition that can be used for describing herein, method, technology and strategy or the restriction of kind, Figure 11 oblatio can with the non-natural amino acid polypeptides reaction that contains carbonyl other illustrative example with the PEG reagent that contains azanol that forms the non-natural amino acid polypeptides that contains oxime of binding to the PEG group, and can with the non-natural amino acid polypeptides that contains oxime or the non-natural amino acid polypeptides reaction that contains azanol to form the example of the new PEG reagent that contains carbonyl that contains oxime non-natural amino acid polypeptides of binding to the PEG group.Figure 12 oblatio is used to form the PEG reagent that contains azanol, or contain azanol PEG reagent through the protection form, or contain four illustrative example through the synthetic method of the form of sheltering of the PEG reagent of azanol.Figure 13 oblatio is used to form the PEG reagent that contains azanol through the acid amides binding, or through the PEG reagent that contains azanol of acid amides binding through the protection form, or through the illustrative example through the synthetic method of the form of sheltering of the PEG reagent that contains azanol of acid amides binding.Figure 14 and Figure 15 oblatio are used to form the PEG reagent that contains azanol through the carbamate binding; or through the PEG reagent that contains azanol of carbamate binding through the protection form, or through the illustrative example through the synthetic method of the form of sheltering of the PEG reagent that contains azanol of carbamate binding.Figure 16 oblatio is used to form the PEG reagent that simply contains azanol, or simply contain azanol PEG reagent through the protection form, or simply contain the illustrative example through the synthetic method of the form of sheltering of the PEG reagent of azanol.In addition, Figure 17 oblatio has the illustrative example of the reagent that contains azanol of the side chain of a plurality of binding PEG groups, and shows that further a kind of a plurality of PEG side chain reagent that this contains azanol and the non-natural amino acid polypeptides that contains carbonyl form the reaction that contains the oxime non-natural amino acid polypeptides of a plurality of PEG branched groups with binding.
In the time differing molecular need being connected to each terminal going up of polymkeric substance, the Heterobifunctional derivative also is especially to be suitable for.For instance, ω-N-amino-N-azido-PEG can allow that the molecule that will have activation electrophilic group (such as aldehyde, ketone, Acibenzolar, activated carbonate etc.) is connected on the end of PEG and the molecule that will have an inferior ethanoyl is connected on another end of PEG.
In certain embodiments, strong nucleophilic reagent (including (but not limited to) azanol) can with the aldehyde radical that exists in the alpha-non-natural amino acid or ketone group reaction to form oxime, it can further reduce by handling with suitable reductive agent in some cases.Perhaps, strong nucleophilic reagent ketone group or the aldehyde radical preferential reaction that can incorporate in the polypeptide and be used for existing by alpha-non-natural amino acid with water-soluble polymers.Usually, at least one end of PEG molecule can be used for reacting with alpha-non-natural amino acid.
Therefore, in certain embodiments, comprise that the polypeptide of alpha-non-natural amino acid passes through the side chain binding of alpha-non-natural amino acid to water-soluble polymers (such as polyoxyethylene glycol (PEG)).The alpha-non-natural amino acid method and composition of Miao Shuing is provided for the height effective ways with PEG derivatives selectively modifying protein herein, it comprises alpha-non-natural amino acid (naturally incorporating non-existent functional group or substituent those amino acid in the amino acid into including (but not limited to) containing 20 kinds) selectivity incorporated in the protein selects codon to respond, and modifies those amino acid to be fit to reactive PEG derivative subsequently.Multiple known chemistry is applicable to that the alpha-non-natural amino acid method and composition of describing is to incorporate water-soluble polymers in the protein into herein.
Main polymer chain can be linear or branched.It is known in the field under the branched polymers main chain is generally.Usually, branched polymers has center branch nuclear part and a plurality of linear polymer chain that is connected on the branch nuclear of center.PEG uses with the branch form, and it can prepare by oxyethane being added on the various polyvalent alcohols (such as glycerol, glycerol oligomer, tetramethylolmethane and Sorbitol Powder).Center branch part also can be derived from some amino acid, such as Methionin.The branch polyoxyethylene glycol can be expressed as R (PEG-OH) by common version m, wherein R is that such as glycerol, glycerol oligomer or tetramethylolmethane, and m represents the number of arm derived from the nuclear part.Multi-arm PEG molecule (such as United States Patent (USP) the 5th, 932, No. 462; The 5th, 643, No. 575; The 5th, 229, No. 490; The 4th, 289, No. 872; U.S. patent application case 2003/0143596; WO 96/21469; And those PEG molecules of describing among the WO 93/21259, each of these documents is all to be incorporated herein by reference) also can be used as main polymer chain.
Branch PEG also can be for by PEG (YCHZ 2) nThe form of forked PEG of expression, wherein to be binding group and Z be the activation end group of the atomchain binding by determining length to the CH to Y.Another branch form PEG that dangles has reactive group such as carboxyl along the PEG main chain but not in the end of PEG chain.
Except that these forms of PEG, also can prepare the polymkeric substance that in main chain, has weak bond or degradable linkage.For instance, can prepare the PEG that has ester bond in main polymer chain, it experiences hydrolysis.Shown in herein, this hydrolysis makes that polymer cracking is the fragment of lower molecular weight:
-PEG-CO 2-PEG-+H 2O→PEG-CO 2H+HO-PEG-。
It will be understood by one of ordinary skill in the art that term polyoxyethylene glycol or PEG represent or comprise under known form of ownership in the field, it is including (but not limited to) those forms that disclose herein.The molecular weight of polymkeric substance can be in broad range, and it is including (but not limited to) between about 100Da and about 100,000Da or higher between scope.The molecular weight of polymkeric substance can be at about 100Da and about 100, and between the 000Da, it is including (but not limited to) 100,000Da, 95,000Da, 90,000Da, 85,000Da, 80,000Da, 75,000Da, 70,000Da, 65,000Da, 60,000Da, 55,000Da, 50,000Da, 45,000Da, 40,000Da, 35,000Da, 30,000Da, 25,000Da, 20,000Da, 15,000Da, 10,000Da, 9,000Da, 8,000Da, 7,000Da, 6,000Da, 5,000Da, 4,000Da, 3,000Da, 2,000Da, 1,000Da, 900Da, 800Da, 700Da, 600Da, 500Da, 400Da, 300Da, 200Da and 100Da.In certain embodiments, the molecular weight of polymkeric substance is in about 100Da and 50, between the 000Da.In certain embodiments, the molecular weight of polymkeric substance is in about 100Da and 40, between the 000Da.In certain embodiments, the molecular weight of polymkeric substance is about 1, and 000Da and 40 is between the 000Da.In certain embodiments, the molecular weight of polymkeric substance is about 5, and 000Da and 40 is between the 000Da.In certain embodiments, the molecular weight of polymkeric substance is about 10, and 000Da and 40 is between the 000Da.
Required character for maximization PEG, PEG polymkeric substance or be connected to the total molecular weight of the polymkeric substance on the bioactive molecules and hydration status should be enough high to give usually and the favorable characteristics of PEG polymkeric substance join dependency, increase such as water-soluble and circulating half-life, and can sharp influence the biological activity of parent molecule.
The method and composition of Miao Shuing can be used for preparing the polymkeric substance of homogeneous in fact herein: the preparation of protein joiner." homogeneous in fact " means and observe polymkeric substance as used herein: half of protein joiner molecular ratio gross protein is bigger.Polymkeric substance: protein joiner biologically active and " homogeneous in fact " the of the present invention PEGization polypeptide formulations that provides herein be enough homogeneous with the advantage that shows homogeneous preparation (for example, be easy to clinical application in batch between in the prediction of pharmacokinetics) those preparations.
Also can select to prepare polymkeric substance: the mixture of protein joiner molecule, and the advantage that provides herein is can to select to be included in the single polymkeric substance in the mixture: the ratio of protein joiner.Therefore, in case of necessity, the polymer moieties that can prepare range protein and the various numbers that are connected (promptly, dipolymer part, trimer part, tetramer part etc.) mixture and with these joiners and the single polymkeric substance that uses the method preparation of describing herein: protein joiner combination, and make mixture have single polymkeric substance of predetermined proportion: protein joiner.
The ratio regular meeting of peg molecule and protein molecule along with its in reaction mixture concentration and change.In general, best ratio (being to exist minimum excessive unreacted protein or polymkeric substance with regard to reaction efficiency) can be determined with the number of reactive group by the molecular weight of selected polyoxyethylene glycol and about available.About molecular weight, the molecular weight of polymkeric substance is high more usually, and the number that can be connected to the polymer molecule on the protein is few more.Similarly, when optimizing these parameters, should be taken into account the branch of polymkeric substance.Usually, molecular weight high more (or branch is many more), polymkeric substance: protein rate is high more.
As used herein, and when containing hydrophilic polymer: during the polypeptides joiner, the amount that provides required benefit to increase to the patient further is provided term " treatment significant quantity ".Described amount can change along with Different Individual and should depend on numerous factors, and it comprises patient's the comprehensive physical state and the potential cause of disease of disease, illness or symptom that institute's desire is treated.The treatment significant quantity of composition of the present invention can be easy to use the material and the program that can openly obtain to determine by the those skilled in the art.
The number of the water-soluble polymers of the binding that adjustable joint is described herein to " modified or not modified " non-natural amino acid polypeptides (promptly, PEGization or glycosylated degree) with provide change (including (but not limited to) increase or reduce) pharmacology, pharmacokinetics or pharmacodynamic properties, such as transformation period in vivo.In certain embodiments, the transformation period of polypeptide than increase at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% without modified polypeptides, twice, five times, 10 times, 50 times or at least about 100 times.
In one embodiment, comprise that the polypeptide of the alpha-non-natural amino acid that contains carbonyl or dicarbapentaborane is modified through the PEG derivative that contains terminal azanol part, its direct binding is to the PEG main chain.
In certain embodiments, the terminal PEG derivative of azanol has following structure:
RO-(CH 2CH 2O) n-O-(CH 2) m-O-NH 2
Wherein R is simple alkyl (methyl, ethyl, a propyl group etc.), and m is that 2-10 and n are 100-1,000 (that is, molecular-weight average is between 5kDa-40kDa).The molecular weight of polymkeric substance can be in broad range, and it is including (but not limited to) between about 100Da and about 100,000Da or higher between scope.The molecular weight of polymkeric substance can be at about 100Da and about 100, and between the 000Da, it is including (but not limited to) 100,000Da, 95,000Da, 90,000Da, 85,000Da, 80,000Da, 75,000Da, 70,000Da, 65,000Da, 60,000Da, 55,000Da, 50,000Da, 45,000Da, 40,000Da, 35,000Da, 30,000Da, 25,000Da, 20,000Da, 15,000Da, 10,000Da, 9,000Da, 8,000Da, 7,000Da, 6,000Da, 5,000Da, 4,000Da, 3,000Da, 2,000Da, 1,000Da, 900Da, 800Da, 700Da, 600Da, 500Da, 400Da, 300Da, 200Da and 100Da.In certain embodiments, the molecular weight of polymkeric substance is in about 100Da and 50, between the 000Da.In certain embodiments, the molecular weight of polymkeric substance is in about 100Da and 40, between the 000Da.In certain embodiments, the molecular weight of polymkeric substance is about 1, and 000Da and 40 is between the 000Da.In certain embodiments, the molecular weight of polymkeric substance is about 5, and 000Da and 40 is between the 000Da.In certain embodiments, the molecular weight of polymkeric substance is about 10, and 000Da and 40 is between the 000Da.
In another embodiment, comprise the amino acid whose polypeptide that contains carbonyl or dicarbapentaborane through containing the PEG derivative modification of terminal azanol part, it passes through the amido linkage binding to the PEG main chain.
In certain embodiments, the terminal PEG derivative of azanol has following structure:
RO-(CH 2CH 2O) n-O-(CH 2) 2-NH-C(O)(CH 2) m-O-NH 2
Wherein R is simple alkyl (methyl, ethyl, a propyl group etc.), and m is that 2-10 and n are 100-1,000 (that is, molecular-weight average is between 5kDa-40kDa).The molecular weight of polymkeric substance can be in broad range, and it is including (but not limited to) between about 100Da and about 100,000Da or higher between scope.The molecular weight of polymkeric substance can be at about 100Da and about 100, and between the 000Da, it is including (but not limited to) 100,000Da, 95,000Da, 90,000Da, 85,000Da, 80,000Da, 75,000Da, 70,000Da, 65,000Da, 60,000Da, 55,000Da, 50,000Da, 45,000Da, 40,000Da, 35,000Da, 30,000Da, 25,000Da, 20,000Da, 15,000Da, 10,000Da, 9,000Da, 8,000Da, 7,000Da, 6,000Da, 5,000Da, 4,000Da, 3,000Da, 2,000Da, 1,000Da, 900Da, 800Da, 700Da, 600Da, 500Da, 400Da, 300Da, 200Da and 100Da.In certain embodiments, the molecular weight of polymkeric substance is in about 100Da and 50, between the 000Da.In certain embodiments, the molecular weight of polymkeric substance is in about 100Da and 40, between the 000Da.In certain embodiments, the molecular weight of polymkeric substance is about 1, and 000Da and 40 is between the 000Da.In certain embodiments, the molecular weight of polymkeric substance is about 5, and 000Da and 40 is between the 000Da.In certain embodiments, the molecular weight of polymkeric substance is about 10, and 000Da and 40 is between the 000Da.
In another embodiment, comprise that the amino acid whose polypeptide that contains carbonyl or dicarbapentaborane is through containing the PEG derivative modification of terminal azanol part, wherein each chain of branch PEG has the MW in the 10kDa-40kDa scope, and in other embodiments, in the 5kDa-20kDa scope.The molecular weight of branched polymers can be in broad range, and it is including (but not limited to) between about 100Da and about 100,000Da or higher between scope.The molecular weight of polymkeric substance can be at about 100Da and about 100, and between the 000Da, it is including (but not limited to) 100,000Da, 95,000Da, 90,000Da, 85,000Da, 80,000Da, 75,000Da, 70,000Da, 65,000Da, 60,000Da, 55,000Da, 50,000Da, 45,000Da, 40,000Da, 35,000Da, 30,000Da, 25,000Da, 20,000Da, 15,000Da, 10,000Da, 9,000Da, 8,000Da, 7,000Da, 6,000Da, 5,000Da, 4,000Da, 3,000Da, 2,000Da, 1,000Da, 900Da, 800Da, 700Da, 600Da, 500Da, 400Da, 300Da, 200Da and 100Da.In certain embodiments, the molecular weight of polymkeric substance is in about 100Da and 50, between the 000Da.In certain embodiments, the molecular weight of polymkeric substance is in about 100Da and 40, between the 000Da.In certain embodiments, the molecular weight of polymkeric substance is about 1, and 000Da and 40 is between the 000Da.In certain embodiments, the molecular weight of polymkeric substance is about 5, and 000Da and 40 is between the 000Da.In certain embodiments, the molecular weight of polymkeric substance is about 10, and 000Da and 40 is between the 000Da.
In another embodiment, the polypeptide that comprises alpha-non-natural amino acid is modified through at least a PEG derivative with apparatus derivatorius.In certain embodiments, the PEG derivative that contains the azanol base has following structure:
[RO-(CH 2CH 2O) n-O-(CH 2) 2-C(O)-NH-CH 2-CH 2] 2CH-X-(CH 2) m-O-NH 2
Wherein R is simple alkyl (methyl, ethyl, a propyl group etc.), and X is NH, O, S, C (O) or does not exist that m is that 2-10 and n are 100-1 according to circumstances, 000.The molecular weight of polymkeric substance can be about 1,000Da and about 100, and between the 000Da, it is including (but not limited to) 100,000Da, 95,000Da, 90,000Da, 85,000Da, 80,000Da, 75,000Da, 70,000Da, 65,000Da, 60,000Da, 55,000Da, 50,000Da, 45,000Da, 40,000Da, 35,000Da, 30,000Da, 25,000Da, 20,000Da, 15,000Da, 10,000Da, 9,000Da, 8,000Da, 7,000Da, 6,000Da, 5,000Da, 4,000Da, 3,000Da, 2,000Da and 1,000Da.In certain embodiments, the molecular weight of side chain PEG is about 1, and 000Da and 50 is between the 000Da.In certain embodiments, the molecular weight of side chain PEG is about 1, and 000Da and 40 is between the 000Da.In certain embodiments, the molecular weight of side chain PEG is about 5, and 000Da and 40 is between the 000Da.In certain embodiments, the molecular weight of side chain PEG is about 5, and 000Da and 20 is between the 000Da.
Can obtain some general introductions and monograph about the functionalized of PEG and joint.Referring to (for example) Harris, Macromol.Chem.Phys.C25:325-373 (1985); Scouten, Methods in Enzymology 135:30-65 (1987); People such as Wong, Enzyme Microb.Technol.14:866-874 (1992); People such as Delgado, Critical Reviewsin Therapeutic Drug Carrier Systems 9:249-304 (1992); Zalipsky, Bioconjugate Chem.6:150-165 (1995).
The method that is used for activated polymer also is found in WO 94/17039, United States Patent (USP) the 5th, 324, No. 844, WO94/18247, WO 94/04193, United States Patent (USP) the 5th, 219, No. 564, United States Patent (USP) the 5th, 122, No. 614, WO90/13540, United States Patent (USP) the 5th, 281, No. 698, and among more WO 93/15189, and be used for the method that engages between activated polymer and the enzyme including (but not limited to) blood coagulation factor VIII (WO 94/15625), oxyphorase (WO94/09027), oxygen carrier molecule (United States Patent (USP) the 4th, 412,989), rnase and superoxide-dismutase (people such as Veronese, App.Biochem.Biotech.11:141-152 (1985)), all these documents are all to be incorporated herein by reference.
In case of necessity, the PEGization non-natural amino acid polypeptides of describing herein that obtains from hydrophobic chromatography can be further purified by the known program of one or more those skilled in the art, and these programs comprise (but being not limited to) affinity chromatography; Negatively charged ion or cation-exchange chromatography (using (comprise, but be not limited to) DEAE SEPHAROSE); The silica chromatogram; Reversed-phase HPLC; Gel-filtration (using (comprise, but be not limited to) SEPHADEX G-75); Hydrophobic interaction chromatograph; Size exclusion chromatography; Immobilized metal ion afinity chromatography; Ultrafiltration/diafiltration; Ethanol sedimentation; Ammonium sulfate precipitation; Chromatofocusing; Displcement chromatography; Electrophoretic procedures (including (but not limited to) the isoelectrofocusing of preparation type), differential solvability (including (but not limited to) ammonium sulfate precipitation) or extraction.By GPC by with the comparison of globular preteins standard substance can estimate apparent molecular weight (Preneta AZ, PROTEIN PURIFICATION METHODS, A PRACTICAL APPROACH (Harris compile with Angal) IRL Press 1989,293-306).Non-natural amino acid polypeptides: the purity of PEG joiner can be by proteolytic degradation (including (but not limited to) the trypsinase cracking), then mass spectroscopy is assessed.People such as Pepinsky RB., J.Pharmacol.﹠amp; Exp.Ther.297 (3): 1059-66 (2001).
But the water-soluble polymers without stint of binding to the alpha-non-natural amino acid of the polypeptide of describing herein is through further deriving or replacing.
E. strengthen sero-abluminous affinity
Various molecules also can merge to be adjusted in the transformation period in the serum with the non-natural amino acid polypeptides of describing herein.In certain embodiments, molecule and " modified or not modified " the non-natural amino acid polypeptides binding of describing herein or fusion are to strengthen endogenous sero-abluminous affinity in the animal.
For instance, in some cases, the reorganization of carrying out polypeptide and albumin bound sequence is merged.Exemplary albumin bound sequence including (but not limited to) from the albumin bound territory of streptococcus protein G (referring to people such as (for example) Makrides, people such as J.Pharmacol Exp.Ther.277 (1): 534-542 (1996) and Sjolander, J, Immunol.Methods 201:115-123 (1997)), or albumin binding peptide, such as people such as (for example) Dennis, those albumin binding peptides of describing among J.Biol.Chem.277 (38): the 35035-35043 (2002).
In other embodiments, " modified or not modified " non-natural amino acid polypeptides of describing herein is through the lipid acid acidylate.In some cases, lipid acid promotes to combine with sero-abluminous.Referring to people such as (for example) Kurtzhals, Biochem.J.312:725-731 (1995).
In other embodiments, direct and serum albumin (including (but not limited to) the human serum albumin) fusion of " modified or not modified " non-natural amino acid polypeptides of describing herein.But it will be understood by one of ordinary skill in the art that also binding combining with adjusting and serum albumin or other serum components to the modified or not modified as described herein non-natural amino acid polypeptides of multiple other molecules.
F. the glycosylation of the non-natural amino acid polypeptides of describing herein
The method and composition of Miao Shuing comprises and is combined with the one or more polypeptide that has the alpha-non-natural amino acid of saccharide residue herein.Saccharide residue can be natural (including (but not limited to) the N-acetyl glucosamine) or non-natural (including (but not limited to) 3-fluorine semi-lactosi).Described sugar can be by N binding or O binding glycosidic link (including (but not limited to) N-ethanoyl semi-lactosi-L-Serine) or non-natural key (including (but not limited to) the glucosides of oxime or corresponding C binding or S binding) binding to alpha-non-natural amino acid.
Sugar (including (but not limited to) glycosyl) part can in vivo or in vitro be added on the non-natural amino acid polypeptides.In certain embodiments, comprise that the polypeptide of the alpha-non-natural amino acid that contains carbonyl or dicarbapentaborane is through passing through the corresponding glycosylated polypeptides that the oxime key connects with the sugar-modified of amino oxygen base derivatize to produce.In case be connected on the alpha-non-natural amino acid, sugar can be attached to oligosaccharides on the non-natural amino acid polypeptides by handling with glycosyltransferase and other enzymes further to make with extra care with generation.Referring to people such as (for example) H.Liu, J.Am.Chem.Soc.125:1702-1703 (2003).
G. the binding group uses and comprises polypeptide dimer and polymeric application
Except directly to non-natural amino acid polypeptides adds the functional group, the alpha-non-natural amino acid part of polypeptide can be at first through multifunctional (for example, difunctionality, trifunctional, four senses) connexon molecular modification, and it is with after further modify.That is to say that at least one end of multifunctional connexon molecule reacts with at least one alpha-non-natural amino acid in the polypeptide and at least one other end of multifunctional connexon can be used for further functionalized.If all ends of multifunctional connexon are identical, (decide) to form the homopolymer of non-natural amino acid polypeptides so on stoichiometric condition.If the end of multifunctional connexon has different chemical reactivities, at least one of so much sense connexon group is terminal can react with different functional groups subsequently with non-natural amino acid polypeptides combination and other ends, and these functional groups comprise (only for instance): mark; Dyestuff; Polymkeric substance; Water-soluble polymers; The derivative of polyoxyethylene glycol; Photocrosslinking agent; Cytotoxic compound; Medicine; Affinity marker; The photoaffinity mark; Reactive compounds; Resin; Second protein or polypeptide or polypeptide analog; Antibody or antibody fragment; Metal chelator; Cofactor; Lipid acid; Carbohydrate; Polynucleotide; DNA; RNA; Antisense polynucleotide; Carbohydrate, water-soluble branch-shape polymer, cyclodextrin, biomaterial; Nanoparticle; Spin labeling; Fluorophore, metallic part; Radioactive segment; Novel functional group; With the covalently or non-covalently interactional group of other molecules; The light cage covers part; But the part that actinic radiation excites; Ligand; But photoisomerization part; Vitamin H; The vitamin H analogue; Be combined with the part of heavy atom; But the group of chemical cracking; But the group of photodestruciton; The side chain that prolongs; The sugar of carbon bond connection; Redox active agent; Amino thioic acid sulfoacid; The toxicity part; Through isotope-labeled part; The biophysics probe; The phosphorescence group; Chemiluminescent groups; The electron dense group; The magnetic group; Insert group; Chromophoric group; Energy transfer agent; Biologically active agent; Detectable label; Small molecules; Inhibition Yeast Nucleic Acid, radioactive nuleus thuja acid; Neutron capture agent; The derivative of vitamin H; Quantum dot; Nanometer is transmitted plain; Radioactivity is transmitted plain; Abzyme, activation composite activating agent, virus, adjuvant, aglycone, anaphylactogen, angiostatin, hormone antagonist, antioxidant, fit, guide RNA, Saponin/TSM, shuttle vectors, macromole, plan epitope, acceptor, reverse micelle with and any combination.
Multifunctional connexon group has following universal architecture:
Wherein:
X is NH independently of one another 2,-C (=O) R 9,-SR ' or-J-R, wherein R 9Be H or OR ', wherein J is
Figure A20058004432802282
R is H, alkyl, is substituted alkyl, cycloalkyl or is substituted cycloalkyl; R " independently of one another for H, alkyl, " during group, two R " forms Heterocyclylalkyl according to circumstances to be substituted alkyl or protecting group, maybe when there being more than one R;
R ' is H, alkyl independently of one another or is substituted alkyl;
L is selected from the group that is made up of following each group independently of one another: alkylidene group, be substituted alkylidene group, alkenylene, be substituted alkenylene ,-O-,-O-(alkylidene group or be substituted alkylidene group)-,-S-,-S-(alkylidene group or be substituted alkylidene group)-,-S (O) k-(wherein k is 1,2 or 3) ,-S (O) k(alkylidene group or be substituted alkylidene group)-,-C (O)-,-C (O)-(alkylidene group or be substituted alkylidene group)-,-C (S)-,-C (S)-(alkylidene group or be substituted alkylidene group)-,-N (R ')-,-NR '-(alkylidene group or be substituted alkylidene group)-,-C (O) N (R ')-,-CON (R ')-(alkylidene group or be substituted alkylidene group)-,-(alkylidene group or be substituted alkylidene group) NR ' C (O) O-(alkylidene group or be substituted alkylidene group)-,-O-CON (R ')-(alkylidene group or be substituted alkylidene group)-,-CSN (R ')-,-CSN (R ')-(alkylidene group or be substituted alkylidene group)-,-N (R ') CO-(alkylidene group or be substituted alkylidene group)-,-N (R ') C (O) O-,-N (R ') C (O) O-(alkylidene group or be substituted alkylidene group)-,-S (O) kN (R ')-,-N (R ') C (O) N (R ')-,-N (R ') C (O) N (R ')-(alkylidene group or be substituted alkylidene group)-,-N (R ') C (S) N (R ')-,-N (R ') S (O) kN (R ')-,-N (R ')-N=,-C (R ')=N-,-C (R ')=N-N (R ')-,-C (R ')=N-N=,-C (R ') 2-N=N-and-C (R ') 2-N (R ')-N (R ')-;
L1 is optionally, and when existing is-C (R ') p-NR '-C (O) O-(alkylidene group or be substituted alkylidene group)-, wherein p is 0,1 or 2; W is NH 2,-C (=O) R 9,-SR ' or-J-R; And n is 1 to 3;
Its restricted condition is X and L-L 1-W provides at least one in following each group jointly independently of one another: (a) the azanol base that can react with the carbonyl (comprising dicarbapentaborane) on alpha-non-natural amino acid or " modified or not modified " non-natural amino acid polypeptides; (b) can with the carbonyl (comprising dicarbapentaborane) of azanol radical reaction on alpha-non-natural amino acid or " modified or not modified " non-natural amino acid polypeptides; Or (c) can experience and alpha-non-natural amino acid or " modified or not modified " non-natural amino acid polypeptides on the carbonyl (comprising dicarbapentaborane) of oximido generation permutoid reaction.
Figure 18 oblatio synthesizes illustrative, the limiting examples of difunctionality homotype connexon (wherein said connexon has two same end, that is, azanol base).This connexon can be used for forming the homodimer of the non-natural amino acid polypeptides that contains carbonyl or dicarbapentaborane to form two oxime keys.Perhaps; if an end of this connexon is through protection; this part can be used for by the oxime key azanol end without protection being combined with the non-natural amino acid polypeptides that contains carbonyl or dicarbapentaborane through the connexon of protection so, stays another end through protection and is going to be used for other binding reactions after the protection.Perhaps, can provide similar result (heterodimer), though can obtain the result that wherein required heterodimer can be polluted by some homodimers similarly to the stoichiometric carefully handled of reagent.
Figure 19 oblatio wherein each connexon has illustrative, the limiting examples of two kinds of multifunctional special-shaped connexons of the terminal-reactive group (that is, azanol base, oximido and thioester substrate) of more than one type.Use spreads all over the chemistry based on oxime that this specification sheets is discussed, and this connexon can be used for forming the heterodimer of non-natural amino acid polypeptides.
Figure 20 oblatio special-shaped difunctionality connexon of use in multistep is suddenly synthetic is connected to schematically illustrating property, limiting examples on the non-natural amino acid polypeptides with the PEG group.In a first step, so described in the illustrative drawings, the non-natural amino acid polypeptides that contains carbonyl and the difunctionality connexon reaction that contains azanol form the modified oxime non-natural amino acid polypeptides that contains.Yet, the difunctionality connexon still keep can with the reagent with appropriate reaction (in graphic by the explanation of the object of the matched shape) functional group (herein by visible object explanation) of reaction to form the modified functionalized non-natural amino acid polypeptides that contains oxime.In this certain illustrative was graphic, sense turned to the PEG group, but also can comprise among the aforementioned functional group any one, or under the situation of trifunctional or four sense connexons, comprised functional group or polytype identical functional group of more than one types.The illustrative example of four type the connexon group of the non-natural amino acid polypeptides binding that Figure 21 oblatio is used for containing azanol to the PEG group.As previously mentioned, the PEG functional group only provides for the illustrative purpose.Therefore, the connexon group of describing herein provides the another kind of method of further modifying non-natural amino acid polypeptides in the site selectivity mode.
The method and composition of Miao Shuing also provides polypeptides in combination herein, such as homodimer, heterodimer, homopolymer or heterogeneous polymer (that is, tripolymer, the tetramer etc.).Only for instance, below describe and focus on GH supergene family member, yet, the method of describing in this part, technology and composition can be applicable to be almost any other polypeptide of the provided benefit of dimer and polymer form, and it comprises (only for instance): α-1 antitrypsin, angiostatin, AHF, antibody, lipophorin, apoprotein, atrionatriuretic factor, atrial natriuretic polypeptins, atrial natriuretic peptide, the C-X-C chemokine, T39765, NAP-2, ENA-78, gro-a, gro-b, gro-c, IP-10, GCP-2, NAP-4, SDF-1, PF4, MIG, calcitonin, the c-kit ligand, cytokine, the CC chemokine, MCP-1, MCP-2, monocyte chemoattractant protein-3, monocyte inflammatory protein-1 α, monocyte inflammatory protein-i β, RANTES, 1309, R83915, R91733, HCC1, T58847, D31065, T64262, CD40, the CD40 ligand, the c-kit ligand, collagen protein, G CFS (CSF), complement factor 5a, complement inhibitor, complement receptor 1, cytokine, epithelium neutrophilic granulocyte activation peptide-78, MIP-16, MCP-1, Urogastron (EGF), epithelium neutrophilic granulocyte activation peptide, erythropoietin (EPO), exfoliative toxin,or exfoliatin, factors IX, factor VII, Factor IX, factor X, fibroblast growth factor (FGF), Fibrinogen, fibronectin, four-helix bundle albumen, G-CSF, glp-1, GM-CSF, glucocerebrosidase, gonad-stimulating hormone, somatomedin, growth factor receptors, grf, Shh, hemochrome, pHGF (hGF), r-hirudin, human growth hormone (hGH), the human serum albumin, ICAM-1, the ICAM-1 acceptor, LFA-1, the LFA-1 acceptor, Regular Insulin, insulin-like growth factor (IGF), IGF-I, IGF-II, Interferon, rabbit (IFN), IFN-α, IFN-β, IFN-γ, interleukin-(IL), IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, keratinocyte growth factor (KGF), lactoferrin, leukaemia inhibitory factor, luciferase, neural plain, neutrophil inhibitory factor (nif) (NIF), oncostatin M, bone morphogenic protein, oncoprotein, paracitonin, Rat parathyroid hormone 1-34, PD-ECSF, PDGF, peptide hormone, PTN, albumin A, Protein G, pth, pyrogenic exotoxin A, pyrogenic exotoxin B, pyrogenic exotoxin C, pyy, Relaxin, feritin, SCF, the atom synthetic proteins, soluble complement acceptor I, solubility I-CAM1, soluble interleukin receptor, soluble TNF acceptor, somatomedin, Somat, tethelin, streptokinase, superantigen, staphyloentero-toxin, SEA, SEB, SEC1, SEC2, SEC3, SED, SEE, steroid hormone receptor, superoxide-dismutase, the toxic shock syndrome toxin, thymosin, tissue type plasminogen activator, tumor growth factor (TGF), tumour necrosis factor, tumor necrosis factor alpha, tumor necrosis factor, Tumor Necrosis Factor Receptors (TNFR), VLA-4 albumen, VCAM-1 albumen, vascular endothelial growth factor (VEGF), urokinase, mos, ras, raf, met, p53, tat, fos, myc, jun, myb, rel, estrogen receptor, PgR, the testosterone acceptor, aldosterone receptor, ldl receptor and Kendall compound.
Therefore, contain in method, technology and the composition of describing herein and directly be bonded to the polypeptide main chain or be bonded to another GH supergene family member or its variant or non-GH supergene family member's any other polypeptide or the GH supergene family member polypeptide that contains one or more alpha-non-natural amino acids on its variant by connexon.Because comparing molecular weight with monomer, it increases, so GH supergene family member's dimer or polymer joiner can represent the character of novelty or needs, it is including (but not limited to) with respect to monomer GH supergene family member, different pharmacology, pharmacokinetics, pharmacodynamics, the treatment transformation period through regulating or the plasma half-life through regulating.In certain embodiments, the GH supergene family member dimer of describing herein can be regulated the dimerization of GH supergene family member acceptor.In other embodiments, GH supergene family member's dimer or the polymer of describing herein can serve as GH supergene family member receptor antagonist, agonist or conditioning agent.
In certain embodiments, GH supergene family member polypeptide is through direct binding, and it is including (but not limited to) passing through Asn-Lys amido linkage or Cys-Cys disulfide linkage.In certain embodiments, the non-GH supergene family member of GH supergene family member's polypeptide of binding and/or binding can comprise the different alpha-non-natural amino acid that promotes dimerization, it is including (but not limited to) a GH supergene family member, and/or the non-GH supergene family member of binding, comprise that the polypeptide that contains the ketone alpha-non-natural amino acid and the described polypeptide that engage with the 2nd GH supergene family member polypeptide that comprises the alpha-non-natural amino acid that contains azanol react by forming corresponding oxime.
Perhaps, the non-GH supergene family member of two kinds of GH supergene family member's polypeptide and/or binding connects by connexon.Can use any special-shaped difunctionality connexon or homotype difunctionality connexon to connect two kinds of GH supergene family members, and/or non-GH supergene family member, the polypeptide of binding, it can have identical or different primary sequence.In some cases, be used for, and/or the connexon that non-GH supergene family member, the polypeptide of binding are strapped in together can be difunctionality PEG reagent GH supergene family member.
In certain embodiments, the method and composition of Miao Shuing provides the water-soluble difunctionality connexon with dumbbell structure herein, and described structure comprises: a) trinitride, alkynes, hydrazine, hydrazides, the azanol at least the first end of main polymer chain or contain carbonyl or the part of dicarbapentaborane; And b) at least one second functional group on second end of main polymer chain.Second functional group can be identical or different with first functional group.In certain embodiments, second functional group does not react with first functional group.In certain embodiments, the method and composition of describing herein provides the water-soluble cpds of the arm that comprises at least one branch molecular structure.For instance, the branch molecular structure can be dendroid.
In certain embodiments, the method and composition of describing herein provides by reacting the polymer that comprises one or more GH supergene family members that forms with the water-soluble activated polymer with following structure:
R-(CH 2CH 2O) n-O-(CH 2) m-X
Wherein n is about 5 to 3,000, and m is 2-10, and X can be trinitride, alkynes, hydrazine, hydrazides, amino oxygen base, azanol, ethanoyl, or contains the part of carbonyl or dicarbapentaborane, and R is capping group, functional group, or leavings group that can be identical or different with X.R can be the functional group that (for example) is selected from the group that is made up of following each group: hydroxyl; through the protection hydroxyl; alkoxyl group; N-hydroxyl amber vinegar imido grpup ester; 1-benzotriazole base ester; carbonic acid N-hydroxyl amber vinegar imido grpup ester; carbonic acid 1-benzotriazole base ester; acetal; aldehyde; aldehydrol; thiazolinyl; acrylate; methacrylic ester; acrylamide; active sulfone; amine; the amino oxygen base; through protection amine; hydrazides; through the protection hydrazides; through protection mercaptan; carboxylic acid; through the protection carboxylic acid; isocyanic ester; lsothiocyanates; maleimide; vinyl sulphone; the dithio pyridine; vinyl pyridine; iodo-acid amide; epoxide; dialdehyde; diketone; methanesulfonates; tosylate, and trifluoroethyl sulphonate; alkene and ketone.
Figure 22 oblatio uses the connexon group of describing to form illustrative, the limiting examples of the homodimer of the non-natural amino acid polypeptides of describing herein herein.In this illustrative example, the non-natural amino acid polypeptides that contains carbonyl is contained under the condition of oxime non-natural amino acid polypeptides and the difunctionality connexon radical reaction with two available azanol bases at the homodimer that is suitable for forming binding.In graphic the method for oblatio be not limited to the difunctionality connexon coupling that contains azanol contain the carbonyl non-natural amino acid polypeptides.Non-natural amino acid polypeptides can further contain can with the multifunctional connexon group experience permutoid reaction that contains carbonyl forming the oximido of the homopolymer that is connected by the structure that contains a plurality of oximidos, or non-natural amino acid polypeptides can further contain the azanol base of the homopolymer that can be connected by the structure that contains a plurality of oximidos with formation with the multifunctional connexon group experience reaction that contains carbonyl.Certainly, homopolymer can be homodimer, homotrimer or the homotype tetramer.
Figure 23 oblatio uses special-shaped official's energy connexon group to form illustrative, the limiting examples of the heterodimer of polypeptide, wherein at least one member in the heterodimer is that the non-natural amino acid polypeptides described herein and other members are the non-natural amino acid polypeptides of non-natural amino acid polypeptides, other types as described herein according to circumstances, or the natural amino acid polypeptide that exists.In this is graphic in the example of oblatio, the connexon group contains two identical azanol bases, by other parameters of control stoichiometry, temperature and reaction, the primary product of connexon and the non-natural amino acid polypeptides reaction that contains carbonyl is the modified oxime non-natural amino acid polypeptides that contains that is connected on the connexon with available azanol base.The non-natural amino acid polypeptides reaction that this latter group can be further contains carbonyl or dicarbapentaborane with another kind contains the difunctionality heterodimer of the non-natural amino acid polypeptides of oxime with formation.Certainly, the functional group on the connexon needn't be identical, and it needn't be the azanol base.Use spreads all over the chemistry of this description details, and the those skilled in the art can design the connexon that at least one functional group wherein can form oximido with non-natural amino acid polypeptides; Other functional groups on the connexon can utilize other known chemical, and it comprises the chemistry of knowing in the organic chemistry filed based on nucleophilic reagent/electrophilic reagent.
H. add functional group's example: the separating property of facilitation polypeptide
Natural existence or non-natural amino acid polypeptides may be because of being difficult to separate from sample including (but not limited to) the solvability of polypeptide or in conjunction with the multiple reason of feature.For instance, be used for the treatment of in the process of polypeptide of purposes in preparation, may be from through engineering approaches design to separate this polypeptide the recombination system of excessive production polypeptide.Yet because the solvability of polypeptide or in conjunction with feature, the purity that reaches required degree proves difficulty usually.The method of Miao Shuing, composition, technology and strategy provide the terms of settlement of this situation herein.
Use method, composition, technology and the strategy described herein, the those skilled in the art can prepare the non-natural amino acid polypeptides that contains oxime with required homologous peptide, and the non-natural amino acid polypeptides that wherein contains oxime has the separation characteristic of improvement.In one embodiment, the homology non-natural amino acid polypeptides makes by biosynthesizing and prepares.In another or extra embodiment, alpha-non-natural amino acid has been incorporated herein its structure in one in the alpha-non-natural amino acid of description.In another or extra embodiment, alpha-non-natural amino acid be endways or the place, mid-way incorporate into and further incorporate into for locus specificity.
In one embodiment, had required improvement separation characteristic as gained alpha-non-natural amino acid by the biosynthesizing preparation.In other or extra embodiment, alpha-non-natural amino acid comprises the oxime key with the group that the improvement separation characteristic is provided.In other or extra embodiment, alpha-non-natural amino acid is through further modifying to form the modified oxime non-natural amino acid polypeptides that contains, and wherein said modification provides and provide the oxime key of the group of improvement separation characteristic.In certain embodiments, the direct binding of this group is to alpha-non-natural amino acid, and in other embodiments, this group passes through connexon group binding to alpha-non-natural amino acid.In certain embodiments, this group is connected on the alpha-non-natural amino acid by single chemical reaction, in other embodiments, needs series of chemical that this group is connected on the alpha-non-natural amino acid.The group of preferably giving improvement separation characteristic site specifically on the alpha-non-natural amino acid of binding in the non-natural amino acid polypeptides under the reaction conditions that is utilized, and be not that binding exists on the amino acid to natural.
In other or extra embodiment, gained non-natural amino acid polypeptides and GH supergene family member homology, yet, the method of describing in this part, technology and composition can be applicable to can be from almost any other polypeptide of improvement separation characteristic benefit, and described polypeptide comprises (only for instance): α-1 antitrypsin, angiostatin, AHF, antibody, lipophorin, apoprotein, atrionatriuretic factor, atrial natriuretic polypeptins, atrial natriuretic peptide, the C-X-C chemokine, T39765, NAP-2, ENA-78, gro-a, gro-b, gro-c, IP-10, GCP-2, NAP-4, SDF-1, PF4, MIG, calcitonin, the c-kit ligand, cytokine, the CC chemokine, MCP-1, MCP-2, monocyte chemoattractant protein-3, monocyte inflammatory protein-1 α, monocyte inflammatory protein-i β, RANTES, 1309, R83915, R91733, HCC1, T58847, D31065, T64262, CD40, the CD40 ligand, the c-kit ligand, collagen protein, G CFS (CSF), complement factor 5a, complement inhibitor, complement receptor 1, cytokine, epithelium neutrophilic granulocyte activation peptide-78, MIP-16, MCP-1, Urogastron (EGF), epithelium neutrophilic granulocyte activation peptide, erythropoietin (EPO), exfoliative toxin,or exfoliatin, factors IX, factor VII, Factor IX, factor X, fibroblast growth factor (FGF), Fibrinogen, fibronectin, four-helix bundle albumen, G-CSF, glp-1, GM-CSF, glucocerebrosidase, gonad-stimulating hormone, somatomedin, growth factor receptors, grf, Shh, hemochrome, pHGF (hGF), r-hirudin, human growth hormone (hGH), the human serum albumin, ICAM-1, the ICAM-1 acceptor, LFA-1, the LFA-1 acceptor, Regular Insulin, insulin-like growth factor (IGF), IGF-I, IGF-II, Interferon, rabbit (IFN), IFN-α, IFN-β, IFN-γ, interleukin-(IL), IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, keratinocyte growth factor (KGF), lactoferrin, leukaemia inhibitory factor, luciferase, neural plain, neutrophil inhibitory factor (nif) (NIF), oncostatin M, bone morphogenic protein, oncoprotein, paracitonin, Rat parathyroid hormone 1-34, PD-ECSF, PDGF, peptide hormone, PTN, albumin A, Protein G, pth, pyrogenic exotoxin A, pyrogenic exotoxin B, pyrogenic exotoxin C, pyy, Relaxin, feritin, SCF, the atom synthetic proteins, soluble complement acceptor I, solubility I-CAM1, soluble interleukin receptor, soluble TNF acceptor, somatomedin, Somat, tethelin, streptokinase, superantigen, staphyloentero-toxin, SEA, SEB, SEC1, SEC2, SEC3, SED, SEE, steroid hormone receptor, superoxide-dismutase, the toxic shock syndrome toxin, thymosin, tissue type plasminogen activator, tumor growth factor (TGF), tumour necrosis factor, tumor necrosis factor alpha, tumor necrosis factor, Tumor Necrosis Factor Receptors (TNFR), VLA-4 albumen, VCAM-1 albumen, vascular endothelial growth factor (VEGF), urokinase, mos, ras, raf, met, p53, tat, fos, myc, jun, myb, rel, estrogen receptor, PgR, the testosterone acceptor, aldosterone receptor, ldl receptor and Kendall compound.
In other or extra embodiment, give group improvement polypeptide water-soluble of improvement separation characteristic; In other embodiments, described group improvement polypeptide in conjunction with character; In other embodiments, described group provides new for character (comprising (only for instance) vitamin H group or vitamin H conjugated group) to polypeptide.Among the water miscible embodiment of described therein group improvement polypeptide, group is to be selected from the water-soluble polymers of describing herein, and it comprises in the PEG polymeric groups that (only for instance) describe any one herein.
I. add functional group's example: detect the existence of polypeptide
Natural existence or non-natural amino acid polypeptides can be because of being easy to the multiple former of polypeptide bonded reagent or mark thereby being difficult to detect in sample (comprising in vivo sample and in vitro sample) including (but not limited to) shortage.The method of Miao Shuing, composition, technology and strategy provide the terms of settlement of this situation herein.
Use method, composition, technology and the strategy described herein, the those skilled in the art can prepare the non-natural amino acid polypeptides that contains oxime with required homologous peptide, and the non-natural amino acid polypeptides that wherein contains oxime is allowed and detected sample in vivo and the polypeptide in the sample in vitro.In one embodiment, the homology non-natural amino acid polypeptides is to prepare by biosynthesizing.In another or extra embodiment, alpha-non-natural amino acid has been incorporated herein its structure in one in the alpha-non-natural amino acid of description.In another or extra embodiment, alpha-non-natural amino acid be endways or the place, mid-way incorporate into and further incorporate into for locus specificity.
In one embodiment, had required detected characteristics as gained non-natural amino acid polypeptides by the biosynthesizing preparation.In other or extra embodiment, non-natural amino acid polypeptides comprises that at least one is selected from by the alpha-non-natural amino acid that contains oxime, the alpha-non-natural amino acid that contains the alpha-non-natural amino acid of carbonyl and contain the group that the alpha-non-natural amino acid of azanol forms.In other embodiments, these alpha-non-natural amino acids are incorporated in the polypeptide by biosynthesizing as described herein.In other or alternate embodiment, non-natural amino acid polypeptides comprises that at least one is selected from the amino acid whose alpha-non-natural amino acid of formula I-XVIII, XXX-XXXIV (A and B) or XXXX-XXXXIII.In other or extra embodiment, alpha-non-natural amino acid comprises and the oxime key that the group of improved detection feature is provided.In other or extra embodiment, alpha-non-natural amino acid is through further modifying to form the modified non-natural amino acid polypeptides that contains oxime, and wherein said modification provides and provide the oxime key of the group of improved detection feature.In certain embodiments, the direct binding of this group is to alpha-non-natural amino acid, and in other embodiments, this group passes through connexon group binding to alpha-non-natural amino acid.In certain embodiments, this group is connected on the alpha-non-natural amino acid by single chemical reaction, in other embodiments, needs series of chemical that this group is connected on the alpha-non-natural amino acid.The group of preferably giving improved detection feature site specifically on the alpha-non-natural amino acid of binding in the non-natural amino acid polypeptides under the reaction conditions that is utilized, and be not that binding exists on the amino acid to natural.
In other or extra embodiment, gained non-natural amino acid polypeptides and GH supergene family member homology, yet, the method of describing in this part, almost any other polypeptide that technology and composition can be applicable to sample in vivo and in vitro need in the sample to detect, described polypeptide comprises (only for instance): α-1 antitrypsin, angiostatin, AHF, antibody, lipophorin, apoprotein, atrionatriuretic factor, atrial natriuretic polypeptins, atrial natriuretic peptide, the C-X-C chemokine, T39765, NAP-2, ENA-78, gro-a, gro-b, gro-c, IP-10, GCP-2, NAP-4, SDF-1, PF4, MIG, calcitonin, the c-kit ligand, cytokine, the CC chemokine, MCP-1, MCP-2, monocyte chemoattractant protein-3, monocyte inflammatory protein-1 α, monocyte inflammatory protein-i β, RANTES, 1309, R83915, R91733, HCC1, T58847, D31065, T64262, CD40, the CD40 ligand, the c-kit ligand, collagen protein, G CFS (CSF), complement factor 5a, complement inhibitor, complement receptor 1, cytokine, epithelium neutrophilic granulocyte activation peptide-78, MIP-16, MCP-1, Urogastron (EGF), epithelium neutrophilic granulocyte activation peptide, erythropoietin (EPO), exfoliative toxin,or exfoliatin, factors IX, factor VII, Factor IX, factor X, fibroblast growth factor (FGF), Fibrinogen, fibronectin, four-helix bundle albumen, G-CSF, glp-1, GM-CSF, glucocerebrosidase, gonad-stimulating hormone, somatomedin, growth factor receptors, grf, Shh, hemochrome, pHGF (hGF), r-hirudin, human growth hormone (hGH), the human serum albumin, ICAM-1, the ICAM-1 acceptor, LFA-1, the LFA-1 acceptor, Regular Insulin, insulin-like growth factor (IGF), IGF-I, IGF-II, Interferon, rabbit (IFN), IFN-α, IFN-β, IFN-γ, interleukin-(IL), IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, keratinocyte growth factor (KGF), lactoferrin, leukaemia inhibitory factor, luciferase, neural plain, neutrophil inhibitory factor (nif) (NIF), oncostatin M, bone morphogenic protein, oncoprotein, paracitonin, Rat parathyroid hormone 1-34, PD-ECSF, PDGF, peptide hormone, PTN, albumin A, Protein G, pth, pyrogenic exotoxin A, pyrogenic exotoxin B, pyrogenic exotoxin C, pyy, Relaxin, feritin, SCF, the atom synthetic proteins, soluble complement acceptor I, solubility I-CAM1, soluble interleukin receptor, soluble TNF acceptor, somatomedin, Somat, tethelin, streptokinase, superantigen, staphyloentero-toxin, SEA, SEB, SEC1, SEC2, SEC3, SED, SEE, steroid hormone receptor, superoxide-dismutase, the toxic shock syndrome toxin, thymosin, tissue type plasminogen activator, tumor growth factor (TGF), tumour necrosis factor, tumor necrosis factor alpha, tumor necrosis factor, Tumor Necrosis Factor Receptors (TNFR), VLA-4 albumen, VCAM-1 albumen, vascular endothelial growth factor (VEGF), urokinase, mos, ras, raf, met, p53, tat, fos, myc, jun, myb, rel, estrogen receptor, PgR, the testosterone acceptor, aldosterone receptor, ldl receptor and Kendall compound.
In other or extra embodiment, the group of giving the improved detection feature is to be selected from the group that is made up of following each thing: mark; Dyestuff; Affinity marker; The photoaffinity mark; Spin labeling; Fluorophore; Radioactive segment; Be combined with the part of heavy atom; Through isotope-labeled part; The biophysics probe; The phosphorescence group; Chemiluminescent groups; The electron dense group; The magnetic group; Chromophoric group; Energy transfer agent; Detectable label, with and any combination.
J. add functional group's example: the therapeutic property of improvement polypeptide
Natural existence or non-natural amino acid polypeptides should be able to provide a certain treatment benefit to the patient who suffers from particular disorder, disease or symptom.This treatment benefit should depend on multiple factor, it comprises (only for instance): the security overview of polypeptide, and the pharmacokinetics of polypeptide, pharmacology and/or pharmacodynamics (for example, water-soluble, biological usability, serum half-life, treatment transformation period, immunogenicity, biological activity or cycling time).In addition, it is favourable to provide other functional groups can be to polypeptide, such as the cytotoxic compound or the medicine that connect, maybe may need to be connected to form the homopolymer and the special-shaped polymer of description herein with other polypeptide.Preferred these are modified activity and/or the tertiary structure of not destroying original polypeptide.The method of Miao Shuing, composition, technology and strategy provide the terms of settlement of these problems herein.
Use method, composition, technology and the strategy described herein, the those skilled in the art can prepare the non-natural amino acid polypeptides that contains oxime with required homologous peptide, and the non-natural amino acid polypeptides that wherein contains oxime has the treatment feature of improvement.In one embodiment, the homology non-natural amino acid polypeptides is to prepare by biosynthesizing.In another or extra embodiment, alpha-non-natural amino acid has been incorporated herein its structure in one in the alpha-non-natural amino acid of description.In another or extra embodiment, alpha-non-natural amino acid be endways or the place, mid-way incorporate into and further incorporate into for locus specificity.
In one embodiment, had required improved treatment feature as gained alpha-non-natural amino acid by the biosynthesizing preparation.In other or extra embodiment, alpha-non-natural amino acid comprises and the oxime key that the group of improved treatment feature is provided.In other or extra embodiment, alpha-non-natural amino acid is through further modifying to form the modified oxime non-natural amino acid polypeptides that contains, and wherein said modification provides and provide the oxime key of the group of improved treatment feature.In certain embodiments, the direct binding of this group is to alpha-non-natural amino acid, and in other embodiments, this group passes through connexon group binding to alpha-non-natural amino acid.In certain embodiments, this group is connected on the alpha-non-natural amino acid by single chemical reaction, in other embodiments, needs series of chemical that this group is connected on the alpha-non-natural amino acid.The group of preferably giving improvement separation characteristic site specifically on the alpha-non-natural amino acid of binding in the non-natural amino acid polypeptides under the reaction conditions that is utilized, and be not that binding exists on the amino acid to natural.
In other or extra embodiment, gained non-natural amino acid polypeptides and GH supergene family member homology, yet, the method of describing in this part, technology and composition can be applicable to almost any other polypeptide that can benefit from the treatment feature of improvement, described comprising (only for instance): α-1 antitrypsin, angiostatin, AHF, antibody, lipophorin, apoprotein, atrionatriuretic factor, atrial natriuretic polypeptins, atrial natriuretic peptide, the C-X-C chemokine, T39765, NAP-2, ENA-78, gro-a, gro-b, gro-c, IP-10, GCP-2, NAP-4, SDF-1, PF4, MIG, calcitonin, the c-kit ligand, cytokine, the CC chemokine, MCP-1, MCP-2, monocyte chemoattractant protein-3, monocyte inflammatory protein-1 α, monocyte inflammatory protein-i β, RANTES, 1309, R83915, R91733, HCC1, T58847, D31065, T64262, CD40, the CD40 ligand, the c-kit ligand, collagen protein, G CFS (CSF), complement factor 5a, complement inhibitor, complement receptor 1, cytokine, epithelium neutrophilic granulocyte activation peptide-78, MIP-16, MCP-1, Urogastron (EGF), epithelium neutrophilic granulocyte activation peptide, erythropoietin (EPO), exfoliative toxin,or exfoliatin, factors IX, factor VII, Factor IX, factor X, fibroblast growth factor (FGF), Fibrinogen, fibronectin, four-helix bundle albumen, G-CSF, glp-1, GM-CSF, glucocerebrosidase, gonad-stimulating hormone, somatomedin, growth factor receptors, grf, Shh, hemochrome, pHGF (hGF), r-hirudin, human growth hormone (hGH), the human serum albumin, ICAM-1, the ICAM-1 acceptor, LFA-1, the LFA-1 acceptor, Regular Insulin, insulin-like growth factor (IGF), IGF-I, IGF-II, Interferon, rabbit (IFN), IFN-α, IFN-β, IFN-γ, interleukin-(IL), IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, keratinocyte growth factor (KGF), lactoferrin, leukaemia inhibitory factor, luciferase, neural plain, neutrophil inhibitory factor (nif) (NIF), oncostatin M, bone morphogenic protein, oncoprotein, paracitonin, Rat parathyroid hormone 1-34, PD-ECSF, PDGF, peptide hormone, PTN, albumin A, Protein G, pth, pyrogenic exotoxin A, pyrogenic exotoxin B, pyrogenic exotoxin C, pyy, Relaxin, feritin, SCF, the atom synthetic proteins, soluble complement acceptor I, solubility I-CAM1, soluble interleukin receptor, soluble TNF acceptor, somatomedin, Somat, tethelin, streptokinase, superantigen, staphyloentero-toxin, SEA, SEB, SEC1, SEC2, SEC3, SED, SEE, steroid hormone receptor, superoxide-dismutase, the toxic shock syndrome toxin, thymosin, tissue type plasminogen activator, tumor growth factor (TGF), tumour necrosis factor, tumor necrosis factor alpha, tumor necrosis factor, Tumor Necrosis Factor Receptors (TNFR), VLA-4 albumen, VCAM-1 albumen, vascular endothelial growth factor (VEGF), urokinase, mos, ras, raf, met, p53, tat, fos, myc, jun, myb, rel, estrogen receptor, PgR, the testosterone acceptor, aldosterone receptor, ldl receptor and Kendall compound.
In other or extra embodiment, give group improvement polypeptide water-soluble of improved treatment feature; In other embodiments, described group improvement polypeptide in conjunction with character; In other embodiments, described group provides new for character (comprising (only for instance) vitamin H group or vitamin H conjugated group) to polypeptide.Among the water miscible embodiment of described therein group improvement polypeptide, group is to be selected from the water-soluble polymers of describing herein, and it comprises (only for instance) PEG polymeric groups.In other or extra embodiment, described group is a cytotoxic compound, and in other embodiments, described group is a medicine.In other embodiments, the medicine of binding or cytotoxic compound can be from cracking on the non-natural amino acid polypeptides to be delivered to required treatment position with medicine or cytotoxic compound.In other embodiments, described group is second polypeptide, and it comprises the non-natural amino acid polypeptides that (for instance) contains oxime, and it further comprises the polypeptide that (for instance) has the amino acid structure identical with first non-natural amino acid polypeptides.
In other or extra embodiment, the non-natural amino acid polypeptides that contains oxime is the modified non-natural amino acid polypeptides that contains oxime.In other or extra embodiment, with respect to the natural amino acid polypeptide that exists of homology, the non-natural amino acid polypeptides that contains oxime increases the biological usability of polypeptide.In other or extra embodiment, with respect to the natural amino acid polypeptide that exists of homology, the non-natural amino acid polypeptides that contains oxime increases the security overview of polypeptide.In other or extra embodiment, with respect to the natural amino acid polypeptide that exists of homology, the non-natural amino acid polypeptides that contains oxime increases the water-soluble of polypeptide.In other or extra embodiment, with respect to the natural amino acid polypeptide that exists of homology, the non-natural amino acid polypeptides that contains oxime increases the treatment transformation period of polypeptide.In other or extra embodiment, with respect to the natural amino acid polypeptide that exists of homology, the non-natural amino acid polypeptides that contains oxime increases the serum half-life of polypeptide.In other or extra embodiment, with respect to the natural amino acid polypeptide that exists of homology, the non-natural amino acid polypeptides that contains oxime prolongs the cycling time of polypeptide.In other or extra embodiment,, contain the activity that the non-natural amino acid polypeptides of oxime is regulated polypeptide with respect to the natural amino acid polypeptide that exists of homology.In other or extra embodiment,, contain the immunogenicity that the non-natural amino acid polypeptides of oxime is regulated polypeptide with respect to the natural amino acid polypeptide that exists of homology.
XI. the therepic use of modified polypeptide
For simplicity, " modified or not modified " non-natural polypeptide of describing in this part is general and/or describe with particular instance.Yet, general description or particular instance that " modified or not modified " non-natural polypeptide of describing in this part should not only limit to provide in this part, but " modified or not modified " non-natural polypeptide of describing in this part is applicable to that equally fully all comprise that at least one is in formula I-XVIII, amino acid whose " modified or not modified " non-natural polypeptide in the category of XXX-XXXIV (A and B) and XXXX-XXXXIII, it comprises the specification sheets that is in herein, claim and the graphic middle formula I-XVIII that describes, any minor or specific compound in the category of XXX-XXXIV (A and B) and XXXX-XXXXIII.
There is multiple use in " the modified or not modified " non-natural amino acid polypeptides (comprising its homopolymer and special-shaped polymer) of Miao Shuing herein, and it is including (but not limited to): therepic use, diagnostic uses, based on purposes, industrial use, cosmetic use, plant biology purposes, environmental application, energy generation purposes and/or the military use of calibrating.As non-limitative illustration, provide the following therepic use of " modified or not modified " non-natural amino acid polypeptides.
" the modified or not modified " non-natural amino acid polypeptides of Miao Shuing is applicable to treatment various disease conditions, symptom or disease herein.Throw with " modified or not modified " non-natural amino acid polypeptides product of describing herein and in the mankind, produce any activity that confirms by commercially available polypeptide formulations.The mean vol of " modified or not modified " non-natural amino acid polypeptides product can change and especially should change according to the suggestion and the prescription of qualified physicians.The exact amount of " modified or not modified " non-natural amino acid polypeptides should be preferentially selected according to following factor: such as the definite type of the symptom of being treated, the patient's that treated symptom, and other compositions in the composition.The amount of desiring to give can be easy to be determined according to the therapy of using " modified or not modified " non-natural amino acid polypeptides by the those skilled in the art.
A. offer medicine and medical composition
There is multiple use in " the modified or not modified " non-natural amino acid polypeptides (comprising its homopolymer and special-shaped polymer) of Miao Shuing herein, and it is including (but not limited to): therepic use, diagnostic uses, based on purposes, industrial use, cosmetic use, plant biology purposes, environmental application, energy generation purposes and/or the military use of calibrating.As non-limitative illustration, provide the following therepic use of " modified or not modified " non-natural amino acid polypeptides.
" the modified or not modified " non-natural amino acid polypeptides of Miao Shuing is applicable to the treatment various disease conditions herein.Throw with " modified or not modified " non-natural amino acid polypeptides product of describing herein and in the mankind, produce any activity that confirms by commercially available polypeptide formulations.The mean vol of " modified or not modified " non-natural amino acid polypeptides product can change and especially should change according to the suggestion and the prescription of qualified physicians.The exact amount of " modified or not modified " non-natural amino acid polypeptides should preferentially be selected according to following factor: the definite type of the symptom of being treated, the patient's who is treated symptom, and other compositions in the composition.The amount of desiring to give can be easy to be determined according to the therapy of using " modified or not modified " non-natural amino acid polypeptides by the those skilled in the art.
Modified as described herein or not modified non-natural amino acid polypeptides (including (but not limited to) synthetic enzyme, comprise the protein of one or more alpha-non-natural amino acids etc.) is used for the treatment of purposes according to circumstances, its including (but not limited to) be fit to medical supporting agent combination.These compositions (for example) comprise the modified as described herein or not modified non-natural amino acid polypeptides for the treatment of significant quantity, and pharmaceutically acceptable supporting agent or vehicle.Described supporting agent or vehicle are including (but not limited to) salt solution, buffer saline, dextrose, water, glycerol, ethanol and/or its combination.Allocate to adapt to the dispensing pattern.In general, throwing with method of protein is to know in the affiliated field and it can be applicable to the dispensing of modified as described herein or not modified non-natural amino acid polypeptides.
According to the method for knowing in the affiliated field, the therapeutic composition that comprises the non-natural amino acid polypeptides that one or more are modified as described herein or not modified is according to circumstances in one or more ill suitably in vitro and/or in vivo testing to determine effect, tissue metabolism and estimation dosage in the animal model.Specifically, dosage just begins to be fit to measure (that is, in relevant calibrating) by the activity of the relative natural amino acid homologue of alpha-non-natural amino acid (including (but not limited to) being compared with polypeptide and the natural amino acid polypeptide that comprises one or more alpha-non-natural amino acids modified), stability or other and measures.
Dispensing be by be generally used for introducing molecule with finally with approach that blood or histocyte contact in any one carry out.Modified as described herein or not modified non-natural amino acid polypeptides be according to circumstances with one or more pharmaceutically acceptable supporting agents with any suitable mode throw with.Can obtain to throw and the appropriate methodology of modified or not modified non-natural amino acid polypeptides as described herein to the patient, although and can use more than one approach to throw and particular composition, the common comparable another kind of approach of particular approach provides more direct and more effective effect or reaction.
Pharmaceutically acceptable supporting agent part throw by institute and particular composition and definite by the ad hoc approach that is used to throw with composition.Therefore, the suitable prescription that has the multiple medical composition of describing herein.
Herein the non-natural amino acid polypeptides of Miao Shuing and comprise these polypeptide composition can by any conventional route that is applicable to protein or peptide throw with, its including (but not limited to) non-through intestines throw with, for example injection, it is including (but not limited to) any other form of subcutaneous injection or intravenous injection or injection or transfusion.Polypeptide medical composition (comprise herein describe various non-natural amino acid polypeptides) can by numerous approach throw with, it is including (but not limited to) per os, intravenously, intraperitoneal, intramuscular, through skin, subcutaneous, local, hypogloeeis or rectal.The composition that comprises modified as described herein or not modified non-natural amino acid polypeptides also can by liposome throw with.It is known that these dosing ways and proper formulation are generally the those skilled in the art.The non-natural amino acid polypeptides of Miao Shuing also can be used in combination separately or with other suitable components (including (but not limited to) medical supporting agent) herein.
Separately or with the modified as described herein or not modified non-natural amino acid polypeptides of other suitable combination of components also can be made into can by suck throw and aerosol formulations (that is, it can " atomize ").Aerosol formulations can be inserted in the acceptable propelling agent of pressurization, such as Refrigerant 12, propane, nitrogen with and analogue.
Be applicable to non-through intestines dispensings (such as, by intraarticular (at intraarticular), intravenously, intramuscular, intracutaneous, intraperitoneal and subcutaneous route) prescription comprise water-based and the aseptic injectable solution of opening such as non-aqueous, the solute that it can contain antioxidant, buffer reagent, fungistat and the blood etc. of prescription and predetermined acceptor is opened; And water-based and non-aqueous sterile suspensions, it can comprise suspension agent, solubilizing agent, thickening material, stablizer and sanitas.The prescription of the nucleic acid through packing can be present in unitary dose or the multiple doses sealed vessel (such as ampoule and bottle).
Non-is preferred medication administration method through intestines dispensing and intravenously dispensing.Specifically, the dosing way (sending proteinic those dosing ways) of natural amino acid homologue therapeutical agent and preferred dosing way and the prescription that the present prescription that uses is provided for modified as described herein or not modified non-natural amino acid polypeptides have been used for including (but not limited to) being generally used for EPO, IFN, GH, G-CSF, GM-CSF, IFN, interleukin-, antibody and/or any other medicine.
Under the situation of composition of Miao Shuing and method, throwing and the dosage in the patient are enough to produce in time useful therapeutic response in the patient in this article.Dosage is by the effect of special formulation and activity, stability or the serum half-life of employed modified or not modified non-natural amino acid polypeptides and patient's symptom, and the patient's of institute's desire treatment body weight or surface-area are determined.Existence, the nature and extent of any harmful side effect that the dosage size is also followed by throwing in particular patient and special formulation or its analogue are determined.
Determine treatment or preventing disease (including (but not limited to) cancer, genetic diseases, diabetes, AIDS or its similar disease) time institute's desire is thrown and the significant quantity of prescription the time, the progress of doctor's assessments blood plasma level, prescription toxicity, disease and/or (if relevant) anti-non-natural amino acid polypeptides production of antibodies.
Throw with dosage to the patient of (for example) 70 kilograms usually in the scope of the proteinic dosage of present used treatment that is equivalent to the activity that is suitable for compositions related change or serum half-life.Herein the medicine prescription of Miao Shuing can by any known conventional therapy (its comprise antibody dispensing, vaccine dispensing, cytotoxic agent, natural amino acid polypeptide, nucleic acid, nucleotide analog, biological response modifier with and the dispensing of analogue) come the supplement therapy situation.
For dispensing, herein the medicine prescription of Miao Shuing be with by the LD-50 of relevant prescription or ED-50 and/or to throw in the definite speed of observation of various concentration (its including (but not limited to) when quality that is applied to the patient and general health time) any side effect of modified or not modified non-natural amino acid polypeptides down and.Dispensing can realize by single-dose or gradation administration.
If the patient of experience prescription transfusion heating occurs, feels cold or myalgia, make him receive Asprin (aspirin), Ibuprofen BP/EP (ibuprofen), acetaminophen (acetaminophen) or other pain/heating control medicine of suitable dosage so.The patient who makes experience infusion reaction (such as heating, myalgia and feel cold) future with Asprin, acetaminophen or (comprise, but be not limited to) diphenhydramine (diphenhydramine) transfusion before medication in 30 minutes in advance.For more serious the feeling cold and myalgia of febrifuge and antihistaminic agent not being had rapid reaction, usage degree suddenly (Meperidine).The cell transfusion is slowed down or stopped to the seriousness of visual response.
Modified as described herein or not modified non-natural amino acid polypeptides can directly throw with to the Mammals person under inspection.Offer medicine by being generally used for that polypeptide is introduced in the approach among the person under inspection any.Modified as described herein or not modified non-natural amino acid polypeptides comprises and is applicable to per os, per rectum, local, suck (including (but not limited to) passing through aerosol), oral cavity (including (but not limited to) the hypogloeeis), transvaginal, non-through intestines (including (but not limited to) subcutaneous, intramuscular, intracutaneous, intraarticular, in the pleura, intraperitoneal, in the brain, intra-arterial or intravenously), the part (promptly, skin and mucous membrane surface, it comprises the tracheae surface) and through the those polypeptides of skin dispensing, although optimal approach should be decided on the character and the seriousness of the symptom of being treated under any given situation.Dispensing can be part or general.Prescription can be present in unitary dose or the multiple doses sealed vessel (such as ampoule and bottle).Modified as described herein or not modified non-natural amino acid polypeptides can be prepared in the mixture with the unitary dose injectable forms (including (but not limited to) solution, suspension or emulsion) of pharmaceutically acceptable supporting agent.Modified as described herein or not modified non-natural amino acid polypeptides also can by the continuous transfusion minipump of (comprise, but be not limited to) such as osmotic pump (use), single bolus or slowly discharge storage tank formula prescription throw with.
The prescription that is suitable for offeing medicine comprise water-based and non-aqueous solution, etc. open aseptic injectable solution, the solute that it can contain antioxidant, buffer reagent, fungistat and prescription etc. is opened; And water-based and non-aqueous sterile suspensions, it can comprise suspension agent, solubilizing agent, thickening material, stablizer and sanitas.Solution and suspension can be prepared by sterilized powder, particle and the tablet of before having described kind.
Freeze-drying is the normally used proteinic technology that presents, and it is used for removing water from the protein formulation of being paid close attention to.Lyophilize (Freeze-drying) or freeze-drying (lyophilization) by at first with desire exsiccant material freezing and remove the ice or the process of chilled solvent by distillation in vacuum environment subsequently.Vehicle can be contained in the freeze dried in advance prescription to increase stability and/or the stability of improvement lyophilized products when storing in the freeze-drying process.Pikal, people such as M.Biopharm.3 (9) 26-30 (1990) and Arakawa, Pharm.Res.8 (3): 285-291 (1991).
The spraying drying of medicine also is that the those skilled in the art is known.For instance, referring to Broadhead, people such as J., " The Spray Drying of Pharmaceuticals, " Drug Dev.Ind.Pharm, 18 (11 and 12), 1169-1206 (1992).Except that small-molecule drug, spray-dried and these materials of multiple biomaterial comprise: enzyme, serum, microorganism and yeast.The technology of spraying drying for being suitable for is because it can single stage method change liquid pharmaceutical formulation into powder meticulous, dustless or cohesion.Basic fundamental comprises following four steps: a) material solution is atomized into spraying; B) spraying-air contact; C) make spraying drying; And d) product with drying separates with dry air.United States Patent (USP) the 6th, 235, No. 710 and the 6th, 001, No. 800 (it is all to be incorporated herein by reference) described and prepared recombiant erythropoietin by spraying drying.
The medical composition of Miao Shuing can comprise pharmaceutically acceptable supporting agent, vehicle or stablizer herein.Pharmaceutically acceptable supporting agent part throw by institute and the ad hoc approach thrown with composition of particular composition and being used to definite.Therefore, the suitable prescription that has the medical composition (comprising pharmaceutically acceptable supporting agent, vehicle or stablizer according to circumstances) of the multiple modified or not modified non-natural amino acid polypeptides of describing herein, (referring to (for example) Remington:The Science and Practice of Pharmacy, the 19th edition (Easton, Pa.:Mack Publishing Company, 1995); Hoover, John E., Remington ' s Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania 1975; Liberman, H.A. and Lachman, L. compiles, Pharmaceutical Dosage Forms, MarcelDecker, New York, N.Y., 1980; And Pharmaceutical Dosage Forms and Drug DeliverySystems, the 17th edition (Lippincott Williams ﹠amp; Wilkins, 1999)).Suitable supporting agent comprises buffer reagent, and it contains succinate, phosphoric acid salt, borate, HEPES, Citrate trianion, imidazoles, acetate, supercarbonate and other organic acids; Antioxidant, it is including (but not limited to) xitix; Low molecular weight polypeptide, it is including (but not limited to) the those polypeptides less than about 10 residues; Protein, it is including (but not limited to) serum albumin, gelatin or immunoglobulin (Ig); Hydrophilic polymer, it is including (but not limited to) polyvinylpyrrolidone; Amino acid, it is including (but not limited to) glycine, glutamine, l-asparagine, arginine, Histidine or histidine derivative, methionine(Met), L-glutamic acid or Methionin; Monose, disaccharides and other carbohydrate, it is including (but not limited to) trehalose, sucrose, glucose, seminose or dextrin; Sequestrant, it is including (but not limited to) EDTA; Divalent-metal ion, it is including (but not limited to) zinc, cobalt or copper; Sugar alcohol, it is including (but not limited to) mannitol or Sorbitol Powder; The salify counter ion, it is including (but not limited to) sodium; And/or nonionogenic tenside, it is including (but not limited to) Tween TM(including (but not limited to) Tween 80 (polysorbate80) and Tween 20 (polysorbate20)), Pluronics TMAnd other general Lip river nicotinic acids (pluronic acid) (including (but not limited to) general Lip river nicotinic acid F68 (Pa Luoshamu 188 (poloxamer 188))) or PEG.Suitable tensio-active agent for example comprises (but being not limited to) based on polyoxyethylene-polyoxytrimethylene-polyoxyethylene (promptly, (PEO-PPO-PEO)), or polyoxytrimethylene-polyoxyethylene-polyoxytrimethylene (that is, (PPO-PEO-PPO)), or the polyethers of its combination.PEO-PPO-PEO and PPO-PEO-PPO are with trade(brand)name PluronicsTM, R-PluronicsTM, TetronicsTM and R-TetronicsTM (BASF WyandotteCorp., Wyandotte, Mich.) commercially available and be further described in United States Patent (USP) the 4th, in 820, No. 352 (they are all to be incorporated herein by reference).Other ethylene/polypropylene block polymers can be suitable tensio-active agent.The combination of tensio-active agent or tensio-active agent can be used for resisting one or more stress (including (but not limited to) from the stress that stirs) and stablizes the PEGization non-natural amino acid polypeptides.Some above-mentioned tensio-active agents can be known as " swelling agent ".Some also can be known as " tension regulator ".
Modified as described herein or not modified non-natural amino acid polypeptides (comprising binding those non-natural amino acid polypeptides to the water-soluble polymers (such as PEG)) also can by sustained release system or as the part of sustained release system throw with.Sustained-release composition comprises (comprise, but be not limited to) and is the semipermeability polymeric matrix of formed article (it is including (but not limited to) film or microcapsule) form.Lasting release matrix comprises biocompatible materials, such as poly-(2-hydroxyethyl meth acrylate) (people such as Langer, J.Biomed.Mater.Res., 15:167-277 (1981); Langer, Chem.Tech., 12:98-105 (1982)), ethylene vinyl acetate people such as (, as preceding) Langer or poly--D-(-)-3-hydroxybutyric acid (EP 133,988), polylactide (poly(lactic acid)) (United States Patent (USP) the 3rd, 773, No. 919; EP 58,481), poly-glycollide (polymkeric substance of oxyacetic acid), polylactide-altogether-glycollide (multipolymer of lactic acid and oxyacetic acid), polyanhydride, the multipolymer of L-L-glutamic acid and γ-ethyl-L-glutamate (people such as U.Sidman, Biopolymers, 22,547-556 (1983)), poly-(former) ester, polypeptide, hyaluronic acid, collagen protein, chondroitin sulfate, carboxylic acid, lipid acid, phosphatide, polysaccharide, nucleic acid, polyamino acid, amino acid is (such as phenylalanine, tyrosine, Isoleucine), polynucleotide, the polyvinyl propylene, polyvinylpyrrolidone and poly-silica.Sustained-release composition also comprises liposomal encapsulated compound.The liposome that contains compound is by known method preparation itself: DE 3,218, and 121; People such as Epstein, Proc.Natl.Acad.Sci.U.S.A., 82:3688-3692 (1985); People such as Hwang, Proc.Natl Acad.Sci.U.S.A., 77:4030-4034 (1980); EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP 142,641; Japanese patent application case 83-118008; United States Patent (USP) the 4th, 485, No. 045 and the 4th, 544, No. 545; And EP 102,324.
Liposomal encapsulated polypeptide can be prepared by the method for describing in the following document: for example, DE 3,218, and 121; People such as Epstein, Proc.Natl Acad.Sci.U.S.A., 82:3688-3692 (1985); People such as Hwang, Proc.Natl Acad.Sci.U.S.A., 77:4030-4034 (1980); EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP 142,641; Japanese patent application case 83-118008; United States Patent (USP) the 4th, 485, No. 045 and the 4th, 544, No. 545; And EP 102,324.The composition of liposome and size for know or can rule of thumb easily determine by the those skilled in the art.Some case descriptions of liposome are in people such as (for example) Park JW, Proc.Natl.Acad.Sci.USA 92:1327-1331 (1995); Lasic D and Papahadjopoulos D (volume): MEDICAL APPLICATIONS OF LIPOSOMES (1998); People such as Drummond DC, Liposomal drug delivery systems for cancer therapy is in Teicher B (volume): among the CANCER DRUG DISCOVERY AND DEVELOPMENT (2002); People such as Park JW, Clin.Cancer Res.8:1172-1181 (2002); People such as Nielsen UB, Biochim.Biophys.Acta1591 (1-3): 109-118 (2002); People such as Mamot C, Cancer Res.63:3154-3161 (2003).
Throw under the situation of the composition of Miao Shuing, prescription and method in this article with dosage and should be enough in the person under inspection, produce in time useful reaction to the patient.Usually, every dosage non-through intestines throw and the total medical significant quantity of modified as described herein or not modified non-natural amino acid polypeptides at the about 0.01 μ g of per kilogram weight in patients every day to about 100 μ g, or about 0.05mg judges although it should stand treatment to the scope of about 1mg.Administration frequency also stands treatment to be judged, and comparable to be used for the frequency of human commercially available prod through approval higher or frequency is lower.Usually, polymkeric substance: polypeptide joiner (comprise (only for instance) PEGization polypeptide) as described herein can by in the above-mentioned dosing way any throw with.
Example
Example 1
The amino acid whose of carbonyl that contain of oblatio synthesizes among this example detailed description Fig. 4.The alpha-non-natural amino acid that contains carbonyl is preparation as shown in Figure 4.
Example 2
Oblatio contains the amino acid whose synthetic of azanol through protection among this example detailed description Fig. 5 a.The alpha-non-natural amino acid that contains azanol through protection is to prepare described in Fig. 5 a.
Example 3
The amino acid whose of azanol that contain of oblatio synthesizes among this example detailed description Fig. 5 b.The alpha-non-natural amino acid that contains azanol is to prepare described in Fig. 5 b.
Example 4
The amino acid whose of azanol that contain of oblatio synthesizes among this example detailed description Fig. 5 c.The alpha-non-natural amino acid that contains azanol is to prepare described in Fig. 5 c.
Example 5
The amino acid whose of oxime that contain of oblatio synthesizes among this example detailed description Fig. 5 d.The alpha-non-natural amino acid that contains oxime is to prepare described in Fig. 5 d.
Example 6
The amino acid whose of oxime that contain of oblatio synthesizes among this example detailed description Fig. 6 a.The alpha-non-natural amino acid that contains oxime is to prepare described in Fig. 6 a.
Example 7
The amino acid whose of oxime that contain of oblatio synthesizes among this example detailed description Fig. 6 b.The alpha-non-natural amino acid that contains oxime is to prepare described in Fig. 6 b.
Example 8
The amino acid whose of oxime that contain of oblatio synthesizes among this example detailed description Fig. 6 c.The alpha-non-natural amino acid that contains oxime is to prepare described in Fig. 6 c.
Example 9
The amino acid whose of carbonyl that contain of oblatio synthesizes among this example detailed description Figure 24.
Synthetic
Under 0 ℃, in NaOH solution (40mL, 25 volume %), add ether (60mL).The blast shield thing is placed the reaction flask front.Through 3 parts of 3 minutes branches in the gained mixture, add N-nitroso-group-N-methyl ureas (6.0g, 57.9mmol).0 ℃ of following stirring reaction 10 minutes.Ether layer is separated with the sodium hydroxide layer.Through 5 minutes with organic layer by part (about 6 times add) add to N-Boc-4-hydroxymethyl phenylalanine (7.5g, 25.4mmol) in the solution in anhydrous THF (20mL) until initial substance completely dissolve (by the TLC monitoring).Add 5 glacial acetic acids subsequently with stopped reaction.Remove after the organic solvent by rotary evaporation, add ethyl acetate.With organic layer with NaHCO 3Saturated solution, H 2O and salt solution wash successively, with after anhydrous MgSO 4Dry, filter and concentrate with the generation pulverous product (5.9g, 75%) that is white in color.
Figure A20058004432802452
Synthetic
Under 0 ℃ to alcohol (6.0g, 19.4mmol) and pyridine (12mL is 150mmol) in CH 2Cl 2(400mL) in stirred solution, add Dai Si-Martin cross iodine alkane (Dess-Martin periodinane) (14.2g, 33.4mmol).At room temperature mixture is stirred and spend the night.To react subsequently with Na 2S 2O 3-NaHCO 3Saturated aqueous solution (1: 1,300mL) end and use CH 2Cl 2Extraction.With the organic layer combination and with H 2O and salt water washing are with after anhydrous Na 2SO 4Dry, filtration and concentrated in a vacuum.By flash chromatography (silica, 1: 100-1: 1 hexane: EtOAc) the purifying resistates aldehyde product (5.48g, 92%) of solid state that obtains being white in color.
Figure A20058004432802461
Synthetic
To aldehyde (3.07g, 10mmol) add in the solution in EtOH (40mL) acetic acid hydrazides (1.7g, 20mmol).Reaction mixture was at room temperature stirred 30 minutes and concentrate.In resistates, add H 2O (200mL) then adds CH 2Cl 2With organic layer separation and concentrated in a vacuum.By flash chromatography (silica, 3: 7-1: 9 hexanes: EtOAc) the purifying resistates product (3.29g, 90%) of solid state that obtains being white in color.
Figure A20058004432802462
Synthetic
Under 0 ℃ to above-mentioned methyl esters (3.29g, 9.1mmol) add in the solution in diox (10mL) LiOH (10mL, 1N).Mixture stirred 1 hour under uniform temp and subsequently by adding citric acid (5g) stopped reaction and with H 2The O dilution.With EtOAc extraction mixture.With organic layer with H 2O and salt solution wash successively, with after anhydrous Na 2SO 4Dry, filtration and concentrated to obtain white solid (3.05g, 96%).
Figure A20058004432802463
Synthetic
(3.02g is 8.6mmol) in CH to above-mentioned acid under 0 ℃ 2Cl 2Add trifluoroacetic acid (20mL) in the solution (20mL).Reaction mixture was stirred 2 hours down and concentrates at 0 ℃.In resistates, add MeOH (1mL), then add HCl (2.0mL, 4N is in the Yu diox).Add ether (200mL) is the yellow solid shape with precipitation product (2.07g, 83%) subsequently.
Example 10
The amino acid whose of dicarbapentaborane that contain of oblatio synthesizes among this example detailed description Figure 25.
Figure A20058004432802471
Synthetic
Under 0 ℃ to amine (10g, 34mmol) in DMF (70mL), in stirred solution, add pyruvic acid (5mL, 72mmol), 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride (EDC, 20g, 104mmol), I-hydroxybenzotriazole hydrate (HOBt, 85g, 71mmol) and N, the N-diisopropyl ethyl amine (DIEA, 35mL, 200mmol).Mixture at room temperature stirred 6 hours and subsequently with aqueous citric acid solution (5%, 500mL) end and extract with EtOAc (500mL).With organic layer with H 2O and salt solution wash successively, with after anhydrous Na 2SO 4Dry, filtration and concentrated in a vacuum.(silica, 3: 1-1: 1 hexane: EtOAc) the purifying resistates obtains being the product (4.78g, 40%) of solid state by flash chromatography.
Figure A20058004432802472
Synthetic
Under 0 ℃ to above-mentioned methyl esters (2.96g, 8.1mmol) add in the solution in diox (10mL) LiOH (10mL, 1N).Mixture was stirred 3 hours under uniform temp.To react subsequently with aqueous citric acid solution (5%) termination and with EtOAc and dilute.With the organic layer separation and with H 2O and salt solution wash successively, with after anhydrous Na 2SO 4Drying, filtration and concentrated product (2.87g, 100%) to obtain being the yellow solid shape.
Figure A20058004432802481
Synthetic
(2.05g is 5.9mmol) in CH to above-mentioned acid under 0 ℃ 2Cl 2Add trifluoroacetic acid (10mL) in the solution (10mL).Mixture was stirred 2 hours and concentrated in a vacuum.In resistates, add HCl (1mL, 4N is in the Yu diox), then add ether (400mL).The be white in color throw out (1.38g, 82%) of solid state of collection.
Example 11
The amino acid whose of dicarbapentaborane that contain of oblatio synthesizes among this example detailed description Figure 26.
Figure A20058004432802482
Synthetic
Under 90 ℃ to 4 '-methyl phenyl ketone (20g, 122mmol) and N-bromosuccinimide (NBS, 23g 130mmol) add 2,2 in the solution in benzene (300mL) '-azobis isobutyronitrile (AIBN, 0.6g, 3.6mmol).Gained solution is heated to backflow to spend the night.Subsequently reaction is cooled to room temperature.With brown solution with H 2O and salt solution wash successively, with after anhydrous Na 2SO 4Dry, filtration and concentrated in a vacuum.With resistates crystallization product (27g, 87%) from hexane to obtain being the light yellow solid shape.
Synthetic
(14.5g, (39g 180mmol), then adds above-mentioned bromide (27g, 119mmol) solution in EtOH (100mL) 203mmol) to add diethyl acetamido in the solution in EtOH (400mL) to EtONa under 0 ℃.The gained mixture heating up is lasted 1 hour and ended and use H with citric acid (30g) to refluxing 2O (300mL) dilution.After removing most of solvent in a vacuum, with the EtOAc extracted residues.With organic layer with H 2O and salt solution wash successively, with after anhydrous Na 2SO 4Dry, filtration and concentrated in a vacuum.(silica, 10: 1-3: 1 hexane: EtOAc) the purifying resistates obtains being the product (37g, 88%) of yellow solid shape by flash chromatography.
Figure A20058004432802491
Synthetic
(5g 13.8mmol) adds Br in the solution in ether (100mL) to ketone under 0 ℃ 2(0.8mL, 15.6mmol).At room temperature stirred the mixture 3 hours and subsequently with NaHCO 3Saturated aqueous solution is ended.With Et 2O extracts mixture.With organic layer with H 2O and salt solution wash successively, with after anhydrous Na 2SO 4Drying, filtration and the concentrated in a vacuum product (5.4g, 88%) to obtain being the yellow solid shape, it need not to be further purified and can be directly used in the next step.
Figure A20058004432802492
Synthetic
To α-bromoketone (5.4g, 12.2mmol) and Na 2CO 3(2.0g, 18.9mmol) add in the solution in DMSO (20mL) KI (2.1g, 13.2mmol).Under nitrogen atmosphere, stirred the mixture 28 hours at 90 ℃.To react subsequently with H 2O ends and dilutes with EtOAc.With the organic layer separation and with H 2O and salt solution wash successively, with after anhydrous Na 2SO 4Dry, filtration and concentrated in a vacuum.By flash chromatography (silica, 6: 1-1: 10 hexanes: the EtOAc) product (1.12g, 24%) of purifying resistates to obtain being solid state.
Figure A20058004432802493
Synthetic
(1.12g, 3.0mmol) (10mL) is with the solution in the diox (10mL) is heated to reflux spends the night in dense HCl with diketone.After removing solvent in a vacuum, add MeOH (3mL) with the dissolving resistates.Add ether (300mL) is the light yellow solid shape with precipitation product (302mg, 42%) subsequently.
Example 12
The amino acid whose of dicarbapentaborane that contain of oblatio synthesizes among this example detailed description Figure 27.
Synthetic
Under 0 ℃ to C 3H 7MgCl (2M, 50mmol) make an addition in the solution in ether (25mL) phenyl aldehyde in the ether (50mL) (5mL, 42.5mmol).Stirred gained solution 30 minutes down at 0 ℃.To react subsequently with NH 4The Cl saturated solution is ended and is diluted with ether.With the organic layer separation and with H 2O and salt solution wash successively, with after anhydrous Na 2SO 4Dry, filtration and concentrated in a vacuum to obtain crude product (7.2g), it need not purifying and can be directly used in the next reaction.
Figure A20058004432802502
Synthetic
Under 0 ℃ to above-mentioned alcohol (7.2g, 43.9mmol) and pyridine (7mL is 86.7mmol) in CH 2Cl 2Add in the solution (300mL) Dai Si-Martin cross iodine alkane (19.2g, 45.3mmol).The gained mixture stirred spend the night and with Na 2S 2O 3Saturated aqueous solution and NaHCO 3Saturated aqueous solution (1: 1) is ended.With organic layer with H 2O and salt solution wash successively, with after anhydrous Na 2SO 4Dry, filtration and concentrated in a vacuum.(silica, 8: 1-4: 1 hexane: EtOAc) the purifying resistates obtains being the product (6.28g is 91% for two steps) of colorless oil by flash chromatography.
Figure A20058004432802503
Synthetic
Under 90 ℃ to above-mentioned ketone (4.43g, 27.3mmol) and N-bromosuccinimide (NBS, 5.5g 30.9mmol) add 2,2 in the solution in benzene (150mL) '-azobis isobutyronitrile (AIBN, 0.2g, 1.2mmol).Gained solution is heated to reflux spends the night and be cooled to room temperature subsequently.With brown solution with H 2O and salt solution wash successively, with after anhydrous Na 2SO 4Dry, filtration and concentrated in a vacuum.With resistates from hexane crystallization with the product (6.21g, 95%) of the solid state that obtains being white in color.
Figure A20058004432802511
Synthetic
(2.5g, (6.7g 30.9mmol), then adds above-mentioned bromide (6.2g, 25.8mmol) solution in EtOH (100mL) 34.9mmol) to add diethyl acetamido in the solution in EtOH (200mL) to EtONa under 0 ℃.The gained mixture heating up lasted 1 hour to refluxing and end with citric acid (9g) subsequently and with H 2The O dilution.After removing most of solvent, with the EtOAc extracted residues.With organic layer with H 2O and salt solution wash successively, with after anhydrous Na 2SO 4Dry, filtration and concentrated in a vacuum.By flash chromatography (silica, 4: 1-2: 1 hexane: the EtOAc) product (8.92g, 92%) of purifying resistates to obtain being the light yellow solid shape.
Figure A20058004432802512
Synthetic
(1.4g 3.71mmol) adds Br in the solution in HOAc (50mL) to above-mentioned ketone 2(0.7mL, 13.6mmol).Mixture at room temperature stirred spend the night and subsequently with NaHCO 3Saturated aqueous solution is ended.With Et 2O extracts mixture.With organic layer with H 2O and salt solution wash successively, with after anhydrous Na 2SO 4Dry, filtration and concentrated in a vacuum.By flash chromatography (silica, 5: 1-3: 2 hexanes: the EtOAc) product (1.23g, 73%) of purifying resistates to obtain being the yellow solid shape.
Figure A20058004432802521
Synthetic
To α-bromoketone (1.12g, 2.46mmol) and Na 2CO 3(0.4g, 3.77mmol) add in the solution in DMSO (30mL) KI (0.45g, 13.2mmol).Mixture stirred down at 90 ℃ spend the night and subsequently with citric acid (2g) and H 2O (200mL) ends.With EtOAc extraction mixture.With organic layer with H 2O and salt solution wash successively, with after anhydrous Na 2SO 4Dry, filtration and concentrated in a vacuum.(silica, 6: 1-1: 10 hexanes: EtOAc) the purifying resistates obtains being buttery alpha-alcohol ketone (0.62g, 64%) by flash chromatography.
Under 0 ℃ to above-mentioned alcohol (0.62g, 1.58mmol) and pyridine (0.5mL is 6.19mmol) in CH 2Add in the solution among the Cl (100mL) Dai Si-Martin cross iodine alkane (0.9g, 2.12mmol).The gained mixture stirred spend the night and subsequently with Na 2S 2O 3Saturated aqueous solution and NaHCO 3Saturated aqueous solution (1: 1) is ended.With organic layer with H 2O and salt solution wash successively, with after anhydrous Na 2SO 4Dry, filtration and concentrated in a vacuum.By flash chromatography (silica, 9: 1-3: 2 hexanes: the EtOAc) product (287mg for two steps be 30%) of purifying resistates to obtain being yellow oily.
Figure A20058004432802522
Synthetic
With above-mentioned diketone (272mg, 0.7mmol) in dense HCl (10mL) He the mixture heating up in the diox (10mL) spend the night to refluxing.After removing solvent in a vacuum, add MeOH (1mL) with the dissolving resistates.Add ether (200mL) is the yellow solid shape with precipitation product (162mg, 81%) subsequently.
Example 13
The amino acid whose of dicarbapentaborane that contain of oblatio synthesizes among this example detailed description Figure 28.These compounds are to come as shown in Figure 28 to synthesize.
Example 14
This example describes clone and the expression of modified polypeptide in intestinal bacteria in detail.Use comprises that the translation system of the being introduced expression of quadrature tRNA (O-tRNA) and quadrature aminoacyl tRNA synthetase (O-RS) contains the polypeptide of alpha-non-natural amino acid.O-RS is preferentially with alpha-non-natural amino acid aminoacyl O-tRNA.Next, translation system is inserted alpha-non-natural amino acid in the polypeptide, selects codon to respond coding.Amino acid and be applicable to the O-tRNA that incorporates alpha-non-natural amino acid into and the polynucleotide sequence of O-RS is described in the U.S. patent application case 10/126th of title for " In vivo Incorporation of Unnatural AminoAcids ", No. 927 and title are the U.S. patent application case the 10/126th of " Methods and Compositions for theProduction of Orthogonal tRNA-Aminoacyl tRNA Synthetase Pairs ", in 931, these documents are to be incorporated herein by reference.Also can use following O-RS and O-tRNA sequence:
SEQ?ID?NO：1 Methanococcus jannaschii mtRNA CUA Tyr tRNA
SEQ?ID?NO：2 HLAD03; The amber of optimizing suppresses tRNA tRNA
SEQ?ID?NO：3 HL325A; The AGGA frameshift suppressor tRNA of optimizing tRNA
SEQ?ID?NO：4 Be used to incorporate into aminoacyl tRNA synthetase p-Az-PheRS (6) to azido--L-phenylalanine RS
SEQ?ID?NO：5 Be used to incorporate into aminoacyl tRNA synthetase p-BpaRS (1) to benzoyl-L-phenylalanine RS
SEQ?ID?NO：6 Be used to incorporate into the aminoacyl tRNA synthetase propargyl-PheRS of propargyl-phenylalanine RS
SEQ?ID?NO：7 Be used to incorporate into the aminoacyl tRNA synthetase propargyl-PheRS of propargyl-phenylalanine RS
SEQ?ID?NO：8 Be used to incorporate into the aminoacyl tRNA synthetase propargyl-PheRS of propargyl-phenylalanine RS
SEQ?ID?NO：9 Be used to incorporate into aminoacyl tRNA synthetase p-Az-PheRS (1) to azido--phenylalanine RS
SEQ?ID?NO：10 Be used to incorporate into aminoacyl tRNA synthetase p-Az-PheRS (3) to azido--phenylalanine RS
SEQ?ID?NO：11 Be used to incorporate into aminoacyl tRNA synthetase p-Az-PheRS (4) to azido--phenylalanine RS
SEQ?ID?NO：12 Be used to incorporate into aminoacyl tRNA synthetase p-Az-PheRS (2) to azido--phenylalanine RS
SEQ?ID?NO：13 Be used to incorporate into to azido--phenylalanine RS
Aminoacyl tRNA synthetase (LW1)
SEQ?ID?NO：14 Be used to incorporate into aminoacyl tRNA synthetase (LW5) to azido--phenylalanine RS
SEQ?ID?NO：15 Be used to incorporate into aminoacyl tRNA synthetase (LW6) to azido--phenylalanine RS
SEQ?ID?NO：16 Be used to incorporate into aminoacyl tRNA synthetase (AzPheRS-5) to azido--phenylalanine RS
SEQ?ID?NO：17 Be used to incorporate into aminoacyl tRNA synthetase (AzPheRS-6) to azido--phenylalanine RS
The plasmid transformation escherichia coli of (required alpha-non-natural amino acid is had specificity) is allowed the alpha-non-natural amino acid locus specificity is incorporated in the polypeptide to contain modified gene and quadrature aminoacyl tRNA synthetase/tRNA.The intestinal bacteria through transforming that grow in the substratum that is containing the specific alpha-non-natural amino acid between 0.01mM-100mM under 37 ℃ are expressed modified polypeptide with the fidelity and the efficient of height.The polypeptide that contains alpha-non-natural amino acid through the His mark is to be produced with inclusion body or aggregate form by e. coli host cell.These aggregates under the sex change condition in the 6M Guanidinium hydrochloride dissolving and through affinity purification.At 50mM TRIS-HCl (pH 8.0), 40 μ M CuSO 4With under 4 ℃, carry out again folding spending the night in 2% (w/v) the N-sarcosyl (Sarkosyl) by dialysis.Subsequently with described material with 20mM TRIS-HCl (pH 8.0), 100mM NaCl, 2mM CaCl 2Dialysis then removes the His-label.Referring to people such as Boissel, J.Biol.Chem., (1993) 268:15983-93.The method that is used for purified polypeptide in affiliated field for know and by SDS-PAGE, west ink dot analysis or electron spray ionisation ion trap mass spectrometry with and similar approach confirm.
Example 15: test alpha-non-natural amino acid
This example provides the result of some illustrative alpha-non-natural amino acid is carried out four times tests, incorporates the character in the non-natural amino acid polypeptides into to help to predict it.
Figure A20058004432802541
Figure A20058004432802551
Figure A20058004432802561
Example 16: test alpha-non-natural amino acid
This example provides the result of the pH value stabilization property testing that some illustrative alpha-non-natural amino acid is carried out, and incorporates the character in the non-natural amino acid polypeptides into to help to predict it.
Figure A20058004432802581
Figure A20058004432802582
Figure A20058004432802591
Figure A20058004432802592
Figure A20058004432802593
Example 17
The amino acid whose of dicarbapentaborane that contain of oblatio synthesizes among this example detailed description Figure 29.
Figure A20058004432802601
Synthetic
(10g is 41.1mmol) in H to amino acid pAF 2Add NaHCO in the solution in the O-diox (300mL, 1: 1) 3(12g, 142.9mmol) and Boc 2O (12g, 55.0mmol).Mixture was at room temperature stirred 7 hours and end with citric acid subsequently.With EtOAc extraction mixture.With organic layer with H 2O and salt solution wash successively, with after anhydrous Na 2SO 4Dry, filter and concentrate N-Boc-pAF with the solid state that obtains being white in color (13.7g, quantitatively).
Figure A20058004432802602
Synthetic
Under 0 ℃, in NaOH (40mL, 25 volume %), add ether (60mL).The blast shield thing is placed the reaction flask front.Through 3 parts of 3 minutes branches in the gained mixture, add N-nitroso-group-N-methyl ureas (6.0g, 57.9mmol).0 ℃ of following stirring reaction 10 minutes.Ether layer is separated with the sodium hydroxide layer.Through 5 minutes with organic layer by part (about 6 times add) add to N-Boc-pAF (5.0g, 16.2mmol) in the solution in anhydrous THF (20mL) until initial substance completely dissolve (by the TLC monitoring).Add 5 glacial acetic acids subsequently with stopped reaction.Remove after the organic solvent by rotary evaporation, add ethyl acetate.With organic layer with NaHCO 3Saturated solution, H 2O and salt solution wash successively, with after anhydrous MgSO 4Dry, filtration and concentrated to produce white powder (4.1g, 80%).
Figure A20058004432802603
Synthetic
(3.82g is 11.9mmol) in new distillatory methyl propionate (20mL, 208mmol) solution in through the pAF of protection in slow interpolation in t-BuOK (60mL, 1.0M is in THF).At room temperature stirred the gained mixture 30 minutes and with citric acid solution (10%, 300mL) end.With EtOAc extraction mixture.With organic layer with H 2O and salt water washing are with after anhydrous Na 2SO 4Dry, filtration and concentrated in a vacuum.(silica, 4: 1-1: 1 hexane: EtOAc) the purifying resistates is with the product (3.89g, 87%) of the solid state that obtains being white in color by flash chromatography.
Figure A20058004432802611
Synthetic
Under 0 ℃ to above-mentioned methyl esters (1.12g, 2.97mmol) add in the solution in diox (4mL) LiOH (4mL, 1N).With mixture 0 ℃ stirred 3 hours down and with aqueous citric acid solution (5%, 200mL) end and dilute with EtOAc.With the organic layer separation and with H 2O and salt solution wash successively, with after anhydrous Na 2SO 4Dry, filtration and concentrated to obtain white solid (1.02g, 94%).
Synthetic
(1.0g is 2.75mmol) in CH to above-mentioned acid under 0 ℃ 2Cl 2Add trifluoroacetic acid (10mL) in the solution (10mL).Mixture was stirred 2 hours down and concentrates subsequently at 0 ℃.In resistates, add MeOH (1mL), then add HCl (1.5mL, 4N is in the Yu diox).Add ether (200mL) subsequently with the be white in color product (701mg, 96%) of solid state of precipitation.
Example 18
The amino acid whose of dicarbapentaborane that contain of oblatio synthesizes among this example detailed description Figure 30.
Figure A20058004432802613
Synthetic
(1.09g is 3.4mmol) in difluoroacetic acid methyl esters (6mL, 68.7mmol) solution in through the pAF of protection in slow interpolation in t-BuOK (15mL, 1.0M is in THF).The gained mixture at room temperature stirred 30 minutes and (5g 25.4mmol) ends and with H with citric acid 2The O dilution.With EtOAc extraction mixture.With organic layer with H 2O and salt solution wash successively, with after anhydrous Na 2SO 4Dry, filtration and concentrated.By flash chromatography (silica, 20: 1-3: 2 hexanes: the EtOAc) product (1.27g, 94%) of purifying resistates to obtain being light brown solid state.
Figure A20058004432802621
Synthetic
Under 0 ℃ to above-mentioned methyl esters (1.26g, 3.17mmol) add in the solution in diox (30mL) LiOH (30mL, 1N).Mixture was stirred 0.5 hour down and (10g 51mmol) ends and with H with citric acid at 0 ℃ 2The O dilution.With EtOAc extraction mixture.With organic layer with H 2O and salt solution wash successively, with after anhydrous Na 2SO 4Dry, filtration and concentrated.By flash chromatography (silica, 100: 1-10: 1CH 2Cl 2: MeOH, 0.5%HOAc) the purifying resistates is to obtain brown oil (1.19g, 98%).
Figure A20058004432802622
Synthetic
(1.19g is 3.1mmol) in CH to above-mentioned acid under 0 ℃ 2Cl 2Add trifluoroacetic acid (15mL) in the solution (15mL).Mixture was stirred 0.5 hour and concentrated.In resistates, add MeOH (2mL), then add HCl (2mL, 4N is in the Yu diox).Add ether (200mL) subsequently with the be white in color product (0.82mg, 82%) of solid state of precipitation.
Example 19
The PEG reagent that contains azanol of oblatio is synthetic among this example detailed description Figure 12 a.The PEG reagent that contains azanol is to prepare described in Figure 12 a.
Example 20
The PEG reagent that contains azanol of oblatio is synthetic among this example detailed description Figure 12 b.The PEG reagent that contains azanol is to prepare described in Figure 12 b.
Example 21
The PEG reagent that contains azanol of oblatio is synthetic among this example detailed description Figure 12 c.The PEG reagent that contains azanol is to prepare described in Figure 12 c.
Example 22
The PEG reagent that contains azanol of oblatio is synthetic among this example detailed description Figure 12 d.The PEG reagent that contains azanol is to prepare described in Figure 12 d.
Example 23
The PEG reagent that contains azanol of oblatio is synthetic among this example detailed description Figure 13.The PEG reagent that contains azanol is preparation as shown in Figure 13.
Example 24
The PEG reagent that contains azanol of oblatio is synthetic among this example detailed description Figure 14.The PEG reagent that contains azanol is preparation as shown in Figure 14.
Example 25
The PEG reagent that contains azanol of oblatio is synthetic among this example detailed description Figure 15.The PEG reagent that contains azanol is preparation as shown in Figure 15.
Example 26
The PEG reagent that contains azanol of oblatio is synthetic among this example detailed description Figure 16 a.The PEG reagent that contains azanol is to prepare described in Figure 16 a.
Example 27
The PEG reagent that contains azanol of oblatio is synthetic among this example detailed description Figure 16 b.The PEG reagent that contains azanol is to prepare described in Figure 16 b.
Example 28
The connexon reagent that contains azanol of oblatio is synthetic among this example detailed description Figure 18.The connexon reagent that contains azanol is preparation as shown in Figure 18.
Example 29
This example describes 1 of oblatio among Figure 36 in detail, two (4-(brooethyl) phenyl) disulphanes (1) of 2-synthetic.Under nitrogen pressure to have stirring rod in the round-bottomed flask of oven drying, add p-methylphenyl disulphide (5.0g, 20.3mmol), N-bromosuccinimide (8.6g, 48.4mmol) and the 60mL dry-out benzene.Solution is heated to 95 ℃.With portion add azobis isobutyronitrile (.106g .64mmol).To react and reflux 16 hours.Remove solvent and brown solid is dissolved in the 100mL ethyl acetate by rotary evaporation.With saturated aqueous solution of sodium bicarbonate (2 * 50mL), deionized water (1 * 50mL) and salt solution (1 * 50mL) washing reaction mixture successively.With the organic layer separation and through anhydrous magnesium sulfate drying, filtration and under reduced pressure concentrated.By using Biotage Inc.HORIZON TMThe silica chromatogram purification crude product of chromatographic system at concentrated suitable cut and after removing trace solvent (vacuum pump), obtains 1 of the solid state that is white in color, two (4-(brooethyl) phenyl) disulphanes (2.1g, 25%) of 2-.Obtain 1H NMR spectroscopic data and mass spectrum.Reaction repeated is to obtain the product of (2.0g, 23%).
Example 30
This example describes 1 of oblatio among Figure 36 in detail, two (4-diethyl-2-acetamidomalonic acid ester) the phenyl disulphanes (2) of 2-synthetic.(6.48g is 30mmol) with the anhydrous EtOH of 50mL to the diethyl acetamido that adds in the round-bottomed flask of oven drying with stirring rod under nitrogen pressure.With portion in solution, add ethoxyquin sodium (2.6g, 38mmol).Reaction is cooled to 0 ℃.With 1, (4.1g 10.1mmol) is dissolved in 20mL 1: add in the cold soln through 1 hour process among the 1EtOH/THF and by feed hopper to two (4-(brooethyl) phenyl) disulphanes of 2-.Remove ice bath and stirring reaction 6 hours at room temperature.Remove solvent and red solid is dissolved in the 100mL ethyl acetate by rotary evaporation.With 5% citric acid solution (2 * 50mL), deionized water (1 * 50mL) and salt solution (1 * 50mL) washing reaction mixture successively.With the organic layer separation and through anhydrous magnesium sulfate drying, filtration and under reduced pressure concentrated.By using Biotage Inc.HORIZON TMThe silica chromatogram purification crude product of chromatographic system at concentrated suitable cut and after removing trace solvent (vacuum pump), obtains being 1 of yellow solid shape, two (4-diethyl-2-acetamidomalonic acid ester) the phenyl disulphanes (5.0g, 73%) of 2-.Obtain 1H NMR spectroscopic data, HPLC trace and mass spectrum.
Example 31
This example describes 1 of oblatio among Figure 36 in detail, two (4-(2-amino-3-propionic acid) phenyl disulphanes (3) synthetic of 2-.Under nitrogen pressure, in the round-bottomed flask of oven drying, add 1 to what have a stirring rod, two (4-diethyl-2-acetamidomalonic acid ester group) the phenyl disulphanes of 2-(1.0g, 1.4mmol), HCl (8mL, 12M) and 8mL 1, the 4-diox.Stirring reaction is 16 hours under refluxing.It is thick 1 to obtain being transparent buttery to remove solvent by rotary evaporation and vacuum pump, two (4-(2-amino-3-propionic acid) the phenyl disulphanes (0.75g, 135%) of 2-.Obtain 1H NMR spectroscopic data and mass spectrum.
Example 32
This example describes the N of oblatio among Figure 36 in detail, N '-two tertbutyloxycarbonyl-1, two (synthesizing of 4-(2-amino-3-propionic acid) phenyl disulphanes (4) of 2-.To in dry round-bottomed flask 1,2-two (4-(2-amino-3-propionic acid) phenyl disulphanes (0.75g, 1.9mmol) middle 5mL 1,4-diox, 5mL deionized water, the tert-Butyl dicarbonate (.65g of adding, 3.0mmol) and sodium bicarbonate (0.98g, 12mmol).At room temperature stirring reaction is 16 hours.Remove solvent and clean oil is dissolved in the 100mL ethyl acetate by rotary evaporation.With 5% citric acid solution (5mL * 2), deionized water (50mL) and salt solution (50mL) washing reaction mixture successively.With the organic layer separation and through anhydrous magnesium sulfate drying, filtration and under reduced pressure concentrated.By using Biotage Inc.HORIZON TMThe silica chromatogram purification crude product of chromatographic system, at concentrated suitable cut and after removing trace solvent (vacuum pump), the N of solid state obtains being white in color, N '-two tertbutyloxycarbonyl-1, two (4-(2-amino-3-propionic acid) phenyl disulphanes (0.5g of 2-, from crude product is 44%, is 52% through 2 steps).Obtain 1H NMR spectroscopic data, HPLC trace and mass spectrum.
Example 33
N-tertbutyloxycarbonyl-2-amino-3-(4-sulfydryl phenyl) propionic acid (5) of oblatio is synthetic among this example detailed description Figure 36.Under nitrogen pressure, in the round-bottomed flask of oven drying, add N to what have a stirring rod, N '-two tertbutyloxycarbonyl-1,2-two (4-(2-amino-3-propionic acid) phenyl disulphanes (and 0.5g, 0.84mmol), the normal-butyl phosphine (0.6mL, 2.44mmol) and the anhydrous THF of 15mL.At room temperature stirring reaction is 2 hours.Remove solvent and clean oil is dissolved in the 50mL ethyl acetate by rotary evaporation.With 5% citric acid solution (2 * 25mL), deionized water (25mL) and salt solution (25mL) washing reaction mixture successively.With the organic layer separation and through anhydrous magnesium sulfate drying, filtration and under reduced pressure concentrated.By using Biotage Inc.HORIZON TMThe silica chromatogram purification crude product of chromatographic system, at concentrated suitable cut and after removing trace solvent (vacuum pump), N-tertbutyloxycarbonyl-2-amino-3-(4-sulfydryl phenyl) propionic acid (0.5g, 100%) of the solid state that obtains being white in color.Obtain 1H NMR spectroscopic data, HPLC trace and mass spectrum.
Example 34
2-amino-3-(4-sulfydryl phenyl) propionic salt hydrochlorate (6) of oblatio is synthetic among this example detailed description Figure 36.To have stirring rod in the round-bottomed flask of oven drying, add N-tertbutyloxycarbonyl-2-amino-3-(4-sulfydryl phenyl) propionic acid (.5g, 1.6mmol), 10mL anhydrous methylene chloride and 3mL trifluoroacetic acid.At room temperature stirring reaction is 2 hours.Remove solvent and add 1mL in 1, the 4.0M hydrogenchloride in the 4-diox by rotary evaporation.Simple turn round-bottomed flask adds the 100mL anhydrous diethyl ether subsequently be white in color 2-amino-3-(4-sulfydryl phenyl) propionic salt hydrochlorate (0.39g, 100%) of solid state of precipitation.Obtain 1H NMR spectroscopic data, HPLC trace and mass spectrum.
Example 35
2-(4-(2-oxo rosickyite base) the benzyl)-2-diethyl acetamido (7) of oblatio is synthetic among this example detailed description Figure 37.Under nitrogen pressure to have in the round-bottomed flask of oven drying, the adding of stirring rod (2) (1.1g, 1.6mmol), n-Bu 3(1.2mL is 4.8mmol) with the anhydrous THF of 25mL (25mL) for P.At room temperature stirring reaction is 2 hours.In reaction, add monochloroacetone (0.16mL, 2.0mmol) and NaHCO 3(0.98g, 12mmol).At room temperature stirring reaction is 2 hours.Remove solvent and white solid is dissolved in the 100mL ethyl acetate by rotary evaporation.With saturated aqueous solution of sodium bicarbonate (50mL * 2), deionized water (50mL) and salt solution (50mL) washing reaction mixture successively.With the organic layer separation and through anhydrous magnesium sulfate drying, filtration and under reduced pressure concentrated.By using Biotage Inc.HORIZON TMThe silica chromatogram purification crude product of chromatographic system, at concentrated suitable cut and after removing trace solvent (vacuum pump), 2-(4-(2-oxo rosickyite base) the benzyl)-2-diethyl acetamido (0.62g, 98%) of the solid state that obtains being white in color.Obtain 1H NMR spectroscopic data, HPLC trace and mass spectrum.
Example 36
3-(4-(2-oxo rosickyite base) the phenyl)-2-alanine (8) of oblatio is synthetic among this example detailed description Figure 37.Under nitrogen pressure to have stirring rod in the round-bottomed flask of oven drying, add 7 (0.62g, 1.5mmol), 10mL1,4-diox and 10mL 12M HCl.Reaction backflow and stirring are spent the night.Remove solvent to produce 3-(4-(2-oxo rosickyite base) phenyl)-2-alanine (0.40g, 99% crude product) by rotary evaporation.
Example 37
N-tertbutyloxycarbonyl-3-(4-(2-oxo rosickyite base) the phenyl)-2-alanine (9) of oblatio is synthetic among this example detailed description Figure 37.Under nitrogen pressure in 8 (0.35g in the round-bottomed flask of oven drying with stirring rod, 1.3mmol) the middle tert-Butyl dicarbonate (0.63g that adds, 3.0mmol), sodium bicarbonate (0.98g, 12mmol), 8mL 1,4-diox and 8mL deionized water.At room temperature stirring reaction is 16 hours.Remove solvent and clean oil is dissolved in the 100mL ethyl acetate by rotary evaporation.With 5% citric acid solution (50mL * 2), deionized water (50mL) and salt solution (50mL) washing reaction mixture successively.With the organic layer separation and through anhydrous magnesium sulfate drying, filtration and under reduced pressure concentrated.By using Biotage Inc.HORIZON TMThe silica chromatogram purification crude product of chromatographic system, at concentrated suitable cut and after removing trace solvent (vacuum pump), N-tertbutyloxycarbonyl-3-(4-(2-oxo rosickyite base) the phenyl)-2-alanine (0.30g is 66% from crude product) of solid state obtains being white in color.Obtain 1H NMR spectroscopic data, HPLC trace and mass spectrum.
Example 38
N-tertbutyloxycarbonyl-3-(4-(2-oxopropyl sulfinyl) the phenyl)-2-alanine (10) of oblatio is synthetic among this example detailed description Figure 37.Under nitrogen pressure to have stirring rod in the round-bottomed flask of oven drying, add 9 (150mg .4mmol), 8mL glacial acetic acid and the 30%v/v hydrogen peroxide of 2mL in water.At room temperature stirring reaction is 2 hours.Remove solvent and clean oil is dissolved in the 50mL ethyl acetate by rotary evaporation.With 5% citric acid solution (25mL * 2), deionized water (25mL) and salt solution (25mL) washing reaction mixture successively.With the organic layer separation and through anhydrous magnesium sulfate drying, filtration and under reduced pressure concentrated.By using Biotage Inc.HORIZON TMThe silica chromatogram purification crude product of chromatographic system at concentrated suitable cut and after removing trace solvent (vacuum pump), obtains N-tertbutyloxycarbonyl-3-(4-(2-oxopropyl sulfinyl) phenyl)-2-alanine (0.13g, 86% crude product).Obtain HPLC trace and mass spectrum.
Example 39
3-(4-(2-oxopropyl sulfinyl) the phenyl)-2-alanine (11) of oblatio is synthetic among this example detailed description Figure 37.To have stirring rod in the round-bottomed flask of oven drying, add N-tertbutyloxycarbonyl-3-(4-(2-oxopropyl sulfinyl) phenyl)-2-alanine 10 (0.13g, 0.35mmol), 10mL anhydrous methylene chloride and 3mL trifluoroacetic acid.At room temperature stirring reaction is 2 hours.Remove solvent and add 1mL in 1, the 4.0M hydrogenchloride in the 4-diox by rotary evaporation.Simple turn round-bottomed flask adds the 100mL anhydrous diethyl ether subsequently be white in color 3-(4-(2-oxopropyl sulfinyl) the phenyl)-2-alanine (0.072g is 74% from crude product) of solid state of precipitation.Obtain 1H NMR spectroscopic data, HPLC trace and mass spectrum.
Example 40
N-tertbutyloxycarbonyl-3-(4-(2-oxopropyl alkylsulfonyl) the phenyl)-2-alanine (12) of oblatio is synthetic among this example detailed description Figure 37.Under nitrogen pressure to have stirring rod in the round-bottomed flask of oven drying, add 9 (150mg, 0.4mmol), 8mL glacial acetic acid and the 30%v/v hydrogen peroxide of 2mL in water.At room temperature stirring reaction is 24 hours.Remove solvent and clean oil is dissolved in the 50mL ethyl acetate by rotary evaporation.With 5% citric acid solution (25mL * 2), deionized water (25mL) and salt solution (25mL) washing reaction mixture successively.With the organic layer separation and through anhydrous magnesium sulfate drying, filtration and under reduced pressure concentrated.By using Biotage Inc.HORIZON TMThe silica chromatogram purification crude product of chromatographic system at concentrated suitable cut and after removing trace solvent (vacuum pump), obtains N-tertbutyloxycarbonyl-3-(4-(2-oxopropyl alkylsulfonyl) phenyl)-2-alanine (12) (0.13g, 86% crude product).Obtain HPLC trace and mass spectrum.
Example 41
3-(4-(2-oxopropyl alkylsulfonyl) the phenyl)-2-alanine (13) of oblatio is synthetic among this example detailed description Figure 37.To have stirring rod in the round-bottomed flask of oven drying, add N-tertbutyloxycarbonyl-3-(4-(2-oxopropyl alkylsulfonyl) phenyl)-2-alanine 12 (0.13g, 0.35mmol), 10mL anhydrous methylene chloride and 3mL trifluoroacetic acid.At room temperature stirring reaction is 2 hours.Remove solvent and add 1mL in 1, the 4.0M hydrogenchloride in the 4-diox by rotary evaporation.Simple turn round-bottomed flask adds the 100mL anhydrous diethyl ether subsequently be white in color 3-(4-(2-oxopropyl alkylsulfonyl) the phenyl)-2-alanine (0.067g is 65% from crude product) of solid state of precipitation.Obtain 1H NMR spectroscopic data, HPLC trace and mass spectrum.
Example 42
3-(4-(2-oxo rosickyite base) the phenyl)-2-alanine (14) of oblatio is synthetic among this example detailed description Figure 37.To have stirring rod in the round-bottomed flask of oven drying, add N-tertbutyloxycarbonyl-3-(4-(2-oxo rosickyite base) phenyl)-2-alanine 9 (0.10g .28mmol), 10mL anhydrous methylene chloride and 3mL trifluoroacetic acid.At room temperature stirring reaction is 2 hours.Remove solvent and add 1mL in 1, the 4.0M hydrogenchloride in the 4-diox by rotary evaporation.Simple turn round-bottomed flask adds the 100mL anhydrous diethyl ether subsequently be white in color 3-(4-(2-oxo rosickyite base) the phenyl)-2-alanine (0.062g is 85% from crude product) of solid state of precipitation.Obtain 1H NMR spectroscopic data, HPLC trace and mass spectrum.
Example 43
N-tertbutyloxycarbonyl-3-(4-(2-oxocyclopentyl sulfenyl) the phenyl)-2-alanine (15) of oblatio is synthetic among this example detailed description Figure 38.Under nitrogen pressure to have stirring rod in the round-bottomed flask of oven drying, add 5 (0.15g, 0.76mmol), 2-chlorine cyclopentanone (0.12mL, 1.25mmol), sodium bicarbonate (0.98g, 12mmol), the anhydrous THF of 15mL.At room temperature stirring reaction is 16 hours.Remove solvent and white solid is dissolved in the 100mL ethyl acetate by rotary evaporation.With saturated aqueous solution of sodium bicarbonate (50mL * 2), deionized water (50mL) and salt solution (50mL) washing reaction mixture successively.With the organic layer separation and through anhydrous magnesium sulfate drying, filtration and under reduced pressure concentrated.By using Biotage Inc.HORIZON TMThe silica chromatogram purification crude product of chromatographic system, at concentrated suitable cut and after removing trace solvent (vacuum pump), N-tertbutyloxycarbonyl-3-(4-(2-oxocyclopentyl sulfenyl) the phenyl)-2-alanine (0.15g, 51%) of the solid state that obtains being white in color.Obtain HPLC trace and mass spectrum.
Example 44
3-(4-(2-oxocyclopentyl sulfenyl) the phenyl)-2-alanine (16) of oblatio is synthetic among this example detailed description Figure 38.To have stirring rod in the round-bottomed flask of oven drying, add N-tertbutyloxycarbonyl-3-(4-(2-oxocyclopentyl sulfenyl) phenyl)-2-alanine 15 (0.15g, 0.39mmol), 10mL anhydrous methylene chloride and 3mL trifluoroacetic acid.At room temperature stirring reaction is 2 hours.Remove solvent and add 1mL in 1, the 4.0M hydrogenchloride in the 4-diox by rotary evaporation.Simple turn round-bottomed flask adds the 100mL anhydrous diethyl ether subsequently be white in color 3-(4-(2-oxocyclopentyl sulfenyl) the phenyl)-2-alanine (0.108g, 100%) of solid state of precipitation.Obtain 1H NMR spectroscopic data, HPLC trace and mass spectrum.
Example 45
N-tertbutyloxycarbonyl-3-(4-(2-oxo butylthio) the phenyl)-2-alanine (18) of oblatio is synthetic among this example detailed description Figure 38.Under nitrogen pressure to have stirring rod in the round-bottomed flask of oven drying, add 5 (0.15g, 0.76mmol), 1-bromo-2-butanone (0.12mL, 1.25mmol), sodium bicarbonate (0.98g, 12mmol), the anhydrous THF of 15mL.At room temperature stirring reaction is 16 hours.Remove solvent and white solid is dissolved in the 100mL ethyl acetate by rotary evaporation.With saturated aqueous solution of sodium bicarbonate (50mL * 2), deionized water (50mL) and salt solution (50mL) washing reaction mixture successively.With the organic layer separation and through anhydrous magnesium sulfate drying, filtration and under reduced pressure concentrated.By using Biotage Inc.HORIZON TMThe silica chromatogram purification crude product of chromatographic system, at concentrated suitable cut and after removing trace solvent (vacuum pump), N-tertbutyloxycarbonyl-3-(4-(2-oxo butylthio) the phenyl)-2-alanine (0.15g, 51%) of the solid state that obtains being white in color.Obtain HPLC trace and mass spectrum.
Example 46
The compound 19-22's of oblatio is synthetic among this example detailed description Figure 38.Compound 19-22 is to use to be similar to about the described method of compound 10-14 and synthesizes.
Example 47
3-(4-(2-oxo butylthio) the phenyl)-2-alanine (23) of oblatio is synthetic among this example detailed description Figure 38.To have stirring rod in the round-bottomed flask of oven drying, add N-tertbutyloxycarbonyl-3-(4-(2-oxo butylthio) phenyl)-2-alanine 18 (0.15g .56mmol), 10mL anhydrous methylene chloride and 3mL trifluoroacetic acid.At room temperature stirring reaction is 2 hours.Remove solvent and add 1mL in 1, the 4.0M hydrogenchloride in the 4-diox by rotary evaporation.Simple turn round-bottomed flask adds the 100mL anhydrous diethyl ether subsequently be white in color 3-(4-(2-oxo butylthio) the phenyl)-2-alanine (0.149g, 100%) of solid state of precipitation.Obtain 1H NMR spectroscopic data, HPLC trace and mass spectrum.
Example 48
The compound 24-27's of oblatio is synthetic among this example detailed description Figure 39.Compound 24-27 is to use to be similar to about the described method of compound 10-14 and synthesizes.
Example 49
The amino acid whose of dicarbapentaborane that contain of oblatio synthesizes among this example detailed description Figure 31.
Figure A20058004432802691
Synthetic
(1.0g is 3.1mmol) in trifluoro-acetate (5mL, 50mmol) solution in through the pAF of protection in slow interpolation in t-BuOK (15mL, 1.0M is in THF).Reaction mixture at room temperature stirred 30 minutes and (5g 25.4mmol) ends and dilutes with EtOAc with citric acid.With organic layer with H 2O and salt solution wash successively, with after anhydrous Na 2SO 4Dry, filtration and concentrated.By flash chromatography (silica, 20: 1-3: 2 hexanes: the EtOAc) product (1.07g, 83%) of purifying resistates to obtain being light brown solid state.
Figure A20058004432802692
Synthetic
Under 0 ℃ to above-mentioned methyl esters (1.0g, 2.4mmol) add in the solution in diox (30mL) LiOH (30mL, 1N).Mixture was stirred 0.5 hour down and (10g 51mmol) ends and with H with citric acid at 0 ℃ 2The O dilution.With EtOAc extraction mixture.With organic layer with H 2O and salt solution wash successively, with after anhydrous Na 2SO 4Dry, filtration and concentrated.By flash chromatography (silica, 100: 1-10: 1CH 2Cl 2: MeOH, 0.5%HOAc) the purifying resistates is to obtain brown oil (0.87g, 98%).
Figure A20058004432802701
Synthetic
(1.0g is 2.5mmol) in CH to above-mentioned acid under 0 ℃ 2Cl 2Add trifluoroacetic acid (15mL) in the solution (15mL).The gained mixture was stirred 0.5 hour and concentrated in a vacuum.In resistates, add MeOH (2mL), then add HCl (2mL, 4N is in the Yu diox).Add ether (200mL) subsequently with the be white in color product (0.56g, 75%) of solid state of precipitation.
Example 50
The amino acid whose of dicarbapentaborane that contain of oblatio synthesizes among this example detailed description Figure 32.
Synthetic
(1.0g is 3.1mmol) in five fluorine methyl propionates (8mL, 62mmol) solution in through the pAF of protection in slow interpolation in t-BuOK (15mL, 1.0M is in THF).The gained mixture at room temperature stirred 1 hour and (5g 25.4mmol) ends and with H with citric acid 2O (100mL) dilution.After removing most of solvent, with the EtOAc extracted residues.With organic layer with H 2O and salt solution wash successively, with after anhydrous Na 2SO 4Dry, filtration and concentrated.By flash chromatography (silica, 20: 1-3: 2 hexanes: the EtOAc) product (1.1g, 76%) of purifying resistates to obtain being light brown solid state.
Synthetic
Under 0 ℃ to above-mentioned methyl esters (1.0g, 2.1mmol) add in the solution in diox (30mL) LiOH (30mL, 1N).Under 0 ℃, the gained mixture stirred 0.5 hour and with citric acid (10g, 51mmol) and H 2O ends.With EtOAc extraction mixture.With organic layer with H 2O and salt solution wash successively, with after anhydrous Na 2SO 4Dry, filtration and concentrated.By flash chromatography (silica, 100: 1-10: 1CH 2Cl 2: MeOH, 0.5%HOAc) product (0.8g, 84%) of purifying resistates to obtain being the yellow solid shape.
Figure A20058004432802711
Synthetic
(0.7g is 2.5mmol) in CH to above-mentioned acid under 0 ℃ 2Cl 2Add trifluoroacetic acid (15mL) in the solution (15mL).Mixture was stirred 0.5 hour under uniform temp and concentrate.In resistates, add MeOH (2mL), then add HCl (2mL, 4N is in the Yu diox).Add ether (200mL) subsequently with the be white in color product (0.62g, 70%) of solid state of precipitation.
Example 51
The amino acid whose of dicarbapentaborane that contain of oblatio synthesizes among this example detailed description Figure 33.
Figure A20058004432802712
Synthetic
Under 0 ℃ to alcohol 1 (2.35g, 7.6mmol) and pyridine (1.5mL is 18.6mmol) in CH 2Cl 2(150mL) in stirred solution, add Dai Si-Martin cross iodine alkane (3.5g, 8.3mmol).Mixture at room temperature stirred spend the night and with Na 2S 2O 3-NaHCO 3Saturated aqueous solution (1: 1,100mL) end and with CH 2Cl 2Dilution.With the organic layer separation and with H 2O and salt water washing are with after anhydrous Na 2SO 4Dry, filtration and concentrated in a vacuum.(silica, 20: 1-3: 1 hexane: EtOAc) the purifying resistates is with the aldehyde 2 (2.15g, 92%) of the solid state that obtains being white in color by flash chromatography.ESI-MS，m/z：292(M +-OH)，232，204，175，131，115(100)。
Figure A20058004432802721
Synthetic
Under 0 ℃ to Diisopropylamine (0.33mL, 2.33mmol) in THF (60mL) in stirred solution, add n-Butyl Lithium (1.46mL, 2.34mmol).Under 0 ℃, mixture stirred 20 minutes and was cooled to-78 ℃ and add cyclopentanone subsequently.Under-78 ℃ the mixture stirring after 20 minutes, is being added aldehyde 2 (0.5g, 1.63mmol) solution in THF (20mL is with the 20mL washing).Under-78 ℃, the gained mixture stirred 1.0 hours and with NH 4The Cl saturated aqueous solution is ended.After removing most of solvent, with the EtOAc extracted residues.With organic layer with H 2O and salt water washing are with after anhydrous Na 2SO 4Dry, filtration and concentrated in a vacuum.(silica, 10: 1-1: 1 hexane: EtOAc) the purifying resistates obtains being 3 (482mg, 80%) of colorless oil by flash chromatography.
Figure A20058004432802722
Synthetic
Under 0 ℃ to alcohol 3 (0.44g, 1.13mmol) and pyridine (0.6mL is 7.44mmol) in CH 2Cl 2(150mL) in stirred solution, add Dai Si-Martin cross iodine alkane (0.6g, 1.41mmol).Mixture at room temperature stirred spend the night.To react with Na 2S 2O 3-NaHCO 3Saturated aqueous solution (1: 1,100mL) end and with CH 2Cl 2Extraction.With the organic layer combination and with H 2O and salt water washing are with after anhydrous Na 2SO 4Dry, filtration and concentrated in a vacuum.(silica, 20: 1-2: 1 hexane: EtOAc) the purifying resistates obtains being the diketone 4 (342mg, 78%) of colorless oil by flash chromatography.ESI-MS，m/z：412(M ++Na)，356，312，230，212，184，146(100)。
Figure A20058004432802723
Synthetic
Under 0 ℃ to methyl esters 4 (330mg, 0.85mmol) add in the solution in the Yu diox (4mL) LiOH (4mL, 1N).Under 0 ℃, the gained mixture stirred 30 minutes and with aqueous citric acid solution (5%, 100mL) end.With EtOAc extraction mixture.With organic layer with H 2O and salt solution wash successively, with after anhydrous Na 2SO 4Dry, filtration and concentrated to obtain white solid (310mg, 97%).ESI-MS，m/z：342，330(M +-COOH)，298，230，185，119(100)。
Figure A20058004432802731
Synthetic
(310mg is 0.83mmol) in CH to acid 5 under 0 ℃ 2Cl 2Add trifluoroacetic acid (4mL) in the solution (4mL).Mixture was stirred 30 minutes down and concentrates in a vacuum at 0 ℃.In resistates, add MeOH (1mL), then add HCl (1mL, 4N is in the Yu diox).Add ether (100mL) subsequently with the be white in color product (241mg, 94%) of solid state of precipitation.ESI-MS，m/z：298(M ++Na)，276(M ++1)，230(M +-COOH)，184，119(100)。
Example 52
The amino acid whose of dicarbapentaborane that contain of oblatio synthesizes among this example detailed description Figure 34.
Figure A20058004432802732
Synthetic
Under 0 ℃ to alcohol (6.0g, 19.4mmol) and pyridine (12mL is 150mmol) in CH 2Cl 2(400mL) in stirred solution, add Dai Si-Martin cross iodine alkane (14.2g, 33.4mmol).Mixture at room temperature stirred spend the night.To react with Na 2S 2O 3-NaHCO 3Saturated aqueous solution (1: 1,300mL) end and with CH 2Cl 2Extraction.With the organic layer combination and with H 2O and salt water washing are with after anhydrous Na 2SO 4Dry, filtration and concentrated in a vacuum.By flash chromatography (silica, 1: 100-1: 1 hexane: EtOAc) the purifying resistates aldehyde (5.48g, 92%) of solid state that obtains being white in color.
Figure A20058004432802741
Synthetic
(3.41g 11.1mmol) makes an addition to H in the solution in acetone (70mL) to above-mentioned aldehyde 2KMnO among the O (10mL) 4(2.5g, 15.8mmol).The gained mixture at room temperature stirred spend the night.After removing most of solvent, with resistates be dissolved in aqueous citric acid solution (5%, 300mL) in and extract with EtOAc.With the organic layer combination and with H 2O and salt water washing are with after anhydrous Na 2SO 4Dry, filter and concentrate in a vacuum product (2.83g, 79%) with the solid state that obtains being white in color, it need not to be further purified and can be directly used in the next step.
Figure A20058004432802742
Synthetic
Under 0 ℃ to above-mentioned acid (2.83g, 8.76mmol) add 1-amino-2-propyl alcohol (1.4mL in the solution in DMF (60mL), 17.9mmol), 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride (EDC, 4.1g, 21.4mmol), I-hydroxybenzotriazole hydrate (HOBt, 2.2g, 18.5mmol) and N, the N-diisopropyl ethyl amine (DIEA, 9mL, 51.6mmol).With mixture at room temperature stir spend the night and subsequently with aqueous citric acid solution (5%, 200mL) end and extract with EtOAc.With the organic layer combination and with H 2O and salt water washing are with after anhydrous Na 2SO 4Dry, filtration and concentrated in a vacuum.By flash chromatography (silica, 10: 1-1: 1 hexane: EtOAc) the purifying resistates foamed product (2.45g, 74%) that obtains being white in color.
Figure A20058004432802743
Synthetic
Under 0 ℃ to above-mentioned alcohol (2.44g, 6.4mmol) and pyridine (4mL is 49.6mmol) in CH 2Cl 2(100mL) in stirred solution, add Dai Si-Martin cross iodine alkane (4.1g, 9.7mmol).Mixture at room temperature stirred spend the night.To react with Na 2S 2O 3-NaHCO 3Saturated aqueous solution (1: 1,300mL) end and with CH 2Cl 2Extraction.With organic layer with H 2O and salt water washing are with after anhydrous Na 2SO 4Dry, filtration and concentrated in a vacuum.(silica, 1: 1-1: 3 hexanes: EtOAc) the purifying resistates obtains being the product (1.84g, 76%) of yellow solid shape by flash chromatography.
Figure A20058004432802751
Synthetic
Under 0 ℃ to above-mentioned methyl esters (1.72g, 4.6mmol) add in the solution in diox (10mL) LiOH (10mL, 1N).Mixture was stirred 3 hours under uniform temp and end with aqueous citric acid solution (5%).With EtOAc extraction mixture.With organic layer with H 2O and salt solution wash successively, with after anhydrous Na 2SO 4Drying, filtration and the concentrated in a vacuum product (1.7g) to obtain being solid state, it need not purifying and can be directly used in the next step.
(1.7g is 4.7mmol) in CH to above-mentioned acid under 0 ℃ 2Cl 2Add trifluoroacetic acid (15mL) in the solution (15mL).Mixture was stirred 2 hours under uniform temp and concentrate in a vacuum.In resistates, add HCl (1.5mL, 4N is in the Yu diox), then add ether (400mL).The be white in color precipitated product (1.52g is 90% for 2 steps) of solid state of collection.
Example 53
The amino acid whose of hydrazides that contain of oblatio synthesizes among this example detailed description Figure 44.
Figure A20058004432802752
Synthetic
To aldehyde (410mg, 1.34mmol) add in the solution in EtOH (15mL) the formic acid hydrazides (170mg, 2.83mmol).Reaction mixture was at room temperature stirred 1 hour.After removing most of solvent, with CH 2Cl 2Extracted residues.With organic layer combination and concentrated in a vacuum.(silica, 1: 6-1: 1 hexane: EtOAc) the purifying resistates obtains white solid (390mg, 83%) by flash chromatography.
Figure A20058004432802761
Synthetic
Under 0 ℃ to above-mentioned methyl esters (349mg, 1mmol) add in the solution in diox (7mL) LiOH (7mL, 1N).Mixture stirred 10 minutes under uniform temp and end and with H with citric acid (2.5g) 2The O extraction.With EtOAc extraction mixture.With organic layer with H 2O and salt solution wash successively, with after anhydrous Na 2SO 4Dry, filtration and concentrated to obtain white solid (290mg, 87%).
Figure A20058004432802762
Synthetic
(290mg is 0.87mmol) in CH to above-mentioned acid under 0 ℃ 2Cl 2Add trifluoroacetic acid (20mL) in the solution (20mL).Mixture was stirred 20 minutes and concentrated.In resistates, add MeOH (1mL), then add HCl (1.0mL, 4N is in the Yu diox).Add ether (200mL) is the light yellow solid shape with precipitation product (195mg, 83%) subsequently.
Example 54
The amino acid whose of hydrazides that contain of oblatio synthesizes among this example detailed description Figure 45.
Figure A20058004432802763
Synthetic
To N-tertbutyloxycarbonyl-4-hydroxymethyl phenylalanine (11.73g, 39.8mmol) add alanine methyl ester hydrochloride (9.0g in the solution in DMF (100mL), 64.5mmol), 1-(3-dimethylaminopropyl) 3-ethyl-carbodiimide hydrochloride (EDC, 15.4g, 80.3mmol), N, N-diisopropyl ethyl amine (DIEA, 30mL, 172mmol) and 1-hydroxy benzo azoles hydrate (HOBt, 8.4g, 70.6mmol).Reaction mixture at room temperature stirred spend the night and dilute with EtOAc subsequently.With the organic layer separation and with H 2O, citric acid (5%), H 2O, NaHCO 3, H 2O and salt solution wash successively, with after anhydrous Na 2SO 4Dry, filter and concentrate with the solid state that obtains being white in color through protection dipeptides (13.74g, 91%).
Figure A20058004432802771
Synthetic
Under 0 ℃ to above-mentioned through the protection dipeptides (10.33g is 27.2mmol) in CH 2Cl 2Add in the solution (300mL) pyridine (8mL, 99.1mmol) and Dai Si-Martin cross iodine alkane (14g, 33.0mmol).Reaction mixture stirred spend the night and subsequently with NaHCO 3/ Na 2S 2O 3Saturated aqueous solution (1: 1) is ended.With organic layer with H 2O, citric acid, H 2O and salt solution wash successively, with after anhydrous Na 2SO 4Dry, filtration and concentrated.(silica, 9: 1-1: 1 hexane: EtOAc) the purifying resistates is with the product (10.12g, 98%) of the solid state that obtains being white in color by flash chromatography.
Synthetic
To dipeptides aldehyde (10.1g, 26.7mmol) add in the solution in EtOH (200mL) acetic acid hydrazides (3.7g, 45mmol).Reaction mixture was at room temperature stirred 30 minutes and concentrate.In resistates, add H 2O (1L) and CH 2Cl 2(500mL).With organic layer separation and concentrated to obtain white solid (11.21g, 97%).
Figure A20058004432802773
Synthetic
Under 0 ℃ to above-mentioned methyl esters (11.1g, 25.6mmol) add in the solution in diox (50mL) LiOH (50mL, 1N).Mixture stirred 30 minutes under uniform temp and end with citric acid (20g) subsequently and with H 2O (200mL) dilution.With EtOAc extraction mixture.With organic layer with H 2O and salt solution wash successively, with after anhydrous Na 2SO 4Dry, filtration and concentrated to obtain white solid (9.52g, 88%).
Figure A20058004432802781
Synthetic
(9.5g is 22.6mmol) in CH to above-mentioned acid under 0 ℃ 2Cl 2Add trifluoroacetic acid (50mL) in the solution (50mL).Mixture was stirred 1 hour down and concentrates in a vacuum at 0 ℃.In resistates, add HCl (7mL, 4N is in the Yu diox), then add ether (500mL).The be white in color throw out (7.25g, 90%) of solid state of collection.
Example 55
The amino acid whose of oxime that contain of oblatio synthesizes among this example detailed description Figure 46 A.
Figure A20058004432802782
Synthetic
To aldehyde (3.0g) in MeOH/H +In solution in add 2 equivalent hydroxylamine hydrochlorides.Reaction mixture was at room temperature stirred 2 hours and concentrate.In resistates, add H 2O (200mL) then adds CH 2Cl 2With organic layer separation and concentrated in a vacuum.(silica, 3: 7-1: 9 hexanes: EtOAc) the purifying resistates produces the product (96%) that is solid state by flash chromatography.
Figure A20058004432802783
Synthetic
In the solution of above-mentioned methyl esters (3.0g) in diox (10mL), add under 0 ℃ LiOH (10mL, 1N).Mixture stirred 3 hours under uniform temp and subsequently by adding that citric acid (5g) is ended and with H 2The O dilution.With EtOAc extraction mixture.With organic layer with H 2O and salt solution wash successively, with after anhydrous Na 2SO 4Dry, filtration and concentrated to obtain solid.Subsequently, under 0 ℃ to gained acid in CH 2Cl 2Add trifluoroacetic acid (20mL) in the solution (20mL).Under 0 ℃, reaction mixture was stirred 1 hour and concentrated.In resistates, add MeOH (1mL), then add HCl (2.0mL, 4N is in the Yu diox).Add ether (200mL) is solid state with precipitation product (87%) subsequently.
Example 56
The amino acid whose of oxime that contain of oblatio synthesizes among this example detailed description Figure 46 B.
Figure A20058004432802791
Synthetic
To aldehyde (3.0g) in MeOH/H +In solution in add 2 equivalent methoxy amine hydrochlorates.Reaction mixture was at room temperature stirred 2 hours and concentrate.In resistates, add H 2O (200mL) then adds CH 2Cl 2With organic layer separation and concentrated in a vacuum.(silica, 3: 7-1: 9 hexanes: EtOAc) the purifying resistates produces the product (93%) that is solid state by flash chromatography.
Figure A20058004432802792
Synthetic
In the solution of above-mentioned methyl esters (3.0g) in diox (10mL), add under 0 ℃ LiOH (10mL, 1N).Mixture stirred 3 hours under uniform temp and subsequently by adding that citric acid (5g) is ended and with H 2The O dilution.With EtOAc extraction mixture.With organic layer with H 2O and salt solution wash successively, with after anhydrous Na 2SO 4Dry, filtration and concentrated to obtain solid.Subsequently, under 0 ℃ to gained acid in CH 2Cl 2Add trifluoroacetic acid (20mL) in the solution (20mL).Reaction mixture was stirred 1 hour down and concentrates at 0 ℃.In resistates, add MeOH (1mL), then add HCl (2.0mL, 4N is in the Yu diox).Add ether (200mL) is solid state with precipitation product (89%) subsequently.
Example 57
The amino acid whose of hydrazine that contain of oblatio synthesizes among this example detailed description Figure 46 C.
Synthetic
To aldehyde (3.0g) in MeOH/H +In solution in add 2 equivalent methyl hydrazines.Reaction mixture was at room temperature stirred 2 hours and concentrate.In resistates, add H 2O (200mL) then adds CH 2Cl 2With organic layer separation and concentrated in a vacuum.(silica, 3: 7-1: 9 hexanes: EtOAc) the purifying resistates produces the product (93%) that is solid state by flash chromatography.
Synthetic
In the solution of above-mentioned methyl esters (3.0g) in diox (10mL), add under 0 ℃ LiOH (10mL, 1N).Mixture stirred 3 hours under uniform temp and subsequently by adding that citric acid (5g) is ended and with H 2The O dilution.With EtOAc extraction mixture.With organic layer with H 2O and salt solution wash successively, with after anhydrous Na 2SO 4Dry, filtration and concentrated to obtain solid.Subsequently, under 0 ℃ to gained acid in CH 2Cl 2Add trifluoroacetic acid (20mL) in the solution (20mL).Reaction mixture was stirred 1 hour down and concentrates at 0 ℃.In resistates, add MeOH (1mL), then add HCl (2.0mL, 4N is in the Yu diox).Add ether (200mL) is solid state with precipitation product (89%) subsequently.
Example 58
The mPEG-azanol of oblatio is synthetic among this example detailed description Figure 48 A.
Figure A20058004432802803
Synthetic
(1.0g is 0.033mmol) in anhydrous CH to mPEG (30K)-OH 2Cl 2Interpolation p-NP chloro-formic ester in the solution (10mL) (60mg, 0.28mmol).Mixture was at room temperature stirred 15 hours.Add ether (200mL).With the throw out filtration, with ether washing and dry in a vacuum to obtain the pulverous product (1.0g, 100%) that is white in color.
Figure A20058004432802811
Synthetic
To 3-hydroxyethylamino t-butyl formate (1.75g, 10mmol) add in the solution in THF (60mL) the N-hydroxyphthalimide (3.2g, 20mmol), triphenylphosphine (2.0g, 15mmol).Reaction mixture was at room temperature stirred 10 minutes and be cooled to 0 ℃ subsequently.Through 1 hour by syringe dropwise add the di-isopropyl azodiformate (DIAD, 2.0mL, 10.5mmol).Remove ice bath and mixture stirred and spend the night and concentrate.White solid is dissolved in the ethyl acetate (100mL).With reaction mixture with saturated aqueous solution of sodium bicarbonate (100mL), H 2O (100mL) and salt solution (100mL) wash successively, with after anhydrous MgSO 4Dry, filtration and concentrated in a vacuum.(silica, 100: 1-10: 1 hexane: EtOAc) the purifying crude product is with the title compound (2.6g, 81%) of the solid state that obtains being white in color by flash chromatography.
Figure A20058004432802812
Synthetic
(2.0g is 9.1mmol) in CH to the connexon through the tertbutyloxycarbonyl protection 2Cl 2Add trifluoroacetic acid (5mL) in the solution (5mL).The gained mixture was at room temperature stirred 1 hour and concentrate.(4N in the Yu diox, 1.5mL), then adds Et to add HCl in resistates 2O (150mL).With throw out filter, with ether washing and dry in a vacuum amine connexon (1.1g, 85%) with the solid state that obtains being white in color.
Figure A20058004432802813
Synthetic
(1.0g, 0.033mmol) (53mg is 0.21mmol) in DMF-CH with the amine connexon to mPEG (30K) p-NP carbonic ether 2Cl 2In (10mL, 1: 2) mixture in add diisopropyl ethyl amine (50 μ L, 0.28mmol) and DMAP (5mg, 0.041mmol).At room temperature stirred the gained mixture 15 hours.Add ether (200mL).With the throw out filtration, with ether washing and dry in a vacuum to obtain the pulverous product (0.83g, 83%) that is white in color.
Figure A20058004432802821
Synthetic
To the mPEG phthalic imidine (30K, 0.8g, 0.0266mmol) add in the solution in MeOH (5mL) hydrazine (8.5 μ L, 0.27mmol).Stirred the gained mixture 1.0 hours down at 45 ℃.After reaction is cooled to room temperature, add CH 2Cl 2(150mL) and with the HCl aqueous solution (0.1N, 100mL) washing soln.With CH 2Cl 2(150mL) aqueous layer extracted.With the organic layer combination and with H 2O (100mL) washing is with after anhydrous Na 2SO 4Dry, filtration and concentrated.Resistates is dissolved in CH 2Cl 2(5mL).Add ether (200mL) with the precipitation pulverous azanol product (0.72g, 90%) that is white in color.
Example 59
The mPEG-azanol of oblatio is synthetic among this example detailed description Figure 48 B.
Figure A20058004432802822
Synthetic
(1.0g is 0.033mmol) in anhydrous CH to mPEG (30K)-OH 2Cl 2Interpolation p-NP chloro-formic ester in the solution (10mL) (60mg, 0.28mmol).At room temperature stirred the mixture 15 hours.Add ether (200mL).With the throw out filtration, with ether washing and dry in a vacuum to obtain the pulverous product (1.0g, 100%) that is white in color.
Synthetic
To 2-hydroxyethylamino t-butyl formate (2.8mL, 18mmol) add in the solution in THF (60mL) the N-hydroxyphthalimide (5.8g, 36mmol), triphenylphosphine (3.6g, 27mmol).Reaction mixture was at room temperature stirred 10 minutes and be cooled to 0 ℃ subsequently.Through 1 hour by syringe dropwise add the di-isopropyl azodiformate (DIAD, 3.6mL, 19mmol).Remove ice bath and mixture stirred and spend the night and concentrate.White solid is dissolved in the ethyl acetate (100mL).With reaction mixture with saturated aqueous solution of sodium bicarbonate (2 * 50mL), H 2O (50mL) and salt solution (50mL) wash successively, with after anhydrous MgSO 4Dry, filtration and concentrated in a vacuum.By using Biotage Inc.HORIZON TMThe purification by flash chromatography crude product of chromatographic system is with the title compound that contains impurity (12g, 206%) of the solid state that obtains being white in color.
Figure A20058004432802831
Synthetic
To thick connexon (12g) through the tertbutyloxycarbonyl protection in CH 2Cl 2Add trifluoroacetic acid (5mL) in the solution (5mL).The gained mixture was at room temperature stirred 1 hour and concentrate.(4N in the Yu diox, 1.5mL), then adds Et to add HCl in resistates 2O (150mL).With throw out filter, with ether washing and dry in a vacuum amine connexon (3.0g is 68% for two steps) with the solid state that obtains being white in color.
Figure A20058004432802832
Synthetic
(1.0g, 0.033mmol) (50mg is 0.21mmol) in DMF-CH with the amine connexon to mPEG (30K) p-NP carbonic ether 2Cl 2Add in the mixture in (10mL, 1: 2) diisopropyl ethyl amine (50 μ L, 0.28mmol) and 4-dimethylaminopyridine (4mg, 0.033mmol).At room temperature stirred the gained mixture 15 hours.Add ether (200mL).With the throw out filtration, with ether washing and dry in a vacuum to obtain the pulverous product (0.81g, 81%) that is white in color.
Figure A20058004432802841
Synthetic
To mPEG (30K) phthalic imidine (0.8g, 0.0266mmol) add in the solution in MeOH (5mL) hydrazine (8.5 μ L, 0.27mmol).Stirred the gained mixture 1.0 hours down at 45 ℃.After reaction is cooled to room temperature, add CH 2Cl 2(150mL) and with the HCl aqueous solution (0.1N, 100mL) washing soln.With CH 2Cl 2(150mL) aqueous layer extracted.With the organic layer combination and with H 2O (100mL) washing is with after anhydrous Na 2SO 4Dry, filtration and concentrated.Resistates is dissolved in CH 2Cl 2(5mL).Add ether (200mL) with the precipitation pulverous azanol product (0.68g, 85%) that is white in color.
Example 60
The mPEG-azanol of oblatio is synthetic among this example detailed description Figure 49 A.
Figure A20058004432802842
Synthetic
(4.0g 15.0mmol) adds K in the solution in DMF (50mL) to N-(3-bromopropyl) phthalic imidine under 0 ℃ 2CO 3(10g, 73mmol) and N-hydroxyl amino t-butyl formate (2.5g, 18.8mmol).At room temperature stirred reaction mixture is 3 hours.With mixture with H 2O dilutes (200mL) and extracts with EtOAc (200mL).With organic layer with H 2O and salt water washing are with after anhydrous Na 2SO 4Dry, filtration and concentrated in a vacuum.(silica, 20: 1-3: 1 hexane: EtOAc) the purifying resistates obtains being the product (3.5g, 72%) of colorless oil by flash chromatography.
Figure A20058004432802843
Synthetic
To above-mentioned phthalic imidine (500mg, 1.6mmol) add in the solution in EtOH (10mL) hydrazine (0.25mL, 8.0mmol).At room temperature stirred the gained mixture 3 hours.After removing throw out, concentrated filtrate.Resistates stayed in the high vacuum spend the night.By flash chromatography (silica, 3: 1-1: 1EtOAc: MeOH) the purifying resistates amine connexon (252mg, 85%) of solid state that obtains being white in color.
Synthetic
(1.0g is 0.033mmol) in anhydrous CH to mPEG (30K)-OH 2Cl 2Interpolation p-NP chloro-formic ester in the solution (10mL) (60mg, 0.28mmol).At room temperature stirred the mixture 15 hours.Add ether (200mL).With throw out filter, with ether washing and dry in a vacuum product (1.0g, 100%) with the solid state that obtains being white in color.
Synthetic
(3.0g is 0.1mmol) in anhydrous CH to above-mentioned activation mPEG (30K) 2Cl 2Add in the solution (30mL) diisopropyl ethyl amine (88 μ L, 0.5mmol) and the amine connexon (76mg, 0.4mmol).At room temperature stirred the gained mixture 15 hours.Add ether (700mL) to reaction mixture.With the throw out filtration, with ether washing and dry in a vacuum to obtain white powder (2.8g, 93%).
Figure A20058004432802853
Synthetic
(2.0g is 0.067mmol) in anhydrous CH to the mPEG (30K) through the tertbutyloxycarbonyl protection under 0 ℃ 2Cl 2Add trifluoroacetic acid (10mL) in the solution (10mL).At room temperature stirred the gained mixture 5 hours.Add ether (500mL) to reaction mixture.With the throw out filtration, with ether washing and dry in a vacuum to obtain white powder (1.8g, 90%).
Example 61
The mPEG-azanol of oblatio is synthetic among this example detailed description Figure 49 B.
Synthetic
(5.0g 37.6mmol) adds K in the solution in DMF (30mL) to N-hydroxyl amino t-butyl formate under 0 ℃ 2CO 3(12g, 87.6mmol) and N-(2-bromotrifluoromethane) phthalic imidine (10.0g, 39.7mmol).At room temperature stirred reaction mixture is 3 hours.With mixture with H 2O dilutes (200mL) and extracts with EtOAc (200mL).With organic layer with H 2O and salt water washing are with after anhydrous Na 2SO 4Dry, filtration and concentrated in a vacuum.By flash chromatography (silica, 20: 1-1: 1 hexane: EtOAc) the purifying resistates product (5.2g, 55%) of solid state that obtains being white in color.
Figure A20058004432802861
Synthetic
To above-mentioned phthalic imidine (500mg, 1.6mmol) add in the solution in EtOH (10mL) hydrazine (0.25mL, 8.0mmol).At room temperature stirred the gained mixture 3 hours.Remove after the throw out concentrated filtrate.Resistates stayed in the high vacuum spend the night.By flash chromatography (silica, 3: 1-1: 1EtOAc: MeOH) the purifying resistates amine connexon (301mg, 86%) of solid state that obtains being white in color.
Figure A20058004432802862
Synthetic
(1.0g is 0.033mmol) in anhydrous CH to above-mentioned activation mPEG (30K) 2Cl 2Add in the solution (10mL) diisopropyl ethyl amine (58 μ L, 0.33mmol), 4-dimethylaminopyridine (4mg, 0.033mmol) and above-mentioned amine connexon (64mg, 0.31mmol).At room temperature stirred the gained mixture 15 hours.Add ether (200mL) to reaction mixture.With the throw out filtration, with ether washing and dry in a vacuum to obtain white powder (0.85g, 85%).
Figure A20058004432802863
Synthetic
(0.85g is 0.028mmol) in anhydrous CH to the mPEG (30K) through the tertbutyloxycarbonyl protection under 0 ℃ 2Cl 2Add trifluoroacetic acid (10mL) in the solution (10mL).At room temperature stirred the gained mixture 5 hours.Add ether (200mL) to reaction mixture.With the throw out filtration, with ether washing and dry in a vacuum to obtain white powder (0.68g, 80%).
Example 62
The mPEG-azanol of oblatio is synthetic among this example detailed description Figure 50 A.
Figure A20058004432802871
Synthetic
(2.1g 6.9mmol) adds Boc in the solution in pyridine (50mL) to single tertbutyloxycarbonyl phthalic imidine under 0 ℃ 2O (3.3g, 15.1mmol).Gained mixture heating up to 60 ℃ is spent the night.After removing solvent in a vacuum, with resistates with EtOAc (200mL) dilution and with citric acid (5%, 200mL), water (200mL) and salt solution (200mL) washs, with after anhydrous Na 2SO 4Dry, filtration and concentrated in a vacuum.(silica, 20: 1-3: 1 hexane: EtOAc) the purifying resistates obtains being the product (2.37g, 85%) of yellow oily by flash chromatography.
Figure A20058004432802872
Synthetic
Under 0 ℃ to two tertbutyloxycarbonyl phthalic imidines (1.21g, 2.98mmol) make an addition in the solution in MeOH (15mL) ammonia among the MeOH (15mL, 7N, 105mmol).At room temperature stir the gained mixture overnight.Leach throw out and concentrated filtrate in a vacuum.By flash chromatography (silica, 10: 1-6: 4EtOAc: MeOH) the purifying resistates amine connexon (0.61g, 74%) of solid state that obtains being white in color.
Synthetic
(1.0g is 0.033mmol) in anhydrous CH to mPEG (30K)-OH 2Cl 2Interpolation p-NP chloro-formic ester in the solution (10mL) (60mg, 0.28mmol).At room temperature stirred the mixture 15 hours.Add ether (200mL) with precipitation mPEG (30K) product.With product filter, with ether washing and dry (1.0g, 100%) in a vacuum.
Figure A20058004432802881
Synthetic
(1.0g is 0.033mmol) in anhydrous CH to above-mentioned activation mPEG (30K) 2Cl 2Add in the solution (10mL) diisopropyl ethyl amine (58 μ L, 0.33mmol), 4-dimethylaminopyridine (5mg, 0.041mmol) and above-mentioned two tertbutyloxycarbonyl amine connexons (90mg, 0.33mmol).At room temperature stirred the gained mixture 15 hours.Add ether (200mL) to reaction mixture.With the throw out filtration, with ether washing and dry in a vacuum to obtain white powder (0.82g, 82%).
Figure A20058004432802882
Synthetic
(0.82g is 0.027mmol) in anhydrous CH to the mPEG (30K) through the tertbutyloxycarbonyl protection under 0 ℃ 2Cl 2Add trifluoroacetic acid (8mL) in the solution (8mL).At room temperature stirred the gained mixture 5 hours.Add ether (200mL) to reaction mixture.With the throw out filtration, with ether washing and dry in a vacuum to obtain white powder (0.57g, 70%).
Example 63
The mPEG-azanol of oblatio is synthetic among this example detailed description Figure 50 B.
Figure A20058004432802883
Synthetic
(1.5g 4.7mmol) adds Boc in the solution in pyridine to single tertbutyloxycarbonyl phthalic imidine under 0 ℃ 2O (2.2g, 10.0mmol).Gained mixture heating up to 60 ℃ is spent the night.After removing solvent in a vacuum, with resistates with EtOAc (200mL) dilution and with citric acid (5%, 200mL), water (200mL) and salt solution (200mL) washs, with after anhydrous Na 2SO 4Dry, filtration and concentrated in a vacuum.(silica, 20: 1-3: 1 hexane: EtOAc) the purifying resistates obtains being buttery product (1.6g, 81%) by flash chromatography.
Figure A20058004432802891
Synthetic
Under 0 ℃ to two tertbutyloxycarbonyl phthalic imidines (1.5g, 3.6mmol) make an addition in the solution in MeOH (15mL) ammonia among the MeOH (15mL, 7N, 105mmol).At room temperature stir the gained mixture overnight.Leach throw out and concentrated filtrate in a vacuum.By flash chromatography (silica, 10: 1-6: 4EtOAc: MeOH) the purifying resistates amine connexon (0.85g, 82%) of solid state that obtains being white in color.
Figure A20058004432802892
Synthetic
(1.0g is 0.033mmol) in anhydrous CH to mPEG (30K)-OH 2Cl 2Interpolation p-NP chloro-formic ester in the solution (10mL) (60mg, 0.28mmol).At room temperature stirred the mixture 15 hours.Add ether (200mL).With the throw out filtration, with ether washing and dry in a vacuum to obtain the pulverous product (1.0g, 100%) that is white in color.
Figure A20058004432802893
Synthetic
(1.0g is 0.033mmol) in anhydrous CH to above-mentioned activation mPEG (30K) 2Cl 2Add in the solution (10mL) diisopropyl ethyl amine (58 μ L, 0.33mmol), 4-dimethylaminopyridine (5mg, 0.041mmol) and above-mentioned two tertbutyloxycarbonyl amine connexons (100mg, 0.34mmol).At room temperature stirred the gained mixture 15 hours.Add ether (200mL) to reaction mixture.With the throw out filtration, with ether washing and dry in a vacuum to obtain white powder (0.89g, 89%).
Synthetic
(0.89g is 0.030mmol) in anhydrous CH to the mPEG (30K) through the tertbutyloxycarbonyl protection under 0 ℃ 2Cl 2Add trifluoroacetic acid (8mL) in the solution (8mL).At room temperature stirred the gained mixture 5 hours.Add ether (200mL) to reaction mixture.With the throw out filtration, with ether washing and dry in a vacuum to obtain white powder (0.65g, 73%).
Example 64
The mPEG-azanol of oblatio is synthetic among this example detailed description Figure 51 A.
Figure A20058004432802901
Synthetic
(0.5g, 0.0166mmol) (73mg 0.25mmol) adds NaCNBH in the mixture in MeOH (10mL) with the amine connexon to mPEG (30K) propionic aldehyde 3(20mg, 0.30mmol).At room temperature stirred the gained mixture 48 hours.After removing most of solvent, add ether (200mL).With the throw out filtration, with ether washing and dry in a vacuum to obtain white powder (0.43g, 86%).
Figure A20058004432802902
Synthetic
(0.42g is 0.014mmol) in anhydrous CH to the mPEG (30K) through the protection of two tertbutyloxycarbonyls under 0 ℃ 2Cl 2Add trifluoroacetic acid (4mL) in the solution (4mL).At room temperature stirred the gained mixture 8 hours.Add ether (100mL) to reaction mixture.With the throw out filtration, with ether washing and dry in a vacuum to obtain white powder (0.35g, 83%).
Example 65
The mPEG-azanol of oblatio is synthetic among this example detailed description Figure 51 B.
Figure A20058004432802903
Synthetic
Under 0 ℃ to mPEG (30K) (6.0g, 0.2mmol) and pyridine (0.1mL is 1.2mmol) in CH 2Cl 2(60mL) in stirred solution, add Dai Si-Martin cross iodine alkane (0.2g, 0.47mmol).At room temperature stir the mixture and spend the night.To react subsequently with Na 2S 2O 3-NaHCO 3Saturated aqueous solution (1: 1,100mL) end and with CH 2Cl 2(500mL * 2) extraction.With organic layer combination and Yi Shui and salt water washing, with after anhydrous Na 2SO 4Dry, filtration and concentrated in a vacuum.Resistates is dissolved in CH 2Cl 2(50mL).Add ether (1L) to solution.With the throw out filtration, with ether washing and dry in a vacuum to obtain white powder (4.9g, 82%).
Figure A20058004432802911
Synthetic
(0.5g, 0.0166mmol) (73mg 0.25mmol) adds NaCNBH in the mixture in MeOH (10mL) with the amine connexon to mPEG (30K) acetaldehyde 3(20mg, 0.30mmol).At room temperature stirred the gained mixture 48 hours.After removing most of solvent, add ether (200mL).With the throw out filtration, with ether washing and dry in a vacuum to obtain white powder (0.43g, 85%).
Figure A20058004432802912
Synthetic
(0.42g is 0.014mmol) in anhydrous CH to the mPEG (30K) through the protection of two tertbutyloxycarbonyls under 0 ℃ 2Cl 2Add trifluoroacetic acid (4mL) in the solution (4mL).At room temperature stirred the gained mixture 8 hours.Add ether (100mL) to reaction mixture.With the throw out filtration, with ether washing and dry in a vacuum to obtain white powder (0.35g, 83%).
Example 66
The mPEG-azanol of oblatio is synthetic among this example detailed description Figure 52 A.
Synthetic
To mPEG (30K)-NH 2(6.0g, 0.2mmol) add in the solution in DMF (60mL) Boc-Ser-OH (205mg, 1.0mmol), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC, 190mg, 1.0mmol) and N, and N '-diisopropyl ethyl amine (0.17mL, 1.0mmol).At room temperature stirred the mixture 10 hours and dilute with EtOAc (500mL).With the gained mixture with NaHCO 3Saturated aqueous solution (300mL), H 2O (300mL) and salt solution (300mL) wash successively, with after anhydrous Na 2SO 4Dry, filtration and concentrated in a vacuum.Resistates is dissolved in CH 2Cl 2(50mL).Add ether (700mL).With the throw out filtration, with ether washing and dry in a vacuum to obtain white powder (5.1g, 82%).
Figure A20058004432802921
Synthetic
(3.0g is 0.1mmol) in CH to above-mentioned mPEG (30K) under 0 ℃ 2Cl 2Add trifluoroacetic acid (15mL) in the solution (15mL).At room temperature stirred the gained mixture 3 hours and concentrate.In resistates, add CH 2Cl 2(5mL), (4N is in the Yu diox, 2mL) then to add HCl.Add ether (400mL) with the be white in color dihydroxylamine (2.6g, 85%) of solid state of precipitation.
Figure A20058004432802922
Synthetic
(2.0g is 0.067mmol) in H to above-mentioned mPEG (30K) 2O-CH 3CN (1: 1,20mL) add NaIO in the solution in 4(15mg, 0.07mmol).Mixture at room temperature stirred 4.0 hours and with CH 2Cl 2(500mL) dilution.With the gained mixture with the washing of water (100mL) and salt solution (100mL), with after anhydrous Na 2SO 4Dry, filtration and concentrated in a vacuum.Resistates is dissolved in CH 2Cl 2(50mL).Add ether (700mL).With the throw out filtration, with ether washing and dry in a vacuum to obtain white powder (1.8g, 90%).
Figure A20058004432802923
Synthetic
(0.5g, 0.0166mmol) (73mg 0.25mmol) adds NaCNBH in the mixture in MeOH (10mL) with the amine connexon to mPEG (30K) acetaldehyde 3(20mg, 0.30mmol).At room temperature stirred the gained mixture 48 hours.After removing most of solvent, add ether (200mL).With the throw out filtration, with ether washing and dry in a vacuum to obtain white powder (0.43g, 86%).
Figure A20058004432802924
Synthetic
(0.42g is 0.014mmol) in anhydrous CH to the mPEG (30K) through the protection of two tertbutyloxycarbonyls under 0 ℃ 2Cl 2Add trifluoroacetic acid (4mL) in the solution (4mL).At room temperature stirred the gained mixture 8 hours.Add ether (100mL) to reaction mixture.With the throw out filtration, with ether washing and dry in a vacuum to obtain white powder (0.35g, 83%).
Example 67
The mPEG4-azanol of oblatio is synthetic among this example detailed description Figure 52 B.
Figure A20058004432802931
Synthetic
(0.0166mmol) (70mg 0.25mmol) adds NaCNBH in the mixture in MeOH (10mL) with the amine connexon for 30K, 0.5g to the mPEG propionic aldehyde 3(20mg, 0.30mmol).At room temperature stirred the gained mixture 48 hours.After removing most of solvent, add ether (200mL).With the throw out filtration, with ether washing and dry in a vacuum to obtain white powder (0.40g, 80%).
Figure A20058004432802932
Synthetic
(0.40g is 0.013mmol) in anhydrous CH to the mPEG (30K) through the protection of two tertbutyloxycarbonyls under 0 ℃ 2Cl 2Add trifluoroacetic acid (4mL) in the solution (4mL).At room temperature stirred the gained mixture 8 hours.Add ether (100mL) to reaction mixture.With the throw out filtration, with ether washing and dry in a vacuum to obtain white powder (0.35g, 87%).
Example 68
The mPEG-azanol of oblatio is synthetic among this example detailed description Figure 53 A.
Synthetic
Under 0 ℃ to mPEG (30K) (6.0g, 0.2mmol) and pyridine (0.1mL is 1.2mmol) in CH 2Cl 2(60mL) in stirred solution, add Dai Si-Martin cross iodine alkane (0.2g, 0.47mmol).At room temperature stir the mixture and spend the night.To react with Na 2S 2O 3-NaHCO 3Saturated aqueous solution (1: 1,100mL) end and with CH 2Cl 2(500mL * 2) extraction.With the organic layer combination and with H 2O and salt water washing are with after anhydrous Na 2SO 4Dry, filtration and concentrated in a vacuum.Resistates is dissolved in CH 2Cl 2(50mL).Add ether (1L) to solution.With the throw out filtration, with ether washing and dry in a vacuum to obtain white powder (4.9g, 82%).
Figure A20058004432802941
Synthetic
(0.5g, 0.0166mmol) (70mg 0.25mmol) adds NaCNBH in the mixture in MeOH (10mL) with the amine connexon to mPEG (30K) acetaldehyde 3(20mg, 0.30mmol).At room temperature stirred the gained mixture 48 hours.After removing most of solvent, add ether (200mL).With the throw out filtration, with ether washing and dry in a vacuum to obtain white powder (0.40g, 80%).
Figure A20058004432802942
Synthetic
(0.40g is 0.013mmol) in anhydrous CH to the mPEG (30K) through the protection of two tertbutyloxycarbonyls under 0 ℃ 2Cl 2Add trifluoroacetic acid (4mL) in the solution (4mL).At room temperature stirred the gained mixture 8 hours.Add ether (100mL) to reaction mixture.With the throw out filtration, with ether washing and dry in a vacuum to obtain white powder (0.35g, 87%).
Example 69
The mPEG-azanol of oblatio is synthetic among this example detailed description Figure 53 B.
Figure A20058004432802943
Synthetic
To mPEG (30K)-NH 2(6.0g, 0.2mmol) add in the solution in DMF (60mL) Boc-Ser-OH (205mg, 1.0mmol), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC, 190mg, 1.0mmol) and N, and N '-diisopropyl ethyl amine (0.17mL, 1.0mmol).Mixture was at room temperature stirred 10 hours and dilute with EtOAc (500mL).With the gained mixture with NaHCO 3Saturated aqueous solution (300mL), H 2O (300mL) and salt solution (300mL) wash successively, with after anhydrous Na 2SO 4Dry, filtration and concentrated in a vacuum.Resistates is dissolved in CH 2Cl 2(50mL).Add ether (700mL).With the throw out filtration, with ether washing and dry in a vacuum to obtain white powder (5.1g, 82%).
Figure A20058004432802951
Synthetic
(3.0g is 0.1mmol) in CH to above-mentioned mPEG (30K) under 0 ℃ 2Cl 2Add trifluoroacetic acid (15mL) in the solution (15mL).The gained mixture was at room temperature stirred 3 hours and concentrate.In resistates, add CH 2Cl 2(5mL), (4N is in the Yu diox, 2mL) then to add HCl.Add ether (400mL) with the be white in color dihydroxylamine (2.6g, 85%) of solid state of precipitation.
Synthetic
(2.0g is 0.067mmol) in H to above-mentioned mPEG (30K) 2O-CH 3CN (1: 1,20mL) add NaIO in the solution in 4(15mg, 0.07mmol).Mixture at room temperature stirred 4.0 hours and with CH 2Cl 2(500mL) dilution.With the gained mixture with H 2The washing of O (100mL) and salt solution (100mL) is with after anhydrous Na 2SO 4Dry, filtration and concentrated in a vacuum.Resistates is dissolved in CH 2Cl 2(50mL).Add ether (700mL).With the throw out filtration, with ether washing and dry in a vacuum to obtain white powder (1.8g, 90%).
Figure A20058004432802953
Synthetic
(0.5g, 0.0166mmol) (70mg 0.25mmol) adds NaCNBH in the mixture in MeOH (10mL) with the amine connexon to mPEG (30K) acetaldehyde 3(20mg, 0.30mmol).At room temperature stirred the gained mixture 48 hours.After removing most of solvent, add ether (200mL).With the throw out filtration, with ether washing and dry in a vacuum to obtain white powder (0.40g, 80%).
Figure A20058004432802954
Synthetic
(0.40g is 0.013mmol) in anhydrous CH to the mPEG (30K) through the protection of two tertbutyloxycarbonyls under 0 ℃ 2Cl 2Add trifluoroacetic acid (4mL) in the solution (4mL).At room temperature stirred the gained mixture 8 hours.Add ether (100mL) to reaction mixture.With the throw out filtration, with ether washing and dry in a vacuum to obtain white powder (0.35g, 87%).
Example 70
The mPEG-azanol of oblatio is synthetic among this example detailed description Figure 54 A.
Figure A20058004432802961
Synthetic
(0.5g, 0.0166mmol) (40mg 0.16mmol) adds NaCNBH in the mixture in MeOH (10mL) with the amine connexon to mPEG (30K) propionic aldehyde 3(12mg, 0.17mmol).At room temperature stirred the gained mixture 60 hours.After removing most of solvent, resistates is dissolved in CH 2Cl 2(200mL) and with citric acid (5%, 100mL) washing.With organic layer through anhydrous Na 2SO 4Dry, filtration and concentrated in a vacuum to obtain white solid (0.42g, 84%).
Figure A20058004432802962
Synthetic
(0.4g 0.013mmol) adds H in the mixture in MeOH (4mL) to mPEG (30K) phthalic imidine 2NNH 2(4.2 μ L, 0.13mmol).Mixture was stirred 1.0 hours down and concentrates at 45 ℃.Resistates is dissolved in CH 2Cl 2(100mL) and with HCl (0.1N, 100mL) washing.With CH 2Cl 2(100mL) aqueous layer extracted.With the organic layer combination and with H 2O washing is with after anhydrous Na 2SO 4Dry, filtration and concentrated.Resistates is dissolved in CH 2Cl 2(5mL).Add ether (200mL) with the precipitation pulverous azanol product (0.34g, 85%) that is white in color.
Example 71
The mPEG-azanol of oblatio is synthetic among this example detailed description Figure 54 B.
Figure A20058004432802963
Synthetic
Under 0 ℃ to mPEG (30K) (6.0g, 0.2mmol) and pyridine (0.1mL is 1.2mmol) in CH 2Cl 2(60mL) in stirred solution, add Dai Si-Martin cross iodine alkane (0.2g, 0.47mmol).At room temperature stir the mixture and spend the night.To react subsequently with Na 2S 2O 3-NaHCO 3Saturated aqueous solution (1: 1,100mL) end and with CH 2Cl 2(500mL * 2) extraction.With the organic layer combination and with H 2O and salt water washing are with after anhydrous Na 2SO 4Dry, filtration and concentrated in a vacuum.Resistates is dissolved in CH 2Cl 2(50mL).Add ether (1L) to solution.With the throw out filtration, with ether washing and dry in a vacuum to obtain white powder (4.9g, 82%).
Figure A20058004432802971
Synthetic
(0.5g, 0.0166mmol) (40mg 0.16mmol) adds NaCNBH in the mixture in MeOH (10mL) with the amine connexon to mPEG (30K) acetaldehyde 3(12mg, 0.17mmol).At room temperature stirred the gained mixture 60 hours.After removing most of solvent, resistates is dissolved in CH 2Cl 2(200mL) and with citric acid (5%, 100mL) washing.With organic layer through anhydrous Na 2SO 4Dry, filtration and concentrated in a vacuum to obtain white solid (0.42g, 84%).
Figure A20058004432802972
Synthetic
(0.4g 0.013mmol) adds H in the mixture in MeOH (4mL) to mPEG (30K) phthalic imidine 2NNH 2(4.2 μ L, 0.13mmol).Mixture was stirred 1.0 hours down and concentrates at 45 ℃.Resistates is dissolved in CH 2Cl 2(100mL) and with HCl (0.1N, 100mL) washing.With CH 2Cl 2(100mL) aqueous layer extracted.With the organic layer combination and with H 2O washing is with after anhydrous Na 2SO 4Dry, filtration and concentrated.Resistates is dissolved in CH 2Cl 2(5mL).Add ether (200mL) with the precipitation pulverous azanol product (0.34g, 85%) that is white in color.
Example 72
The mPEG-azanol of oblatio is synthetic among this example detailed description Figure 55.
Figure A20058004432802981
Synthetic
To mPEG (30K)-NH 2(6.0g, 0.2mmol) add in the solution in DMF (60mL) Boc-Ser-OH (205mg, 1.0mmol), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC, 190mg, 1.0mmol) and N, and N '-diisopropyl ethyl amine (0.17mL, 1.0mmol).Mixture was at room temperature stirred 10 hours and dilute with EtOAc (500mL).With the gained mixture with NaHCO 3Saturated aqueous solution (300mL), H 2O (300mL) and salt solution (300mL) wash successively, with after anhydrous Na 2SO 4Dry, filtration and concentrated in a vacuum.Resistates is dissolved in CH 2Cl 2(50mL).Add ether (700mL).With the throw out filtration, with ether washing and dry in a vacuum to obtain white powder (5.1g, 82%).
Figure A20058004432802982
Synthetic
(3.0g is 0.1mmol) in CH to above-mentioned mPEG (30K) under 0 ℃ 2Cl 2Add trifluoroacetic acid (15mL) in the solution (15mL).The gained mixture was at room temperature stirred 3 hours and concentrate.In resistates, add CH 2Cl 2(5mL), (4N is in the Yu diox, 2mL) then to add HCl.Add ether (400mL) with the be white in color dihydroxylamine (2.6g, 85%) of solid state of precipitation.
Synthetic
(2.0g is 0.067mmol) in H to above-mentioned mPEG (30K) 2O-CH 3CN (1: 1,20mL) add NaIO in the solution in 4(15mg, 0.07mmol).Mixture at room temperature stirred 4.0 hours and with CH 2Cl 2(500mL) dilution.With the gained mixture with H 2The washing of O (100mL) and salt solution (100mL) is with after anhydrous Na 2SO 4Dry, filtration and concentrated in a vacuum.Resistates is dissolved in CH 2Cl 2(50mL).Add ether (700mL).With the throw out filtration, with ether washing and dry in a vacuum to obtain white powder (1.8g, 90%).
Figure A20058004432802991
Synthetic
(0.5g, 0.0166mmol) (40mg 0.16mmol) adds NaCNBH in the mixture in MeOH (10mL) with the amine connexon to mPEG (30K) acetaldehyde 3(12mg, 0.17mmol).At room temperature stirred the gained mixture 60 hours.After removing most of solvent, resistates is dissolved in CH 2Cl 2(200mL) and with citric acid (5%, 100mL) washing.With organic layer through anhydrous Na 2SO 4Dry, filtration and concentrated in a vacuum to obtain white solid (0.42g, 84%).
Figure A20058004432802992
Synthetic
(0.4g 0.013mmol) adds H in the mixture in MeOH (4mL) to mPEG (30K) phthalic imidine 2NNH 2(4.2 μ L, 0.13mmol).Mixture was stirred 1.0 hours down and concentrates at 45 ℃.Resistates is dissolved in CH 2Cl 2(100mL) and with HCl (0.1N, 100mL) washing.With CH 2Cl 2(100mL) aqueous layer extracted.With the organic layer combination and with H 2O washing is with after anhydrous Na 2SO 4Dry, filtration and concentrated.Resistates is dissolved in CH 2Cl 2(5mL).Add ether (200mL) with the precipitation pulverous azanol product (0.34g, 85%) that is white in color.
Example 73
The mPEG-azanol of oblatio is synthetic among this example detailed description Figure 56 A.
Figure A20058004432802993
Synthetic
(0.5g, 0.0166mmol) (40mg 0.16mmol) adds NaCNBH in the mixture in MeOH (10mL) with the amine connexon to mPEG (30K) propionic aldehyde 3(12mg, 0.17mmol).At room temperature stirred the gained mixture 60 hours.After removing most of solvent, resistates is dissolved in CH 2Cl 2(200mL) and with citric acid (5%, 100mL) washing.With organic layer through anhydrous Na 2SO 4Dry, filtration and concentrated in a vacuum to obtain white solid (0.41g, 82%).
Figure A20058004432803001
Synthetic
(0.4g 0.013mmol) adds H in the mixture in MeOH (4mL) to mPEG (30K) phthalic imidine 2NNH 2(4.2 μ L, 0.13mmol).Mixture was stirred 1.0 hours down and concentrates at 45 ℃.Resistates is dissolved in CH 2Cl 2(100mL) and with HCl (0.1N, 100mL) washing.With CH 2Cl 2(100mL) aqueous layer extracted.With the organic layer combination and with H 2O washing is with after anhydrous Na 2SO 4Dry, filtration and concentrated.Resistates is dissolved in CH 2Cl 2(5mL).Add ether (200mL) with the precipitation pulverous azanol product (0.34g, 83%) that is white in color.
Example 74
The mPEG-azanol of oblatio is synthetic among this example detailed description Figure 56 B.
Figure A20058004432803002
Synthetic
Under 0 ℃ to mPEG (30K) (6.0g, 0.2mmol) and pyridine (0.1mL is 1.2mmol) in CH 2Cl 2(60mL) in stirred solution, add Dai Si-Martin cross iodine alkane (0.2g, 0.47mmol).At room temperature stir the mixture and spend the night.To react subsequently with Na 2S 2O 3-NaHCO 3Saturated aqueous solution (1: 1,100mL) end and with CH 2Cl 2(500mL * 2) extraction.With the organic layer combination and with H 2O and salt water washing are with after anhydrous Na 2SO 4Dry, filtration and concentrated in a vacuum.Resistates is dissolved in CH 2Cl 2(50mL).Add ether (1L) to solution.With the throw out filtration, with ether washing and dry in a vacuum to obtain white powder (4.9g, 82%).
Synthetic
(0.5g, 0.0166mmol) (40mg 0.16mmol) adds NaCNBH in the mixture in MeOH (10mL) with the amine connexon to mPEG (30K) acetaldehyde 3(12mg, 0.17mmol).At room temperature stirred the gained mixture 60 hours.After removing most of solvent, resistates is dissolved in CH 2Cl 2(200mL) and with citric acid (5%, 100mL) washing.With organic layer through anhydrous Na 2SO 4Dry, filtration and concentrated in a vacuum to obtain white solid (0.41g, 82%).
Figure A20058004432803011
Synthetic
(0.4g 0.013mmol) adds H in the mixture in MeOH (4mL) to mPEG (30K) phthalic imidine 2NNH 2(4.2 μ L, 0.13mmol).Mixture was stirred 1.0 hours down and concentrates at 45 ℃.Resistates is dissolved in CH 2Cl 2(100mL) and with HCl (0.1N, 100mL) washing.With CH 2Cl 2(100mL) aqueous layer extracted.With the organic layer combination and with H 2O washing is with after anhydrous Na 2SO 4Dry, filtration and concentrated.Resistates is dissolved in CH 2Cl 2(5mL).Add ether (200mL) with the precipitation pulverous azanol product (0.34g, 83%) that is white in color.
Example 75
The mPEG-azanol of oblatio is synthetic among this example detailed description Figure 57.
Figure A20058004432803012
Synthetic
To mPEG (30K)-NH 2(6.0g, 0.2mmol) add in the solution in DMF (60mL) Boc-Ser-OH (205mg, 1.0mmol), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC, 190mg, 1.0mmol) and N, and N '-diisopropyl ethyl amine (0.17mL, 1.0mmol).Mixture was at room temperature stirred 10 hours and dilute with EtOAc (500mL).With the gained mixture with NaHCO 3Saturated aqueous solution (300mL), H 2O (300mL) and salt solution (300mL) wash successively, with after anhydrous Na 2SO 4Dry, filtration and concentrated in a vacuum.Resistates is dissolved in CH 2Cl 2(50mL).Add ether (700mL).With the throw out filtration, with ether washing and dry in a vacuum to obtain white powder (5.1g, 82%).
Figure A20058004432803013
Synthetic
(3.0g is 0.1mmol) in CH to above-mentioned mPEG (30K) under 0 ℃ 2Cl 2Add trifluoroacetic acid (15mL) in the solution (15mL).At room temperature stirred the gained mixture 3 hours and concentrate.In resistates, add CH 2Cl 2(5mL), (4N is in the Yu diox, 2mL) then to add HCl.Add ether (400mL) with the be white in color dihydroxylamine (2.6g, 85%) of solid state of precipitation.
Figure A20058004432803021
Synthetic
(2.0g is 0.067mmol) in H to above-mentioned mPEG (30K) 2O-CH 3CN (1: 1,20mL) add NaIO in the solution in 4(15mg, 0.07mmol).Mixture at room temperature stirred 4.0 hours and with CH 2Cl 2(500mL) dilution.With the gained mixture with H 2The washing of O (100mL) and salt solution (100mL) is with after anhydrous Na 2SO 4Dry, filtration and concentrated in a vacuum.Resistates is dissolved in CH 2Cl 2(50mL).Add ether (700mL).With the throw out filtration, with ether washing and dry in a vacuum to obtain white powder (1.8g, 90%).
Figure A20058004432803022
Synthetic
(0.5g, 0.0166mmol) (40mg 0.16mmol) adds NaCNBH in the mixture in MeOH (10mL) with the amine connexon to mPEG (30K) acetaldehyde 3(12mg, 0.17mmol).At room temperature stirred the gained mixture 60 hours.After removing most of solvent, resistates is dissolved in CH 2Cl 2(200mL) and with citric acid (5%, 100mL) washing.With organic layer through anhydrous Na 2SO 4Dry, filtration and concentrated in a vacuum to obtain white solid (0.41g, 82%).
Figure A20058004432803023
Synthetic
(0.4g 0.013mmol) adds H in the mixture in MeOH (4mL) to mPEG (30K) phthalic imidine 2NNH 2(4.2 μ L, 0.13mmol).Mixture was stirred 1.0 hours down and concentrates at 45 ℃.Resistates is dissolved in CH 2Cl 2(100mL) and with HCl (0.1N, 100mL) washing.With CH 2Cl 2(100mL) aqueous layer extracted.With the organic layer combination and with H 2O washing is with after anhydrous Na 2SO 4Dry, filtration and concentrated.Resistates is dissolved in CH 2Cl 2(5mL).Add ether (200mL) with the precipitation pulverous azanol product (0.34g, 83%) that is white in color.
Example 76
The mPEG-azanol of oblatio is synthetic among this example detailed description Figure 58 A.
MPEG (5K)-OMs's is synthetic
(1.0g is 0.2mmol) in CH to mPEG (5K)-OH 2Cl 2Add in the solution (40mL) triethylamine (110 μ L, 0.79mmol) and MsCl (50 μ L, 0.64mmol).Mixture was at room temperature stirred 10 hours and concentrate.Crude product (1.0g) need not purifying and can be directly used in the next step.
MPEG (5K)-O-NHBocSynthetic
(1.0g is 0.2mmol) in CH to thick mPEG (5K)-OMs 2Cl 2Add in the solution (10mL) tertiary butyl-N-hydroxy carbamate (0.3g, 2.2mmol) and triethylamine (0.4mL, 2.9mmol).The gained mixture was descended stirring 10 hours and was cooled to room temperature at 45 ℃.Add ether (200mL).With throw out filter, washing and dry in a vacuum product (0.42,42%) with the solid state that obtains being white in color.
MPEG (5K)-O-NH 3 + Cl - Synthetic
℃ under (0.2g is 0.04mmol) in CH to mPEG (5K)-ONHBoc 2Cl 2Add trifluoroacetic acid (7mL) in the solution (3mL).The gained mixture was at room temperature stirred 1 hour and concentrate.In resistates, add CH 2Cl 2(5mL), (4N is in the Yu diox, 2mL) then to add HCl.Add ether (300mL) with the be white in color PEG dihydroxylamine derivative (170mg, 85%) of solid state of precipitation.
Example 77
The mPEG-azanol of oblatio is synthetic among this example detailed description Figure 58 B.
MPEG (30K)-OTfSynthetic
(3.0g is 0.1mmol) in CH to mPEG (30K)-OH 2Cl 2Add 2 in the solution (30mL), and the 6-lutidine (60 μ L, 0.5mmol) and Tf 2O (65 μ L, 0.4mmol).At room temperature stirred the mixture 10 hours.Add ether (400mL) to mixture.With the throw out filtration, with ether washing and dry in a vacuum to obtain white powder (2.7g, 90%).
MPEG (30K)-O-NHBocSynthetic
(2.5g is 0.083mmol) in CH to mPEG (30K)-OTf 2Cl 2Add in the solution (25mL) N-hydroxyl amino t-butyl formate (110mg, 0.84mmol) and diisopropyl ethyl amine (0.2mL, 1,1mmol).At room temperature stir the mixture and spend the night.Add ether (200mL) with the precipitation pulverous product (2.2g, 88%) that is white in color.
MPEG (30K)-O-NH 3 + Cl - Synthetic
(2.0g is 0.067mmol) in CH to above-mentioned mPEG (30K)-ONHBoc under 0 ℃ 2Cl 2Add trifluoroacetic acid (15mL) in the solution (15mL).The gained mixture was at room temperature stirred 3 hours and concentrate.In resistates, add CH 2Cl 2(5mL), (4N is in the Yu diox, 2mL) then to add HCl.Add ether (300mL) with the be white in color PEG dihydroxylamine derivative (1.72g, 86%) of solid state of precipitation.
Example 78
The mPEG-azanol of oblatio is synthetic among this example detailed description Figure 59 A.
Figure A20058004432803041
Synthetic
(0.5g is 2.4mmol) in CH to 2-(2-hydroxyl-oxethyl) phthalic imidine 2Cl 2Add in the solution (20mL) phosgene (20% (in toluene), 8.0mL, 15.0mmol).Reaction mixture was at room temperature stirred 10 hours and concentrate in a vacuum.Resistates (0.45g, 70%) need not purifying and can be directly used in next reaction.
Figure A20058004432803051
Synthetic
To mPEG (30K)-NH 2(3g, 0.1mmol) (0.27g is 1.0mmol) in CH with the chloro-formic ester connexon 2Cl 2Add in the mixture (30mL) diisopropyl ethyl amine (0.2mL, 1.1mmol).The gained mixture was at room temperature stirred 15 hours.Add ether (500mL) to mixture.With throw out filter, washing and dry in a vacuum product (2.7g, 90%) with the solid state that obtains being white in color.
Figure A20058004432803052
Synthetic
(2.1g, (7N is in methyl alcohol, 15mL) 0.07mmol) to add ammonia in the solution in MeOH (15mL) to mPEG (30K) phthalic imidine.The gained mixture was at room temperature stirred 15 hours and concentrate.In resistates, add CH 2Cl 2(5mL), (4N is in the Yu diox, 1mL) then to add HCl.Add ether (300mL) with the be white in color product (2.4g, 89%) of solid state of precipitation.
Example 79
The mPEG-azanol of oblatio is synthetic among this example detailed description Figure 59 B.
Figure A20058004432803053
Synthetic
(3.0g is 16mmol) in CH to (tertbutyloxycarbonyl-amino oxygen base) acetate 2Cl 2Add N in the solution (80mL), and N '-di-isopropyl carbodiimide (DIC, 1.3mL, 8mmol).Mixture was at room temperature stirred 1 hour and concentrate in a vacuum.Crude product (4.9g, 84%) need not to be further purified and can be directly used in the next step.
Figure A20058004432803054
Synthetic
(7.3g 20mmol) adds mPEG (5K)-NH in the solution in DMF (20mL) to acid anhydride 2(20g, 4mmol).Mixture at room temperature stirred 10 hours and with H 2O dilutes (200mL).With CH 2Cl 2(500mL) extraction mixture.With organic layer with H 2O and salt solution (100mL) washing is with after anhydrous Na 2SO 4Dry, filtration and concentrated in a vacuum.In resistates, add CH 2Cl 2(10mL), then add ether (500mL).With the throw out filtration, with ether washing and dry in a vacuum to obtain the pulverous product (19.8g, 99%) that is white in color.
Synthetic
(1.0g is 0.2mmol) in CH to the mPEG (5K) through the tertbutyloxycarbonyl protection 2Cl 2Add trifluoroacetic acid (5mL) in the solution (5mL).The gained mixture was at room temperature stirred 1 hour and concentrate.In resistates, add CH 2Cl 2(2mL), (4N is in the Yu diox, 0.1mL) and ether (40mL) then to add HCl.With the throw out filtration, with ether washing and dry in a vacuum to obtain product (0.75g, 75%).
Example 80
The mPEG-azanol of oblatio is synthetic among this example detailed description Figure 60 A.
Figure A20058004432803062
Synthetic
(4.5g is 0.15mmol) in CH to mPEG (30K)-OH 2Cl 2Add in the solution (50mL) phosgene (20%, in toluene, 1.6mL, 3.0mmol).Reaction mixture was at room temperature stirred 10 hours and concentrate in a vacuum.Resistates (4.2g, 93%) need not purifying and can be directly used in the next reaction.
Figure A20058004432803063
Synthetic
(4.2g, 0.14mmol) (67mg is 0.28mmol) in DMF-CH with amine connexon VIII to above-mentioned activation mPEG (30K) II 2Cl 2Add in the mixture in (10mL, 1: 2) diisopropyl ethyl amine (75 μ L, 0.42mmol).At room temperature stir the gained mixture after 15 hours, add ether (200mL).With the throw out filtration, with ether washing and dry in a vacuum to obtain the pulverous product (3.8g, 90%) that is white in color.
Figure A20058004432803071
Synthetic
(3.5g, (7N is in methyl alcohol, 15mL) 0.12mmol) to add ammonia in the solution in MeOH (15mL) to mPEG (30K) phthalic imidine III.The gained mixture was at room temperature stirred 15 hours and concentrate.In resistates, add CH 2Cl 2(5mL), (4N is in the Yu diox, 1mL) then to add HCl.Add ether (300mL) with the be white in color product (3.0g, 86%) of solid state of precipitation.
Figure A20058004432803072
Synthetic
To 2-hydroxyethylamino t-butyl formate (2.8mL, 18mmol) add in the solution in THF (60mL) the N-hydroxyphthalimide (5.8g, 36mmol), triphenylphosphine (3.6g, 27mmol).Reaction mixture was at room temperature stirred 10 minutes and be cooled to 0 ℃ subsequently.Through 1 hour by syringe dropwise add the di-isopropyl azodiformate (DIAD, 3.6mL, 19mmol).Remove ice bath and mixture stirred and spend the night and concentrate.White solid is dissolved in the ethyl acetate (100mL).With reaction mixture with saturated aqueous solution of sodium bicarbonate (2 * 50mL), deionized water (50mL) and salt solution (50mL) washs successively, with after anhydrous MgSO 4Dry, filtration and concentrated in a vacuum.By using Biotage Inc.HORIZON TMThe purification by flash chromatography crude product of chromatographic system is with the title compound that contains impurity (12g, 206%) of the solid state that obtains being white in color.
Synthetic
To thick connexon (12g) through the tertbutyloxycarbonyl protection in CH 2Cl 2Add trifluoroacetic acid (5mL) in the solution (5mL).The gained mixture was at room temperature stirred 1 hour and concentrate.(4N in the Yu diox, 1.5mL), then adds Et to add HCl in resistates 2O (150mL).With throw out filter, with ether washing and dry in a vacuum amine connexon (3.0g is 68% for two steps) with the solid state that obtains being white in color.
Example 81
The mPEG-azanol of oblatio is synthetic among this example detailed description Figure 60 B.
Figure A20058004432803081
Synthetic
Under 0 ℃ to mPEG (5K)-OH (10g, 2.0mmol), Ph 3(790mg, 3.0mmol) (0.49g is 3.0mmol) in CH with the N-hydroxyphthalimide for P 2Cl 2-THF (2: 3,45mL) add in the solution in diisopropyl azodiformate (DIAD, 409 μ L, 2.0mmol).After mixture at room temperature stirred 15 hours, add ether (1L) to mixture.With the throw out filtration, with ether washing and dry in a vacuum to obtain pulverous mPEG (the 5K)-phthalic imidine (9.8g, 98%) that is white in color.
Figure A20058004432803082
Synthetic
(3g, (7N is in methyl alcohol, 25mL) 0.6mmol) to add ammonia in the solution in MeOH (25mL) to mPEG (5K) phthalic imidine.The gained mixture was at room temperature stirred 15 hours and concentrate.In resistates, add CH 2Cl 2(5mL), (4N is in the Yu diox, 1mL) then to add HCl.Add ether (300mL) with the be white in color azanol (2.5g, 83%) of solid state of precipitation.
Figure A20058004432803083
Synthetic
Under 0 ℃ to mPEG (30K)-OH (6g, 0.2mmol), Ph 3(80mg, 0.3mmol) (49mg is 0.3mmol) in CH with the N-hydroxyphthalimide for P 2Cl 2-THF (2: 3,45mL) add in the solution in diisopropyl azodiformate (DIAD, 41 μ L, 0.2mmol).At room temperature stirred the mixture 15 hours.Add ether (200mL) to mixture.With throw out with ether washing and dry in a vacuum to obtain pulverous mPEG (the 30K)-phthalic imidine product (5.8g, 96%) that is white in color.
Synthetic
(3g, (7N is in methyl alcohol, 20mL) 0.1mmol) to add ammonia in the solution in MeOH (20mL) to mPEG (30K) phthalic imidine.The gained mixture was at room temperature stirred 15 hours and concentrate.In resistates, add CH 2Cl 2(5mL), (4N is in the Yu diox, 1mL) then to add HCl.Add ether (300mL) be white in color mPEG (the 30K)-ONH of solid state of precipitation 2(2.6g, 87%).
Example 82
The mPEG-azanol of oblatio is synthetic among this example detailed description Figure 61 A.
Figure A20058004432803092
Synthetic
To 2-(2-(2-amino ethoxy) oxyethyl group) ethamine (5.0g, 33.8mmol) add (tertbutyloxycarbonyl-amino oxygen base) acetate (14.2g in the solution in DMF (100mL), 74.2mmol), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC, 28.5g, 0.15mol) and N, N '-diisopropyl ethyl amine (26mL, 0.15mol).Mixture was at room temperature stirred 10 hours and dilute with EtOAc (500mL).With the gained mixture with NaHCO 3Saturated aqueous solution (300mL), H 2O (300mL) and salt solution (300mL) wash successively, with after anhydrous Na 2SO 4Dry, filtration and concentrated in a vacuum.Crude product (14.2g, 85%) need not purifying and can be directly used in the next reaction.
Figure A20058004432803093
Synthetic
(3.0g is 6.1mmol) in CH to above-mentioned two tertbutyloxycarbonyl connexons under 0 ℃ 2Cl 2Add trifluoroacetic acid (15mL) in the solution (15mL).The gained mixture was at room temperature stirred 3 hours and concentrate.In resistates, add CH 2Cl 2(5mL), (4N is in the Yu diox, 2mL) then to add HCl.Add ether (400mL) with the be white in color dihydroxylamine (1.47g, 82%) of solid state of precipitation.
Example 83
The mPEG-azanol of oblatio is synthetic among this example detailed description Figure 61 B.
Synthetic
(1.5g 10mmol) adds Ph in the solution in THF (100mL) to three (ethylene glycol) under 0 ℃ 3P (8.0g, 30mmol) and the N-hydroxyphthalimide (4.9g, 30mmol).Slow interpolation diisopropyl azodiformate in mixture (DIAD, 4.08mL, 20mmol).The gained mixture was stirred 4 hours down and at room temperature stirred 2 days at 0 ℃.Add ether (25mL) to reaction mixture.With throw out with ether washing and dry in a vacuum to obtain the pulverous two phthalic imidine products (3.72g, 82%) that are white in color.
Synthetic
(2.2g, (7N is in methyl alcohol, 20mL) 5.0mmol) to add ammonia in the solution in MeOH (20mL) to two phthalic imidines.The gained mixture was at room temperature stirred 15 hours and concentrate.In resistates, add CH 2Cl 2(5mL), (4N is in the Yu diox, 1mL) then to add HCl.Add ether (300mL) be white in color three (ethylene glycol) dihydroxylamine connexon (1.1g, 87%) of solid state of precipitation.
Example 84
The mPEG-azanol of oblatio is synthetic among this example detailed description Figure 61 C.
Figure A20058004432803103
Synthetic
(1.94g 10mmol) adds Ph in the solution in THF (100mL) to four (ethylene glycol) under 0 ℃ 3P (8.0g, 30mmol) and the N-hydroxyphthalimide (4.9g, 30mmol).Slow interpolation diisopropyl azodiformate in mixture (DIAD, 4.08mL, 20mmol).The gained mixture was stirred 4 hours down and at room temperature stirred 2 days at 0 ℃.Add ether (25mL) to reaction mixture.With throw out with ether washing and dry in a vacuum to obtain the pulverous two phthalic imidine products (3.58g, 74%) that are white in color.
Synthetic
(2.42g, (7N is in methyl alcohol, 20mL) 5.0mmol) to add ammonia in the solution in MeOH (20mL) to two phthalic imidines.The gained mixture was at room temperature stirred 15 hours and concentrate.In resistates, add CH 2Cl 2(5mL), (4N is in the Yu diox, 1mL) then to add HCl.Add ether (300mL) be white in color four (ethylene glycol) dihydroxylamine connexon (1.27g, 85%) of solid state of precipitation.
Example 85
The mPEG-azanol of oblatio is synthetic among this example detailed description Figure 62 A.
Figure A20058004432803112
Synthetic
(2.82g 10mmol) adds Ph in the solution in THF (100mL) to six (ethylene glycol) under 0 ℃ 3P (8.0g, 30mmol) and the N-hydroxyphthalimide (4.9g, 30mmol).Slow interpolation diisopropyl azodiformate in mixture (DIAD, 4.08mL, 20mmol).The gained mixture was stirred 4 hours down and at room temperature stirred 2 days at 0 ℃.Add ether (25mL) to reaction mixture.With throw out with ether washing and dry in a vacuum to obtain the pulverous two phthalic imidine products (4.40g, 77%) that are white in color.
Figure A20058004432803113
Synthetic
(2.86g, (7N is in methyl alcohol, 20mL) 5.0mmol) to add ammonia in the solution in MeOH (20mL) to two phthalic imidines.The gained mixture was at room temperature stirred 15 hours and concentrate.In resistates, add CH 2Cl 2(5mL), (4N is in the Yu diox, 1mL) then to add HCl.Add ether (300mL) be white in color six (ethylene glycol) dihydroxylamine connexon (1.68g, 87%) of solid state of precipitation.
Example 86
The mPEG compound of oblatio is synthetic among this example detailed description Figure 62 B.
Figure A20058004432803121
Synthetic
(30K, 3.0g is 0.1mmol) in anhydrous CH to mPEG-OH 2Cl 2Add in the solution (30mL) trichloromethylchloroformate (63 μ L, 0.5mmol).Mixture at room temperature stirred spend the night.Add ether (700mL) with precipitation mPEG.With product filter, with ether washing and dry (3.0g, 100%) in a vacuum.
Following case description is measured and the active in vitro and in vivo method of the active in vitro and in vivo and therapeutic activity natural amino acid polypeptide of more modified therapeutic activity non-natural amino acid polypeptides.
Example 87: cell is in conjunction with calibrating
Do not exist or exist under 0 ℃ various concentration (under volume: un-marked GH, hGH 10 μ l) or the situation of GM-CSF and 125I-GH (about 100,000cpm or 1ng) exists down cell (3 * 10 6) cultivation (two parts) 90 minutes (cumulative volumes: 120 μ l) in PBS/1%BSA (100 μ l).Subsequently with cell suspension and ice-cold FCS higher slice of 200 μ l and centrifugal (1000g in 350 μ l plastic cement centrifuge tubes; 1 minute).Collect centrifugal and centrifugal and supernatant liquor are counted respectively by the bottom that cuts off pipe with gamma counter (Packard).
Measure specificity in conjunction with (cpm) with the form of the combination (cpm) (non-specific binding) under the situation that deducts the GH that has 100 times of excessive un-marked in the total binding under the situation that does not have the rival (two multiple mean value).Each measurement non-specific binding to employed cell type.Experiment is to use identical 125The I-GH preparation carries out and should show internal consistency at the fate that separates. 125The I-GH confirmation combines with the cell that produces the GH acceptor.Suppress in conjunction with natural GH that is subjected to un-marked in the dose-dependently mode or hGH, but not suppressed by GM-CSF or other negative controls.The hGH competition is natural 125I-GH (being similar to natural GH) bonded ability hint acceptor is discerned two kinds of forms equally well.
Example 88: The in vivo research of PEGization hGH
With PEG-hGH, not modified hGH and buffering solution throwing and mouse or rat.The result should show that the hGH with not modified that significantly increases indication by body weight compares, and PEGization hGH of the present invention has the outstanding activity and the transformation period of prolongation.
Example 89: Engage hGH and do not engage hGH with and the measurement of in vivo transformation period of variant
All experimentation on animalies are in the up-to-standard equipment of AAALAC and to be undertaken by the management of laboratory animal of Saint Louis University (St.LouisUniversity) and the scheme of the use council (Institutional Animal Care and Use Committee) approval.Indoor circulation with 12 hours light/dark with the indivedual stable breedings of rat in cage.Make animal can be arbitrarily near qualified Purina rodent food 5001 and water.For the rat that hypophysectomizes, tap water additionally contains 5% glucose.
Example 90: Pharmacokinetic study
Before entering experimentation on animals, assess the quality of each PEGization mutant hGH by three calibratings.By using MES SDS electrophoretic buffer (Invitrogen, Carlsbad, CA) purity of electrophoresis 4%-12% acrylamide NuPAGE Bis-Tris gel detection PEG-hGH under the irreducibility condition.Gel is dyeed with Xylene Brilliant Cyanine G (Coomassie blue).According to density measurement scanning, the purity of PEG-hGH band is greater than 95%.By using from Charles RiverLaboratories (Wilmington, KTA MA) 2The kinetics LAL of test kit examines and determine the endotoxin content of testing among each PEG-hGH, and every dosage is less than 5EU.With the biological activity of IM-9pSTAT5 bioassay assessment PEG-hGH, and confirm EC 50Value is less than 15nM.
The pharmacokinetic property of the growth hormone compound modified through PEG and itself and non-PEGization tethelin compared is compared to each other in the male Sprague-Dawley rat (261g-425g) available from Charles River Laboratories.By operation conduit is installed on and is used for blood collection in the carotid artery.After conduit is successfully installed, before administration, animal is distributed into treatment group (every group three to six).With the administration volume of 0.41-0.55ml/kg compound to animal subcutaneous administration 1mg/kg.At each time point by the inlying catheter blood sample collection and it is entered in the micro-centrifuge tube of EDTA coating.Collect blood plasma in centrifugal back, and be stored under-80 ℃ until analysis.Use from BioSourceInternational (Camarillo, CA) or Diagnostic Systems Laboratories (Webster, TX) antibody sandwich tethelin ELISA test kit is measured compound concentration.Use comes calculating concentration corresponding to the standard of the analogue of being given.The program that uses a model WinNonlin (Pharsight, edition 4 .1) estimates pharmacokinetic parameter.Use has on the linearity/the non-compartment model analysis (Noncompartmental analysis) of logarithm lower trapezoid integration, and homogeneous weighting concentration data.
Be in the rat after the single subcutaneous administration, obtain plasma concentration with the rule timed interval.Give the single bolus dosage of per kilogram protein 1mg to rat (every group of n=3-6).With hGH wild-type protein (WHO hGH), His mark hGH polypeptide (his-hGH) or each place of being included in six different positionss and the alpha-non-natural amino acid of 30kDa PEG covalent bond to the His mark hGH polypeptide of ethanoyl-phenylalanine and WHO hGH with (his)-hGH compares.Take plasma sample at interval at the appointed time and according to described calibrating injection compound wherein.Following table shows that single dose is thrown and the pharmacokinetic parameter value of various hGH polypeptide.(Pharsight, edition 4 .1) assesses concentration to time curve by non-compartment model analysis.The value that shows is mean value (+/-standard deviation).Cmax: peak concentration; Latter stage T1/2: terminal half-life; AUC 0-＞inf: be extrapolated to the area under the infinitely-great concentration-time curve; MRT: average retention time; Cl/f: apparent total plasma clearance; Vz/f: the apparent volume of distribution during the stage in latter stage.HGH compares with contrast, observes 30KPEG-pAF92 (his) hGH circulation and obviously prolongs, and serum half-life increases and biological usability increases.
Table: the pharmacokinetic parameter value of the 1mg/kg bolus of subcutaneous throwing and single dose in normal male Sprague-Dawley rat.
Figure A20058004432803141
Example 91: Pharmacodynamic study
The male Sprague-Dawley rat that hypophysectomizes is available from Charles River Laboratories.Remove hypophysis by operation at 3-4 during age in week.Animal is shaked down period in 3 weeks, monitor body weight during this period.Will be before beginning one's study through 7 days period weight increase 0g-8g animal put into random groups and be combined into the treatment group.To rat throw with bolus dosage or every day subcutaneous administration.In whole research, every day and successively to rat weigh, anesthesia, bloodletting and administration (suitably time).Use the heparinization kapillary to gather blood and insert through the micro-centrifuge tube of EDTA coating from the socket of the eye hole.By centrifugal separation plasma and be stored under-80 ℃ until analysis.Draw average (+/-S.D.) plasma concentration is to the curve in the timed interval.
Peptide IGF-1 is the member of the family of somatomedin or insulin-like growth factor.The effect of many promotion growths of IGF-1 mediating growth hormone.Use is measured IGF-1 concentration at the competitive desmoenzyme immunoassays test kit (Diagnosic Systems Laboratories) of the rat/mouse IGF-1 standard that provides.The hypophysis of excision rat.To rat (every group of n=5-7) subcutaneous give single dose or every day dosage.Every day successively to animal weigh, anesthesia, bloodletting and administration (suitably time).Obtain placebo treatment, wild-type hGH (hGH), His mark hGH ((his) hGH) and be included in position 35 and the body weight result to the hGH polypeptide of ethanoyl-phenylalanine of 92 places and 30kDa PEG covalent bond.Observe for the weight increase and 30KPEG-pAF92 (his) hGH compound statistically different (p＜0.0005) of 30KPEG-pAF35 (his) hGH compound the 9th day the time, difference is to observe bigger weight increase.Throwing and the comprising behind the hGH polypeptide of the non-naturally encoded amino acids of PEGization of single dose, by use two tails distribute, not in pairs, the effect to circulating plasma IGF-1 level of the t-measurements determination of equal variance has significant difference.
Example 92: comprise the security of PEGization hGH of non-naturally encoded amino acids and/or the human clinical trial of effect.
PurposeFor relatively through subcutaneous throwing and the recombinant human hGH that comprises non-naturally encoded amino acids of PEGization and one or more commercially available hGH product (including (but not limited to) Humatrope TM(Eli Lilly ﹠amp; Co.), Nutropin TM(Genentech), Norditropin TM(Novo-Nordisk), Genotropin TM(Pfizer) and Saizen/Serostim TM(Serono)) security and pharmacokinetics.
The patient Recruit 18 ages in the research in 20 years old-40 years old scope and the healthy volunteer of body weight between 60kg-90kg.The person under inspection should not have clinically significantly unusual hematology or serum chemistry and the screening of negative urine toxicity, HIV screening and hepatitis B surface antigen experimental value.It should not have any following sign: hypertension; Any primary hematologic disease history; Great hepatopathy, ephrosis, cardiovascular disorder, gastrointestinal illness, urogenital disease, metabolic trouble, neuropathy medical history; Anaemia or epilepsy medical history; Known susceptibility to product, PEG or the human serum albumin in bacterium or Mammals source; The habituation and the Heavy User that contain the beverage of caffeine; In entering 30 days of research, participate in any other clinical trial or through blood transfusion or donate blood; Contacted hGH in entering 3 months of research; Ill in entering 7 days of research; And in entering 14 days of research, have significantly unusual to physical examination before studying or clinical labororatory's assessment.All persons under inspection are assessed the whole blood that security and timing acquiring be used for pharmacokinetic analysis gather thing.Agree to carry out all researchs through approval of Ethics Committee of mechanism and patient.
Research and designIts should be the I stage in healthy male volunteers, single center, open-label, at random, two period crossover study.With (every group of 9 persons under inspection) in one group in 18 person under inspection's assigned at random to two treatment sequence set.Use the PEGization hGH that comprises non-naturally encoded amino acids and the selected commercially available prod of equal dose to throw and GH through two individually dosed periods with quick subcutaneous injection form on thigh top.Throwing is according to the explanation in the mark of parcels with the dosage and the frequency of commercially available prod.As required, use other dosage, administration frequency or other parameters of commercially available prod to add in the research by comprising other person under inspection's groups.Each medicine-feeding period was separated by 14 days cleaning phase.For in twice medicine-feeding period each, the person under inspection was limited at the research centre in 72 hours after at least 12 hours and administration before the administration, but need not between medicine-feeding period so.If have other dosage, frequency or other parameters, so also can add extra person under inspection and organize and test PEGization hGH.The multiple GH prescription that is used for human purposes through approval can be used for this research.Humatrope TM(Eli Lilly ﹠amp; Co.), Nutropin TM(Genentech), Norditropin TM(Novo-Nordisk), Genotropin TM(Pfizer) and Saizen/Serostim TM(Serono) for be used for the commercially available GH product of human purposes through approval.The experimental formula of hGH is the PEGization hGH that comprises non-naturally encoded amino acids.
Blood samplingBefore throwing and hGH and afterwards by direct venipuncture continuous blood sampling.Administration precontract 30 minutes, 20 minutes and 10 minutes (3 baseline samples) and after administration following time approximately: 30 minutes and 1 hour, 2 hours, 5 hours, 8 hours, 12 hours, 15 hours, 18 hours, 24 hours, 30 hours, 36 hours, 48 hours, 60 hours and acquisition in 72 hours are used to measure the venous samples can (5mL) of Serum GH concentration.Each serum sample is divided into two aliquots containigs.All serum samples are stored under-20 ℃.Serum sample transports on dry ice.Before the initial administration in the 1st day at once, before the 4th day morning, the 16th day administration at once and carry out fasting clinical experiment test (hematology, serum chemistry, and urine test) the 19th day morning.
Bioanalytical methodUse ELISA test kit program (Diagnostic Systems Laboratory[DSL], Webster TX) mensuration Serum GH concentration.
Security is measuredAt administration each time (the 1st day and the 16th day) 6 hours, 24 hours, 48 hours and 72 hours immediate record vital signs before and after the administration each time.The variation from baseline of the incidence be based on adverse events and type and clinical experiment test is measured in security.In addition, among assessment life sign measurement result (comprising blood pressure) and the physical examination result with the preceding variation of research.
Data analysisThe average baselining GH concentration of the GH level determination by deducting three samples gathering when administration preceding 30 minutes, 20 minutes and 10 minutes by equalization by each that is worth after the administration about administration before serum-concentration value after the administration of baseline GH concentration correction.If Serum GH concentration is lower than the quantitative level of calibrating before the administration, it is not contained in the calculating of mean value so.From serum-concentration data determination pharmacokinetic parameter about baseline GH concentration correction.Use the BIOAVL software of recent release on Digital Equipment Corporation VAX 8600 computer systems, to calculate pharmacokinetic parameter by the method that does not rely on model.Measure following pharmacokinetic parameter: peak serum-concentration (C Max); Arrive the time (t of peak serum-concentration Max); By use that linear trapezoidal rule calculates from time zero blood sampling time (AUC to the end 0-72) concentration-time curve under area (AUC); And eliminate transformation period (t by the end that elimination rate constant calculates 1/2).In the terminal linearity region of logarithm-linear concentration-time curve, estimate elimination rate constant by the linear regression at consecutive numbers strong point.For each treatment, calculate the mean standard deviation (SD) and the variation coefficient (CV) of pharmacokinetic parameter.The ratio of calculating parameter mean value (keeping prescription/non-maintenance prescription).
Safety resultsThe incidence of adverse events distributes identical in the treatment group.Comparing with clinical experiment test or blood pressure before baseline or the research does not have noticeable change clinically, and compares no noticeable change before physical examination result and life sign measurement result and the research.The security overview of two treatment groups should be it seems similar.
Pharmacokinetics resultUnder each measured time point, will accept one or more commercially available hGH product of single dose (including (but not limited to) Humatrope TM(Eli Lilly ﹠amp; Co.), Nutropin TM(Genentech), Norditropin TM(Novo-Nordisk), Genotropin TM(Pfizer) and Saizen/Serostim TM(Serono)) average serum GH concentration-time overview of 18 persons under inspection of all after (not about the correction of baseline GH content) with comprise that the PEGization hGH of non-naturally encoded amino acids compares.All persons under inspection should have baseline GH concentration before the administration in the normal physiological scope.By serum data determination pharmacokinetic parameter and mensuration C about average baselining GH concentration correction before the administration MaxAnd t MaxSelected clinical relatively thing (Humatrope TM(Eli Lilly ﹠amp; Co.), Nutropin TM(Genentech), Norditropin TM(Novo-Nordisk), Genotropin TM(Pfizer), Saizen/Serostim TM(Serono)) average t MaxT than the PEGization hGH that comprises non-naturally encoded amino acids MaxSignificantly shorter.Compare with the terminal transformation period of the PEGization hGH that comprises non-naturally encoded amino acids, the end of the commercially available hGH product of test half sad time value is significantly shorter.
Although research of the present invention is to carry out in the healthy male person under inspection, but be expected in other patient colonies (such as the sex patient who suffers from cancer or chronic renal failure, children's's patients with renal failure, patient in the body stored Procedure, or the patient of predetermined selectivity operation) and can have similar absorption feature and security overview.
On the whole, through subcutaneous throwing and the PEGization hGH that comprises non-naturally encoded amino acids of single dose should be safe and the healthy male person under inspection to its good acceptance.According to the relative incidence of adverse events, the hGH of commercial form and comprise that clinical experiment value, vital sign and physical examination result and the security overview of the PEGization hGH of non-naturally encoded amino acids should be suitable.The PEGization hGH that comprises non-naturally encoded amino acids provides big clinical efficacy to patient and health care supplier potentially.
The water-soluble comparison of example 93:PEGization hGH and non-PEGization hGH
The amount that is dissolvable in water the polypeptide out of the ordinary of 100 μ L water by mensuration obtains hGH wild-type protein (WHO hGH), His mark hGH polypeptide (his-hGH) or is included in 92 places, position and the alpha-non-natural amino acid of 30kDa PEG covalent bond water-soluble to the His mark hGH polypeptide of ethanoyl-phenylalanine.The amount of PEGization hGH is greater than the amount of WHO hGH and hGH, and the PEGization increase of its proof non-natural amino acid polypeptides is water-soluble.
Example 94: the in vivo research of modified therapeutic activity non-natural amino acid polypeptides
The prostate cancer tumor xenogeneic graft is implanted in the mouse, subsequently it is divided into two groups.With therapeutic activity natural amino acid polypeptide treat every day with modified therapeutic activity non-natural amino acid polypeptides treatment and another group one group of every day.Measure the tumour size every day, and as by the tumour size of the group for the treatment of with modified therapeutic activity non-natural amino acid polypeptides reduce indicated, compare with therapeutic activity natural amino acid polypeptide, modified therapeutic activity non-natural amino acid polypeptides has the therapeutic efficiency of improvement.
Example 95: the in vivo research of modified therapeutic activity non-natural amino acid polypeptides
The prostate cancer tumor xenogeneic graft is implanted in the mouse, subsequently it is divided into two groups.With therapeutic activity natural amino acid polypeptide treat every day with modified therapeutic activity non-natural amino acid polypeptides treatment and another group one group of every day.Measure the tumour size every day, and as by the tumour size of the group for the treatment of with modified therapeutic activity non-natural amino acid polypeptides reduce indicated, compare with therapeutic activity natural amino acid polypeptide, modified therapeutic activity non-natural amino acid polypeptides has the therapeutic efficiency of improvement.
Should be appreciated that example of Miao Shuing and embodiment are only for illustrative purpose and its various modifications or change and should propose and be included in the spirit and scope of the application's case and in the category of the claim of enclosing by the those skilled in the art herein.The open case of all that quote, patent and patent application case are all to be incorporated herein by reference for all purposes herein.
Sequence table
?SEQ ?ID# The sequence table sequence Remarks Protein, nucleic acid, tRNA or RS
?1 CCGGCGGTAGTTCAGCAGGGCAGAACGGCGGACTCTAAA TCCGCATGGCGCTGGTTCAAATCCGGCCCGCCGGACCA Methanococcus jannaschii mtRNA CUA Tyr tRNA
?2 CCCAGGGTAGCCAAGCTCGGCCAACGGCGACGGACTCTA AATCCGTTCTCGTAGGAGTTCGAGGGTTCGAATCCCTTCC C TGGGACCA HLAD03; The amber of optimizing suppresses tRNA tRNA tRNA
?3 GCGAGGGTAGCCAAGCTCGGCCAACGGCGACGGACTTCC TAATCCGTTCTCGTAGGAGTTCGAGGGTTCGAATCCCTCC CCTCGCACCA HL325A; The AGGA frameshift suppressor tRNA of optimizing tRNA
?4 MDEFEMIKRNTSEIISEEELREVLKKDEKSAGIGFEPSGKIHL GHYLQIKKMIDLQNAGFDIIILLADLHAYLNQKGELDEIRKIG DYNKKVFEAMGLKAKYVYGSTFQLDKDYTLNVYRLALKT TLKRARRSMELIAREDENPKVAEVIYPIMQVNTYYYLGVDV AVGGMEQRKIHMLARELLPKKVVCIHNPVLTGLDGEGKMS SSKGNFIAVDDSPEEIRAKIKKAYCPAGVVEGNPIMEIAKYFL EYPLTIKRPEKFGGDLTVNSYEELESLFKNKELHPMDLKNA VAEELIKILEPIRKRL Be used to incorporate into aminoacyl tRNA synthetase p-Az-PheRS (6) to azido--L-phenylalanine RS
?5 MDEFEMIKRNTSEIISEEELREVLKKDEKSAGIGFEPSGKIHL GHYLQIKKMIDLQNAGFDIIILLADLHAYLNQKGELDEIRKIG DYNKKVFEAMGLKAKYVYGSSFQLDKDYTLNVYRLALKT TLKRARRSMELIAREDENPKVAEVIYPIMQVNTSHYLGVDV AVGGMEQRKIHMLAPJELLPKKWCIHNPVLTGLDGEGKMS SSKGNFIAVDDSPEEIRAKIKKAYCPAGWEGNPIMEIAKYFL EYPLTIKRPEKFGGDLTVNSYEELESLFKNKELHPMDLKNA VAEELIKILEPIRKRL Be used to incorporate into aminoacyl tRNA synthetase p-BpaRS (1) to benzoyl-L-phenylalanine RS
?6 MDEFEMIKRNTSEIISEEELREVLKKDEKAAIGFEPSGKIHLG HYLQIKKMIDL QNAGFDIIILLADLHAYLNQKGELDEIRKIGDYNKKVFEAMG LKAKYVYGSPFQLDKDYTLNVYRLALKTTLKRARRSMELIA REDENPKVAEVIYPIMQVNAIYLAVDVAVGGMEQRKIHML ARELLPKKWCIHNPVLTGLDGEGKMSSSKGNFIAVDDSPEE IRAKIKKAYCPAGVVEGNPIMEIAKYFLEYPLTIKRPEKFGG DLTVNSYEELESLFKNKELHPMDLKNAVAEELIKILEPIRKR L Be used to incorporate into the aminoacyl tRNA synthetase propargyl-PheRS of propargyl-phenylalanine RS
?7 MDEFE?MIKRN?TSEII?SEEEL?REVLK?KDEKS?AAIGF?EPSGK IHLGH?YLQIK?KMIDL?QNAGF?DIIIL?LADLH?AYLNQ?KGELD EIRKI?GDYNK KVFEA?MGLKA?KYVYG?SPFQL?DKDYT LNVYR?LALKT?TLKRA?RRSME?LIARE?DENPK?VAEVI?YPIMQ VNIPY?LPVD?VAVGG?MEQRK?IHMLA?RELLP?KKVVC?IHNPV LTGLD?GEGKM?SSSKG?NFIAV?DDSPE?EIRAK?IKKAY?CPAGV VEGNP?IMEIA?KYFLE?YPLTIKRPEK?FGGDL?TVNSY?EELES LFKNK?ELHPM?DLKNA?VAEEL?IKILE?PIRKR?L Be used to incorporate into the aminoacyl tRNA synthetase propargyl-PheRS of propargyl-phenylalanine RS
?8 MDEFE?MIKRN?TSEII?SEEEL?REVLK?KDEKS?AAIGF?EPSGK IHLGH?YLQIK?KMIDL?QNAGF?DIIIL?LADLH?AYLNQ?KGELD EIRKI?GDYNK?KVFEA?MGLKA?KYVYG?SKFQL?DKDYT LNVYR?LALKT?TLKRA?RRSME?LIARE?DENPK?VAEVI?YPIMQ VNAIY?LAVD?VAVGG?MEQRK?IHMLA?RELLP?KKWC?IHNPV LTGLD?GEGKM?SSSKG?NFIAV?DDSPE?EIRAK?IKKAY?CPAGV VEGNP?IMEIA?KYFLE?YPLTI?KRPEK?FGGDL?TVNSY?EELES LFKNK?ELHPM?DLKNA?VAEEL?IKILE?PIRKR?L Be used to incorporate into the aminoacyl tRNA synthetase propargyl-PheRS of propargyl-phenylalanine RS
?SEQ ?ID# Sequence Remarks Protein, nucleic acid, tRNA or RS
?9 MDEFEMIKRNTSEIISEEELREVLKKDEKSATIGFEPSGKIHL GHYLQIKKMIDLQNAGFDIIILLADLHAYLNQKGELDEIRKIG DYNKKVFEAMGLKAKYVYGSNFQLDKDYTLNVYRLALKT TLKRARRSMELIAREDENPKVAEVIYPIMQVNPLHYQGVDV AVGGMEQRKIHMLARELLPKKVVCIHNPVLTGLDGEGKMS SSKGNFIAVDDSPEEIRAKIKKAYCPAGWEGNPIMEIAKYFL EYPLTIKRPEKFGGDLTVNSYEELESLFKNKELHPMDLKNA VAEELIKILEPIRKRL Be used to incorporate into aminoacyl tRNA synthetase p-Az-PheRS (1) to azido--phenylalanine RS
?10 MDEFEMIKRNTSEIISEEELREVLKKDEKSATIGFEPSGKIHL GHYLQIKKMIDLQNAGFDIIILLADLHAYLNQKGELDEIRKIG DYNKKVFEAMGLKAKYVYGSSFQLDKDYTLNVYRLALKT TLKRARRSMELLAJREDENPKVAEVIYPIMQVNPLHYQGVDV AVGGMEQRKIHMLARELLPKKVVCIHNPVLTGLDGEGKMS SSKGNFIAVDDSPEEIRAKIKKAYCPAGWEGNPIMEIAKYFL EYPLTIKRPEKFGGDLTVNSYEELESLFKNKELHPMDLKNA VAEELIKILEPIRKRL Be used to incorporate into aminoacyl tRNA synthetase p-Az-PheRS (3) to azido--phenylalanine RS
?11 MDEFEMIKRNTSEIISEEELREVLKKDEKSALIGFEPSGKIHL GHYLQIKKMIDLQNAGFDIIILLADLHAYLNQKGELDEIRKIG DYNKKVFEAMGLKAKYVYGSTFQLDKDYTLNVYRLALKT TLKRARRSMELIAREDENPKVAEVIYPIMQVNPVHYQGVDV AVGGMEQRKIHMLARELLPKKWCIHNPVLTGLDGEGKMS SSKGNFIAVDDSPEEIRAKIKKAYCPAGVVEGNPIMEIAKYFL EYPLTIKRPEKFGGDLTVNSYEELESLFKNKELHPMDLKNA VAEELIKILEPIRKRL Be used to incorporate into aminoacyl tRNA synthetase p-Az-PheRS (4) to azido--phenylalanine RS
?12 MDEFEMIKRNTSEIISEEELREVLKKDEKSATIGFEPSGKIHL GHYLQIKKMIDLQNAGFDIIILLADLHAYLNQKGELDEIRKIG DYNKKVFEAMGLKAKYVYGSSFQLDKDYTLNVYRLALKT TLKRARRSMELIAREDENPKVAEVIYPIMQVNPSHYQGVDV AVGGMEQRKIHMLARELLPKKWCIHNPVLTGLDGEGKMS SSKGNFIAVDDSPEEIRAKIKKAYCPAGWEGNPIMEIAKYFL EYPLTIKRPEKFGGDLTVNSYEELESLFKNKELHPMDLKNA VAEELIKILEPIRKRL Be used to incorporate into aminoacyl tRNA synthetase p-Az-PheRS (2) to azido--phenylalanine RS
?13 MDEFEMIKRNTSEIISEEELREVLKKDEKSALIGFEPSGKIHL GHYLQIKKMIDLQNAGFDIIILLADLHAYLNQKGELDEIRKIG DYNKKVFEAMGLKAKYVYGSEFQLDKDYTLNVYRLALKT TLKRARRSMELIAREDENPKVAEVIYPIMQVNGCHYRGVDV AVGGMEQRKIHMLARELLPKKWCIHNPVLTGLDGEGKMS SSKGNFIAVDDSPEEIRAKIKKAYCPAGVVEGNPIMEIAKYFL EYPLTIKRPEKFGGDLTVNSYEELESLFKNKELHPMDLKNA VAEELIKILEPIRKRL Be used to incorporate into aminoacyl tRNA synthetase (LW1) to azido--phenylalanine RS
?14 MDEFEMIKRNTSEIISEEELREVLKKDEKSALIGFEPSGKIHL GHYLQIKKMIDLQNAGFDIIILLADLHAYLNQKGELDEIRKIG DYNKKVFEAMGLKAKYVYGSEFQLDKDYTLNVYRLALKT TLKRARRSMELIAREDENPKVAEVIYPIMQVNGTFIYRGVDV AVGGMEQRKIHMLARELLPKKWCIHNPVLTGLDGEGKMS SSKGNFIAVDDSPEEIRAKIKKAYCPAGWEGNPIMEIAKYFL EYPLTIKRPEKFGGDLTVNSYEELESLFKNKELHPMDLKNA VAEELIKILEPIRKRL Be used to incorporate into aminoacyl tRNA synthetase (LW5) to azido--phenylalanine RS
?15 MDEFEMIKRNTSEIISEEELREVLKKDEKSAAIGFEPSGKIHL GHYLQIKKMIDLQNAGFDIIILLADLHAYLNQKGELDEIRKIG DYNKKVFEAMGLKAKYVYGSEFQLDKDYTLNVYRLALKT TLKRARRSMELLAREDENPKVAEVIYPIMQVNGGHYLGVDV IVGGMEQRKIHMLARELLPKKWCIHNPVLTGLDGEGKMSS SKGNFIAVDDSPEEIRAKIKKAYCPAGVVEGNPIMEIAKYFL EYPLTIKRPEKFGGDLTVNSYEELESLFKNKELHPMDLKNA VAEELIKILEPIRKRL Be used to incorporate into aminoacyl tRNA synthetase (LW6) to azido--phenylalanine RS
?SEQ ?ID# Sequence Remarks Protein, nucleic acid, tRNA or RS
?16 MDEFEMIKRNTSEIISEEELREVLKKDEKSAAIGFEPSGKIHL GHYLQIKKMIDLQNAGFDIIILLADLHAYLNQKGELDEIRKIG DYNKKVFEAMGLKAKYVYGSRFQLDKDYTLNVYRLALKT TLKRARRSMELIAREDENPKVAEVIYPIMQVNVIHYDGVDV AVGGMEQRKIHMLARELLPKKWCIHNPVLTGLDGEGKMS SSKGNFIAVDDSPEEIRAKIKKAYCPAGWEGNPIMEIAKYFL EYPLTIKRPEKFGGDLTVNSYEELESLFKNKELHPMDLKNA VAEELIKILEPIRKRL Be used to incorporate into aminoacyl tRNA synthetase (AzPheRS-5) to azido--phenylalanine RS
?17 MDEFEMIKRNTSEIISEEELREVLKKDEKSAGIGFEPSGKIHL GHYLQIKKMIDLQNAGFDIIILLADLHAYLNQKGELDEIRKIG DYNKKVFEAMGLKAKYVYGSTFQLDKDYTLNVYRLALKT TLKRARRSMELIAREDENPKVAEVIYPIMQVNTYYYLGVDV AVGGMEQRKIHMLARELLPKKVVCIHNPVLTGLDGEGKMS SSKGNFIAVDDSPEEIRAKIKKAYCPAGVVEGNPIMEIAKYFL EYPLTIKRPEKFGGDLTVNSYEELESLFKNKELHPMDLKNA VAEELIKILEPIRKRL Be used to incorporate into aminoacyl tRNA synthetase (AzPheRS-6) to azido--phenylalanine RS

Claims (91)

1. compound, or its salt, it comprises formula (I):
Figure A2005800443280002C1
Wherein:
A is optionally, and when exist, be low-grade alkylidene, be substituted low-grade alkylidene, rudimentary cycloalkylidene, be substituted rudimentary cycloalkylidene, lower alkenylene, be substituted lower alkenylene, alkynylene, the assorted alkyl in rudimentary Asia, be substituted inferior mix alkyl, rudimentary inferior Heterocyclylalkyl, be substituted rudimentary inferior Heterocyclylalkyl, arylidene, be substituted arylidene, inferior heteroaryl, be substituted inferior heteroaryl, alkarylene, be substituted alkarylene, inferior aralkyl or be substituted inferior aralkyl;
B is optionally, and is the connexon that is selected from the group that is made up of following each group when exist: low-grade alkylidene, be substituted low-grade alkylidene, lower alkenylene, be substituted lower alkenylene, alkyl is mixed in rudimentary Asia, be substituted rudimentary Asia mix alkyl ,-O-,-O-(alkylidene group or be substituted alkylidene group)-,-S-,-S-(alkylidene group or be substituted alkylidene group)-, wherein k be 1,2 or 3-S (O) k-,-S (O) k(alkylidene group or be substituted alkylidene group)-,-C (O)-,-C (O)-(alkylidene group or be substituted alkylidene group)-,-C (S)-,-C (S)-(alkylidene group or be substituted alkylidene group)-,-N (R ')-,-NR '-(alkylidene group or be substituted alkylidene group)-,-C (O) N (R ')-,-CON (R ')-(alkylidene group or be substituted alkylidene group)-,-CSN (R ')-,-CSN (R ')-(alkylidene group or be substituted alkylidene group)-,-N (R ') CO-(alkylidene group or be substituted alkylidene group)-,-N (R ') C (O) O-,-S (O) kN (R ')-,-N (R ') C (O) N (R ')-,-N (R ') C (S) N (R ')-,-N (R ') S (O) kN (R ')-,-N (R ')-N=,-C (R ')=N-,-C (R ')=N-N (R ')-,-C (R ')=N-N=,-C (R ') 2-N=N-and-C (R ') 2-N (R ')-N (R ')-, wherein R ' is H, alkyl independently of one another or is substituted alkyl;
J is
Figure A2005800443280003C1
Figure A2005800443280003C2
R is H, alkyl, is substituted alkyl, cycloalkyl or is substituted cycloalkyl;
R " independently of one another for H, alkyl, " during group, two R " forms Heterocyclylalkyl according to circumstances to be substituted alkyl or protecting group, maybe when there being more than one R;
R 1Be H, amino protecting group, resin, amino acid, polypeptide or polynucleotide; And
R 2Be OH, ester protecting group, resin, amino acid, polypeptide or polynucleotide;
R 3With R 4Be H, halogen, low alkyl group or be substituted low alkyl group independently of one another, or R 3With R 4Or two R 3Group forms cycloalkyl or Heterocyclylalkyl according to circumstances;
Or-common dicyclo or three cyclic rings alkyl or the Heterocyclylalkyls that comprise at least one following group that form of A-B-J-R group: carbonyl, it comprises dicarbapentaborane; Through the protection carbonyl, it comprises through the protection dicarbapentaborane; Or through sheltering carbonyl, it comprises through sheltering dicarbapentaborane;
Or-common monocycle or double-ring cycloalkyl or the Heterocyclylalkyl that comprises at least one following group that form of J-R group: carbonyl, it comprises dicarbapentaborane; At least one is through the protection carbonyl, and it comprises through the protection dicarbapentaborane; At least one is through sheltering carbonyl, and it comprises through sheltering dicarbapentaborane; Or its combination;
Its restricted condition is phenylene and R for working as A 3When respectively doing for oneself H, B exists; And when A is-(CH 2) 4-and R 3When respectively doing for oneself H, B is not-NHC (O) (CH 2CH 2)-; And working as A and B does not exist and R 3When respectively doing for oneself H, R is not a methyl;
Or its active metabolite, or pharmaceutically acceptable prodrug or solvate.
2. compound according to claim 1, wherein A is the low-grade alkylidene that is substituted or is unsubstituted, or the arylidene that is selected from the group that is made up of phenylene, pyridylidene, inferior pyrimidyl or inferior thienyl that is unsubstituted or is substituted.
3. compound according to claim 1, it is corresponding to formula (XXXIII):
Figure A2005800443280004C1
X wherein 1Be C, S or S (O); And L is alkylidene group, be substituted alkylidene group, N (R ') (alkylidene group) or N (R ') (being substituted alkylidene group).
4. compound according to claim 3, it is corresponding to formula (XXXIII-A):
Figure A2005800443280004C2
5. compound according to claim 3, it is corresponding to formula (XXXIII-B):
6. compound according to claim 1, it is corresponding to formula (XXXIV):
Figure A2005800443280004C4
X wherein 1Be C, S or S (O); And n is 0,1,2,3,4 or 5; And each CR 8R 9R on the group 8With R 9Group is selected from the group that is made up of H, alkoxyl group, alkylamine, halogen, alkyl, aryl independently of one another, or any R 8And R 9Can form jointly=O or cycloalkyl, or any and adjacent R 8Group can form cycloalkyl jointly.
7. compound according to claim 6, it is corresponding to formula (XXXIV-A):
Figure A2005800443280005C1
7. compound according to claim 6, it is corresponding to formula (XXXIV-B):
Figure A2005800443280005C2
8. compound according to claim 1, it is corresponding to formula (XXXV):
Figure A2005800443280005C3
X wherein 1Be C, S or S (O); And L is alkylidene group, be substituted alkylidene group, N (R ') (alkylidene group) or N (R ') (being substituted alkylidene group).
9. compound according to claim 8, it is corresponding to formula (XXXV-A):
Figure A2005800443280005C4
10. compound according to claim 8, it is corresponding to formula (XXXV-B):
Figure A2005800443280006C1
11. compound according to claim 1, it is corresponding to formula (XXXX):
Figure A2005800443280006C2
Wherein:
M is-C (R 3)-,
Figure A2005800443280006C3
Figure A2005800443280006C4
Wherein (a) indication and described A group bond and (b) indication and carbonyl bond out of the ordinary; And
T 3Be a key, C (R) (R), O or S, and R is H, halogen, alkyl, is substituted alkyl, cycloalkyl or is substituted cycloalkyl.
12. compound according to claim 11, it is corresponding to formula (XXXXIII):
Figure A2005800443280007C1
13. compound according to claim 12, it is to be selected from:
Figure A2005800443280007C2
14. compound according to claim 1, it is corresponding to formula (III):
Figure A2005800443280007C3
R wherein aBe selected from the group that forms by following each group: H, halogen, alkyl independently of one another, be substituted alkyl ,-N (R ') 2, wherein k be 1,2 or 3-C (O) kR ' ,-C (O) N (R ') 2,-O R ' and-S (O) kR ', wherein R ' is H, alkyl independently of one another or is substituted alkyl.
15. compound according to claim 14, it is to be selected from the group that is made up of following each thing:
Figure A2005800443280008C1
Figure A2005800443280008C2
And
Figure A2005800443280008C3
16. compound according to claim 1, it is corresponding to formula (VI):
R wherein aBe selected from the group that forms by following each group: H, halogen, alkyl independently of one another, be substituted alkyl ,-N (R ') 2, wherein k be 1,2 or 3-C (O) kR ' ,-C (O) N (R ') 2,-OR ' and-S (O) kR ', wherein R ' is H, alkyl independently of one another or is substituted alkyl.
17. compound according to claim 16, it is to be selected from the group that is made up of following each thing:
Figure A2005800443280009C1
And
Figure A2005800443280009C2
18. compound according to claim 1, it is corresponding to formula (IX):
Figure A2005800443280009C3
R wherein aBe selected from the group that forms by following each group: H, halogen, alkyl independently of one another, be substituted alkyl ,-N (R ') 2, wherein k be 1,2 or 3-C (O) kR ' ,-C (O) N (R ') 2,-OR ' and-S (O) kR ', wherein R ' is H, alkyl independently of one another or is substituted alkyl.
19. compound according to claim 18, it is to be selected from the group that is made up of following each thing:
Figure A2005800443280009C4
And
Figure A2005800443280009C5
20. compound according to claim 1, wherein said-A-B-J-R group is common form comprise at least one carbonyl, through protection carbonyl or dicyclo through sheltering carbonyl or three cyclic rings alkyl or Heterocyclylalkyls.
21. compound according to claim 20, it is to be selected from the group that is made up of following each thing:
And
22. compound according to claim 1, wherein said-J-R group is common form comprise at least one carbonyl, through protection carbonyl or the monocycle through sheltering carbonyl or double-ring cycloalkyl or Heterocyclylalkyl.
23. compound according to claim 22, it has following structure:
Figure A2005800443280010C3
24. a peptide species, it also has at least a compound according to claim 1.
25. a compound, or its salt, it comprises formula (XI):
Figure A2005800443280010C4
Wherein:
A is optionally, and when exist, be low-grade alkylidene, be substituted low-grade alkylidene, rudimentary cycloalkylidene, be substituted rudimentary cycloalkylidene, lower alkenylene, be substituted lower alkenylene, alkynylene, the assorted alkyl in rudimentary Asia, be substituted inferior mix alkyl, rudimentary inferior Heterocyclylalkyl, be substituted rudimentary inferior Heterocyclylalkyl, arylidene, be substituted arylidene, inferior heteroaryl, be substituted inferior heteroaryl, alkarylene, be substituted alkarylene, inferior aralkyl or be substituted inferior aralkyl;
B is optionally, and is the connexon that is selected from the group that is made up of following each group when exist: low-grade alkylidene, be substituted low-grade alkylidene, lower alkenylene, be substituted lower alkenylene, alkyl is mixed in rudimentary Asia, be substituted rudimentary Asia mix alkyl ,-O-,-O-(alkylidene group or be substituted alkylidene group)-,-S-,-S-(alkylidene group or be substituted alkylidene group)-, wherein k be 1,2 or 3-S (O) k-,-S (O) k(alkylidene group or be substituted alkylidene group)-,-C (O)-,-C (O)-(alkylidene group or be substituted alkylidene group)-,-C (S)-,-C (S)-(alkylidene group or be substituted alkylidene group)-,-N (R ')-,-NR '-(alkylidene group or be substituted alkylidene group)-,-C (O) N (R ')-,-CON (R ')-(alkylidene group or be substituted alkylidene group)-,-CSN (R ')-,-CSN (R ')-(alkylidene group or be substituted alkylidene group)-,-N (R ') CO-(alkylidene group or be substituted alkylidene group)-,-N (R ') C (O) O-,-S (O) kN (R ')-,-N (R ') C (O) N (R ')-,-N (R ') C (S) N (R ')-,-N (R ') S (O) kN (R ')-,-N (R ')-N=,-C (R ')=N-,-C (R ')=N-N (R ')-,-C (R ')=N-N=,-C (R ') 2-N=N-and-C (R ') 2-N (R ')-N (R ')-, wherein R ' is H, alkyl independently of one another or is substituted alkyl;
R is H, alkyl, is substituted alkyl, cycloalkyl or is substituted cycloalkyl;
R 1Be H, amino protecting group, resin, amino acid, polypeptide or polynucleotide; And
R 2Be OH, ester protecting group, resin, amino acid, polypeptide or polynucleotide;
R 3With R 4Be H, halogen, low alkyl group or be substituted low alkyl group independently of one another, or R 3With R 4Or two R 3Group forms cycloalkyl or Heterocyclylalkyl according to circumstances;
R 5For H, alkyl, be substituted alkyl, thiazolinyl, be substituted thiazolinyl, alkynyl, be substituted alkynyl, alkoxyl group, be substituted alkoxyl group, alkyl alkoxy, be substituted alkyl alkoxy, polyoxyalkylene, be substituted polyoxyalkylene, aryl, be substituted aryl, heteroaryl, be substituted heteroaryl, alkaryl, be substituted alkaryl, aralkyl, be substituted aralkyl ,-(alkylidene group or be substituted alkylidene group)-ON (R ") 2,-(alkylidene group or be substituted alkylidene group)-C (O) SR " ,-(alkylidene group or be substituted alkylidene group)-S-S-(aryl or be substituted aryl) ,-C (O) R " ,-C (O) 2R " or-C (O) N (R ") 2, R wherein " independently of one another for hydrogen, alkyl, be substituted alkyl, thiazolinyl, be substituted thiazolinyl, alkoxyl group, be substituted alkoxyl group, aryl, be substituted aryl, heteroaryl, alkaryl, be substituted alkaryl, aralkyl or be substituted aralkyl;
Or R 5Be L-X, wherein
X is selected from the group that is made up of following each thing: mark; Dyestuff; Polymkeric substance; Water-soluble polymers; The derivative of polyoxyethylene glycol; Photocrosslinking agent; Cytotoxic compound; Medicine; Affinity marker; The photoaffinity mark; Reactive compounds; Resin; Second protein or polypeptide or polypeptide analog; Antibody or antibody fragment; Metal chelator; Cofactor; Lipid acid; Carbohydrate; Polynucleotide; DNA; RNA; Antisense polynucleotide; Carbohydrate, water-soluble branch-shape polymer, cyclodextrin, biomaterial; Nanoparticle; Spin labeling; Fluorophore, metallic part; Radioactive segment; Novel functional group; With the covalently or non-covalently interactional group of other molecules; The light cage covers part; But photoisomerization part; Vitamin H; The vitamin H analogue; Be combined with the part of heavy atom; But the group of chemical cracking; But the group of photodestruciton; The side chain that prolongs; The sugar of carbon bond connection; Redox active agent; Amino thioic acid sulfoacid; The toxicity part; Through isotope-labeled part; The biophysics probe; The phosphorescence group; Chemiluminescent groups; The electron dense group; The magnetic group; Insert group; Chromophoric group; Energy transfer agent; Biologically active agent; Detectable label; And any combination of above-mentioned substance; And
L is optionally, and is the connexon that is selected from the group that is made up of following each group when existing: alkylidene group, be substituted alkylidene group, alkenylene, be substituted alkenylene ,-O-,-O-(alkylidene group or be substituted alkylidene group)-,-S-,-S-(alkylidene group or be substituted alkylidene group)-, wherein k be 1,2 or 3-S (O) k-,-S (O) k(alkylidene group or be substituted alkylidene group)-,-C (O)-,-C (O)-(alkylidene group or be substituted alkylidene group)-,-C (S)-,-C (S)-(alkylidene group or be substituted alkylidene group)-,-N (R ')-,-NR '-(alkylidene group or be substituted alkylidene group)-,-C (O) N (R ')-,-CON (R ')-(alkylidene group or be substituted alkylidene group)-,-CSN (R ')-,-CSN (R ')-(alkylidene group or be substituted alkylidene group)-,-N (R ') CO-(alkylidene group or be substituted alkylidene group)-,-N (R ') C (O) O-,-(alkylidene group or be substituted alkylidene group)-O-N=CR '-,-(alkylidene group or be substituted alkylidene group)-C (O) NR '-(alkylidene group or be substituted alkylidene group)-,-(alkylidene group or be substituted alkylidene group)-S (O) k-(alkylidene group or be substituted alkylidene group)-S-,-(alkylidene group or be substituted alkylidene group)-S-S-,-S (O) kN (R ')-,-N (R ') C (O) N (R ')-,-N (R ') C (S) N (R ')-,-N (R ') S (O) kN (R ')-,-N (R ')-N=,-C (R ')=N-,-C (R ')=N-N (R ')-,-C (R ')=N-N=,-C (R ') 2-N=N-and-C (R ') 2-N (R ')-N (R ')-, wherein R ' is H, alkyl independently of one another or is substituted alkyl;
Its restricted condition is not for when A and B exist, and R is not a methyl;
Or its active metabolite, or pharmaceutically acceptable prodrug or solvate.
26. compound according to claim 25, wherein A is phenylene or is substituted phenylene.
27. compound according to claim 25, it is corresponding to formula (XII):
Figure A2005800443280012C1
28. a peptide species, it also has at least a compound according to claim 25.
29. compound according to claim 25, wherein X is the biologically active agent that is selected from the group that is made up of peptide, protein, enzyme, antibody, medicine, dyestuff, lipid, nucleosides, oligonucleotide, cell, virus, liposome, particulate and micella.
30. compound according to claim 29, wherein X is the medicine that is selected from the group that is made up of microbiotic, mycocide, antiviral agent, antiphlogistic, antineoplastic agent, cardiovascular agents, antianxiety agent, hormone, somatomedin and steroid agent.
31. compound according to claim 29, wherein X is the enzyme that is selected from the group that is made up of horseradish peroxidase, alkaline phosphatase, beta-galactosidase enzymes and glucose oxidase.
32. compound according to claim 25, wherein X is the detectable label that is selected from the group that is grouped into by fluorescence part, phosphorescence part, chemiluminescent moiety, chelating moiety, electron dense part, magnetic part, insertion portion, radioactive segment, chromophoric group part and energy transfer portion.
33. method for preparing the amino acid whose polypeptide of the structure that comprises that at least one has formula (I):
Figure A2005800443280013C1
Described method comprises to be incorporated the amino acid of formula (I) in the polypeptide interior terminal position or interior location, wherein into:
A is optionally, and when exist, be low-grade alkylidene, be substituted low-grade alkylidene, rudimentary cycloalkylidene, be substituted rudimentary cycloalkylidene, lower alkenylene, be substituted lower alkenylene, alkynylene, the assorted alkyl in rudimentary Asia, be substituted inferior mix alkyl, rudimentary inferior Heterocyclylalkyl, be substituted rudimentary inferior Heterocyclylalkyl, arylidene, be substituted arylidene, inferior heteroaryl, be substituted inferior heteroaryl, alkarylene, be substituted alkarylene, inferior aralkyl or be substituted inferior aralkyl;
B is optionally, and is the connexon that is selected from the group that is made up of following each group when exist: low-grade alkylidene, be substituted low-grade alkylidene, lower alkenylene, be substituted lower alkenylene, alkyl is mixed in rudimentary Asia, be substituted rudimentary Asia mix alkyl ,-O-,-O-(alkylidene group or be substituted alkylidene group)-,-S-,-S-(alkylidene group or be substituted alkylidene group)-, wherein k be 1,2 or 3-S (O) k-,-S (O) k(alkylidene group or be substituted alkylidene group)-,-C (O)-,-C (O)-(alkylidene group or be substituted alkylidene group)-,-C (S)-,-C (S)-(alkylidene group or be substituted alkylidene group)-,-N (R ')-,-NR '-(alkylidene group or be substituted alkylidene group)-,-C (O) N (R ')-,-CON (R ')-(alkylidene group or be substituted alkylidene group)-,-CSN (R ')-,-CSN (R ')-(alkylidene group or be substituted alkylidene group)-,-N (R ') CO-(alkylidene group or be substituted alkylidene group)-,-N (R ') C (O) O-,-S (O) kN (R ')-,-N (R ') C (O) N (R ')-,-N (R ') C (S) N (R ')-,-N (R ') S (O) kN (R ')-,-N (R ')-N=,-C (R ')=N-,-C (R ')=N-N (R ')-,-C (R ')=N-N=,-C (R ') 2-N=N-and-C (R ') 2-N (R ')-N (R ')-, wherein R ' is H, alkyl independently of one another or is substituted alkyl;
J is
Figure A2005800443280014C1
Figure A2005800443280014C2
R is H, alkyl, is substituted alkyl, cycloalkyl or is substituted cycloalkyl;
R " independently of one another for H, alkyl, " during group, two R " forms Heterocyclylalkyl according to circumstances to be substituted alkyl or protecting group, maybe when there being more than one R;
R 1Be H, amino protecting group, resin, amino acid, polypeptide or polynucleotide; And
R 2Be OH, ester protecting group, resin, amino acid, polypeptide or polynucleotide;
R 3With R 4Be H, halogen, low alkyl group or be substituted low alkyl group independently of one another, or R 3With R 4Or two R 3Group forms cycloalkyl or Heterocyclylalkyl according to circumstances;
Or-common dicyclo or three cyclic rings alkyl or the Heterocyclylalkyls that comprise at least one following group that form of A-B-J-R group: carbonyl, it comprises dicarbapentaborane; Through the protection carbonyl, it comprises through the protection dicarbapentaborane; Or through sheltering carbonyl, it comprises through sheltering dicarbapentaborane;
Or-common monocycle or double-ring cycloalkyl or the Heterocyclylalkyl that comprises at least one following group that form of J-R group: carbonyl, it comprises dicarbapentaborane; Through the protection carbonyl, it comprises through the protection dicarbapentaborane; Or through sheltering carbonyl, it comprises through sheltering dicarbapentaborane;
Its restricted condition is phenylene and R for working as A 3When respectively doing for oneself H, B exists; And when A is-(CH 2) 4-and R 3When respectively doing for oneself H, B is not-NHC (O) (CH 2CH 2)-; And working as A and B does not exist and R 3When respectively doing for oneself H, R is not a methyl.
34. method according to claim 33, wherein said amino acid are to use translation system to incorporate in the interior specific site of described polypeptide, described translation system comprises:
(i) polynucleotide of coding said polypeptide, wherein said polynucleotide comprise corresponding to incorporating the amino acid whose selection codon of specifying the site in advance of described formula (I) into, and
(ii) comprise described amino acid whose tRNA, wherein said tRNA has specificity to described selection codon.
35. method according to claim 34, wherein said translation system comprise that aminoacylization arrives the tRNA on formula (I) amino acid.
36. method according to claim 35, wherein said translation system are the in vivo translation system that comprises the cell that is selected from the group that is made up of bacterial cell, archeobacteria cell and eukaryotic cell.
37. method according to claim 36, wherein said amino acid has the structure corresponding to formula (III):
Figure A2005800443280015C1
R wherein aBe selected from the group that forms by following each group: H, halogen, alkyl independently of one another, be substituted alkyl ,-N (R ') 2, wherein k be 1,2 or 3-C (O) kR ' ,-C (O) N (R ') 2,-OR ' and-S (O) kR ', wherein R ' is H, alkyl independently of one another or is substituted alkyl.
38. according to the described method of claim 37, wherein said amino acid is to be selected from the group that is made up of following each thing:
Figure A2005800443280016C1
Figure A2005800443280016C2
And
Figure A2005800443280016C3
39. method according to claim 36, wherein said amino acid has the structure corresponding to formula (VI):
Figure A2005800443280016C4
R wherein aBe selected from the group that forms by following each group: H, halogen, alkyl independently of one another, be substituted alkyl ,-N (R ') 2, wherein k be 1,2 or 3-C (O) kR ' ,-C (O) N (R ') 2,-OR ' and-S (O) kR ', wherein R ' is H, alkyl independently of one another or is substituted alkyl.
40. according to the described method of claim 39, wherein said amino acid is to be selected from the group that is made up of following each thing:
Figure A2005800443280017C1
And
Figure A2005800443280017C2
41. method according to claim 36, wherein said amino acid has the structure corresponding to formula (IX):
Figure A2005800443280017C3
R wherein aBe selected from the group that forms by following each group: H, halogen, alkyl independently of one another, be substituted alkyl ,-N (R ') 2, wherein k be 1,2 or 3-C (O) kR ' ,-C (O) N (R ') 2,-OR ' and-S (O) kR ', wherein R ' is H, alkyl independently of one another or is substituted alkyl.
42. according to the described method of claim 41, wherein said amino acid is to be selected from the group that is made up of following each thing:
Figure A2005800443280017C4
And
Figure A2005800443280017C5
43. method according to claim 36, common dicyclo or three cyclic rings alkyl or the Heterocyclylalkyls that comprise at least one following group that form of wherein said-A-B-J-R group: carbonyl, it comprises dicarbapentaborane; Through the protection carbonyl, it comprises through the protection dicarbapentaborane; Or through sheltering carbonyl, it comprises through sheltering dicarbapentaborane.
44. according to the described method of claim 43, wherein said amino acid is to be selected from the group that is made up of following each thing:
Figure A2005800443280018C1
And
Figure A2005800443280018C2
45. method according to claim 36, common monocycle or double-ring cycloalkyl or the Heterocyclylalkyl that comprises at least one following group that form of wherein said-J-R group: carbonyl, it comprises dicarbapentaborane; Through the protection carbonyl, it comprises through the protection dicarbapentaborane; Or through sheltering carbonyl, it comprises through sheltering dicarbapentaborane.
46. according to the described method of claim 45, wherein said amino acid is:
Figure A2005800443280018C3
47. method according to claim 36, wherein said formula (I) amino acid has the structure of formula (XXX):
Figure A2005800443280018C4
X wherein 1Be C, S or S (O); And L is alkylidene group, be substituted alkylidene group, N (R ') (alkylidene group) or N (R ') (being substituted alkylidene group).
48. method according to claim 36, wherein said formula (I) amino acid has the structure of formula (XXXIII):
Figure A2005800443280019C1
X wherein 1Be C, S or S (O); And L is alkylidene group, be substituted alkylidene group, N (R ') (alkylidene group) or N (R ') (being substituted alkylidene group).
49. method according to claim 36, it is corresponding to formula (XXXX):
Wherein:
M is-C (R 3)-,
Figure A2005800443280019C4
Wherein (a) indication and described A group bond and (b) indication and carbonyl bond out of the ordinary; And
T 3Be a key, C (R) (R), O or S, and R is H, halogen, alkyl, is substituted alkyl, cycloalkyl or is substituted cycloalkyl.
50. according to the described method of claim 49, it is corresponding to formula (XXXXIII):
51. one kind is used for the method that derivatize comprises the amino acid whose polypeptide of formula (I), described method comprises makes described polypeptide contact with the reagent of formula (XIX), its Chinese style (I) corresponding to:
Figure A2005800443280020C2
Wherein:
A is optionally, and when existing, be low-grade alkylidene, be substituted low-grade alkylidene, lower alkenylene, be substituted lower alkenylene, arylidene, be substituted arylidene, inferior heteroaryl, be substituted inferior heteroaryl, alkarylene, be substituted alkarylene, inferior aralkyl or be substituted inferior aralkyl;
B is optionally, and is the connexon that is selected from the group that is made up of following each group when existing: low-grade alkylidene, be substituted low-grade alkylidene, lower alkenylene, be substituted lower alkenylene ,-O-,-O-(alkylidene group or be substituted alkylidene group)-,-S-,-S-(alkylidene group or be substituted alkylidene group)-, wherein k be 1,2 or 3-S (O) k-,-S (O) k(alkylidene group or be substituted alkylidene group)-,-C (O)-,-C (O)-(alkylidene group or be substituted alkylidene group)-,-C (S)-,-C (S)-(alkylidene group or be substituted alkylidene group)-,-N (R ')-,-NR '-(alkylidene group or be substituted alkylidene group)-,-C (O) N (R ')-,-CON (R ')-(alkylidene group or be substituted alkylidene group)-,-CSN (R ')-,-CSN (R ')-(alkylidene group or be substituted alkylidene group)-,-N (R ') CO-(alkylidene group or be substituted alkylidene group)-,-N (R ') C (O) O-,-S (O) kN (R ')-,-N (R ') C (O) N (R ')-,-N (R ') C (S) N (R ')-,-N (R ') S (O) kN (R ')-,-N (R ')-N=,-C (R ')=N-,-C (R ')=N-N (R ')-,-C (R ')=N-N=,-C (R ') 2-N=N-and-C (R ') 2-N (R ')-N (R ')-, wherein R ' is H, alkyl independently of one another or is substituted alkyl;
J is
Figure A2005800443280021C1
Figure A2005800443280021C2
R is H, alkyl, is substituted alkyl, cycloalkyl or is substituted cycloalkyl;
R ' is H, alkyl independently of one another or is substituted alkyl;
R 1Be H, amino protecting group, resin, amino acid, polypeptide or polynucleotide; And
R 2Be OH, ester protecting group, resin, amino acid, polypeptide or polynucleotide;
R 3With R 4Be H, halogen, low alkyl group or be substituted low alkyl group independently of one another;
Its Chinese style (XIX) corresponding to:
Figure A2005800443280021C3
Wherein:
X is detectable label, biologically active agent or polymkeric substance independently of one another;
L respectively does for oneself and is independently selected from the connexon of the group that is made up of following each group: alkylidene group, be substituted alkylidene group, alkenylene, be substituted alkenylene ,-O-,-O-(alkylidene group or be substituted alkylidene group)-,-S-,-S-(alkylidene group or be substituted alkylidene group)-, wherein k be 1,2 or 3-S (O) k-,-S (O) k(alkylidene group or be substituted alkylidene group)-,-C (O)-,-C (O)-(alkylidene group or be substituted alkylidene group)-,-C (S)-,-C (S)-(alkylidene group or be substituted alkylidene group)-,-N (R ')-,-NR '-(alkylidene group or be substituted alkylidene group)-,-C (O) N (R ')-,-CON (R ')-(alkylidene group or be substituted alkylidene group)-,-(alkylidene group or be substituted alkylidene group) NR ' C (O) O-(alkylidene group or be substituted alkylidene group)-,-O-CON (R ')-(alkylidene group or be substituted alkylidene group)-,-CSN (R ')-,-CSN (R ')-(alkylidene group or be substituted alkylidene group)-,-N (R ') CO-(alkylidene group or be substituted alkylidene group)-,-N (R ') C (O) O-,-N (R ') C (O) O-(alkylidene group or be substituted alkylidene group)-,-S (O) kN (R ')-,-N (R ') C (O) N (R ')-,-N (R ') C (O) N (R ')-(alkylidene group or be substituted alkylidene group)-,-N (R ') C (S) N (R ')-,-N (R ') S (O) kN (R ')-,-N (R ')-N=,-C (R ')=N-,-C (R ')=N-N (R ')-,-C (R ')=N-N=,-C (R ') 2-N=N-and-C (R ') 2-N (R ')-N (R ')-, wherein R ' is H, alkyl independently of one another or is substituted alkyl;
L 1For optionally, and when existing be-C (R ') p-NR '-C (O) O-(alkylidene group or be substituted alkylidene group)-, wherein p is 0,1 or 2;
W is-ON (R 1) 2Or-C (=O) R 2, R wherein 1Be H or amino protecting group independently of one another, and R 2Be H or OR '; And
N is 1 to 3.
52. according to the described method of claim 51, wherein said amino acid is corresponding to formula (II):
Figure A2005800443280022C1
53. according to the described method of claim 51, wherein said reagent is corresponding to formula (XXVII):
Figure A2005800443280022C2
54. according to the described method of claim 51, what the wherein said polypeptide of deriving comprised structure that at least one has formula (XI) contains oxime amino acid:
Figure A2005800443280022C3
Wherein:
A is optionally, and when existing, be low-grade alkylidene, be substituted low-grade alkylidene, lower alkenylene, be substituted lower alkenylene, arylidene, be substituted arylidene, inferior heteroaryl, be substituted inferior heteroaryl, alkarylene, be substituted alkarylene, inferior aralkyl or be substituted inferior aralkyl;
B is optionally, and is the connexon that is selected from the group that is made up of following each group when existing: low-grade alkylidene, be substituted low-grade alkylidene, lower alkenylene, be substituted lower alkenylene ,-O-,-O-(alkylidene group or be substituted alkylidene group)-,-S-,-S-(alkylidene group or be substituted alkylidene group)-, wherein k be 1,2 or 3-S (O) k-,-S (O) k(alkylidene group or be substituted alkylidene group)-,-C (O)-,-C (O)-(alkylidene group or be substituted alkylidene group)-,-C (S)-,-C (S)-(alkylidene group or be substituted alkylidene group)-,-N (R ')-,-NR '-(alkylidene group or be substituted alkylidene group)-,-C (O) N (R ')-,-CON (R ')-(alkylidene group or be substituted alkylidene group)-,-CSN (R ')-,-CSN (R ')-(alkylidene group or be substituted alkylidene group)-,-N (R ') CO-(alkylidene group or be substituted alkylidene group)-,-N (R ') C (O) O-,-S (O) kN (R ')-,-N (R ') C (O) N (R ')-,-N (R ') C (S) N (R ')-,-N (R ') S (O) kN (R ')-,-N (R ')-N=,-C (R ')=N-,-C (R ')=N-N (R ')-,-C (R ')=N-N=,-C (R ') 2-N=N-and-C (R ') 2-N (R ')-N (R ')-, wherein R ' is H, alkyl independently of one another or is substituted alkyl;
R is H, alkyl, is substituted alkyl, cycloalkyl or is substituted cycloalkyl;
R 1Be H, amino protecting group, resin, amino acid, polypeptide or polynucleotide; And
R 2Be OH, ester protecting group, resin, amino acid, polypeptide or polynucleotide;
R 3With R 4Be H, halogen, low alkyl group or be substituted low alkyl group independently of one another;
R 5For H, alkyl, be substituted alkyl, thiazolinyl, be substituted thiazolinyl, alkynyl, be substituted alkynyl, alkoxyl group, be substituted alkoxyl group, alkyl alkoxy, be substituted alkyl alkoxy, polyoxyalkylene, be substituted polyoxyalkylene, aryl, be substituted aryl, heteroaryl, be substituted heteroaryl, alkaryl, be substituted alkaryl, aralkyl, be substituted aralkyl ,-C (O) R " ,-C (O) 2R " or-C (O) N (R ") 2, R wherein " independently of one another for hydrogen, alkyl, be substituted alkyl, thiazolinyl, be substituted thiazolinyl, alkoxyl group, be substituted alkoxyl group, aryl, be substituted aryl, heteroaryl, alkaryl, be substituted alkaryl, aralkyl or be substituted aralkyl; Or R 5Be L-X, wherein
X is detectable label, biologically active agent or polymkeric substance; And
L is optionally, and is the connexon that is selected from the group that is made up of following each group when existing: alkylidene group, be substituted alkylidene group, alkenylene, be substituted alkenylene ,-O-,-O-(alkylidene group or be substituted alkylidene group)-,-S-,-S-(alkylidene group or be substituted alkylidene group)-, wherein k be 1,2 or 3-S (O) k-,-S (O) k(alkylidene group or be substituted alkylidene group)-,-C (O)-,-C (O)-(alkylidene group or be substituted alkylidene group)-,-C (S)-,-C (S)-(alkylidene group or be substituted alkylidene group)-,-N (R ')-,-NR '-(alkylidene group or be substituted alkylidene group)-,-C (O) N (R ')-,-CON (R ')-(alkylidene group or be substituted alkylidene group)-,-CSN (R ')-,-CSN (R ')-(alkylidene group or be substituted alkylidene group)-,-N (R ') CO-(alkylidene group or be substituted alkylidene group)-,-N (R ') C (O) O-,-S (O) kN (R ')-,-N (R ') C (O) N (R ')-,-N (R ') C (S) N (R ')-,-N (R ') S (O) kN (R ')-,-N (R ')-N=,-C (R ')=N-,-C (R ')=N-N (R ')-,-C (R ')=N-N=,-C (R ') 2-N=N-and-C (R ') 2-N (R ')-N (R ')-, wherein R ' is H, alkyl independently of one another or is substituted alkyl;
Its restricted condition is not for when A and B exist, and R is not a methyl.
55., described polypeptide is contacted in the aqueous solution under appropriate acidic conditions with the reagent of described formula (XIX) according to the described method of claim 51.
56. according to the described method of claim 55, wherein said condition is pH2 to 8.
57. according to the described method of claim 51, described polypeptide is contacted in the presence of promotor with the reagent of described formula (XIX), described promotor being is selected from the group that is made up of following each thing:
Figure A2005800443280024C1
And
Figure A2005800443280024C2
58. according to the described method of claim 51, wherein said formula (I) amino acid has the structure of formula (XXX):
Figure A2005800443280024C3
X wherein 1Be C, S or S (O); And L is alkylidene group, be substituted alkylidene group, N (R ') (alkylidene group) or N (R ') (being substituted alkylidene group).
59. according to the described method of claim 51, wherein said formula (I) amino acid has the structure of formula (XXXIII):
Figure A2005800443280024C4
X wherein 1Be C, S or S (O); And L is alkylidene group, be substituted alkylidene group, N (R ') (alkylidene group) or N (R ') (being substituted alkylidene group).
60. according to the described method of claim 51, it is corresponding to formula (XXXX):
Figure A2005800443280025C1
Wherein:
M is-C (R 3)-,
Figure A2005800443280025C2
Wherein (a) indication and described A group bond and (b) indication and carbonyl bond out of the ordinary; And
T 3Be a key, C (R) (R), O or S, and R is H, halogen, alkyl, is substituted alkyl, cycloalkyl or is substituted cycloalkyl.
61. according to the described method of claim 60, it is corresponding to formula (XXXXIII):
62. method for the treatment of illness, symptom or disease, described method comprise throw with the treatment significant quantity comprise that at least one contains the non-natural amino acid polypeptides of the alpha-non-natural amino acid of oxime, the wherein said alpha-non-natural amino acid that contains oxime is to use translation system to incorporate in the interior specific site of described polypeptide, and described translation system comprises:
(i) polynucleotide of coding said polypeptide, wherein said polynucleotide comprise corresponding to incorporating the described amino acid whose selection codon of specifying the site in advance of oxime that contains into, and
(ii) comprise the described amino acid whose tRNA of oxime that contains, wherein said tRNA has specificity to described selection codon.
63. according to the described method of claim 62, wherein said translation system is the in vivo translation system that comprises the cell of the organism that is selected from following group: prokaryotic organism, eukaryote, Mammals, intestinal bacteria (Escherichia coli), fungi, pseudomonas kind (species of Pseudomonas), yeast, archeobacteria, eubacterium, plant, insect and protobiont.
64. according to the described method of claim 62, wherein said at least one alpha-non-natural amino acid is to contain oxime amino acid and the described alpha-non-natural amino acid that contains oxime to have structure corresponding to formula (XI):
Figure A2005800443280026C1
Wherein:
A is optionally, and when exist, be low-grade alkylidene, be substituted low-grade alkylidene, rudimentary cycloalkylidene, be substituted rudimentary cycloalkylidene, lower alkenylene, be substituted lower alkenylene, alkynylene, the assorted alkyl in rudimentary Asia, be substituted inferior mix alkyl, rudimentary inferior Heterocyclylalkyl, be substituted rudimentary inferior Heterocyclylalkyl, arylidene, be substituted arylidene, inferior heteroaryl, be substituted inferior heteroaryl, alkarylene, be substituted alkarylene, inferior aralkyl or be substituted inferior aralkyl;
B is optionally, and is the connexon that is selected from the group that is made up of following each group when exist: low-grade alkylidene, be substituted low-grade alkylidene, lower alkenylene, be substituted lower alkenylene, alkyl is mixed in rudimentary Asia, be substituted rudimentary Asia mix alkyl ,-O-,-O-(alkylidene group or be substituted alkylidene group)-,-S-,-S-(alkylidene group or be substituted alkylidene group)-, wherein k be 1,2 or 3-S (O) k-,-S (O) k(alkylidene group or be substituted alkylidene group)-,-C (O)-,-C (O)-(alkylidene group or be substituted alkylidene group)-,-C (S)-,-C (S)-(alkylidene group or be substituted alkylidene group)-,-N (R ')-,-NR '-(alkylidene group or be substituted alkylidene group)-,-C (O) N (R ')-,-CON (R ')-(alkylidene group or be substituted alkylidene group)-,-CSN (R ')-,-CSN (R ')-(alkylidene group or be substituted alkylidene group)-,-N (R ') CO-(alkylidene group or be substituted alkylidene group)-,-N (R ') C (O) O-,-S (O) kN (R ')-,-N (R ') C (O) N (R ')-,-N (R ') C (S) N (R ')-,-N (R ') S (O) kN (R ')-,-N (R ')-N=,-C (R ')=N-,-C (R ')=N-N (R ')-,-C (R ')=N-N=,-C (R ') 2-N=N-and-C (R ') 2-N (R ')-N (R ')-, wherein R ' is H, alkyl independently of one another or is substituted alkyl;
R is H, alkyl, is substituted alkyl, cycloalkyl or is substituted cycloalkyl;
R 1Be H, amino protecting group, resin, amino acid, polypeptide or polynucleotide; And
R 2Be OH, ester protecting group, resin, amino acid, polypeptide or polynucleotide;
R 3With R 4Be H, halogen, low alkyl group or be substituted low alkyl group independently of one another, or R 3With R 4Or two R 3Group forms cycloalkyl or Heterocyclylalkyl according to circumstances;
R 5For H, alkyl, be substituted alkyl, thiazolinyl, be substituted thiazolinyl, alkynyl, be substituted alkynyl, alkoxyl group, be substituted alkoxyl group, alkyl alkoxy, be substituted alkyl alkoxy, polyoxyalkylene, be substituted polyoxyalkylene, aryl, be substituted aryl, heteroaryl, be substituted heteroaryl, alkaryl, be substituted alkaryl, aralkyl, be substituted aralkyl ,-(alkylidene group or be substituted alkylidene group)-ON (R ") 2,-(alkylidene group or be substituted alkylidene group)-C (O) SR " ,-(alkylidene group or be substituted alkylidene group)-S-S-(aryl or be substituted aryl) ,-C (O) R " ,-C (O) 2R " or-C (O) N (R ") 2, R wherein " independently of one another for hydrogen, alkyl, be substituted alkyl, thiazolinyl, be substituted thiazolinyl, alkoxyl group, be substituted alkoxyl group, aryl, be substituted aryl, heteroaryl, alkaryl, be substituted alkaryl, aralkyl or be substituted aralkyl;
Or R 5Be L-X, wherein
X is selected from the group that is made up of following each thing: mark; Dyestuff; Polymkeric substance; Water-soluble polymers; The derivative of polyoxyethylene glycol; Photocrosslinking agent; Cytotoxic compound; Medicine; Affinity marker; The photoaffinity mark; Reactive compounds; Resin; Second protein or polypeptide or polypeptide analog; Antibody or antibody fragment; Metal chelator; Cofactor; Lipid acid; Carbohydrate; Polynucleotide; DNA; RNA; Antisense polynucleotide; Carbohydrate, water-soluble branch-shape polymer, cyclodextrin, biomaterial; Nanoparticle; Spin labeling; Fluorophore, metallic part; Radioactive segment; Novel functional group; With the covalently or non-covalently interactional group of other molecules; The light cage covers part; But photoisomerization part; Vitamin H; The vitamin H analogue; Be combined with the part of heavy atom; But the group of chemical cracking; But the group of photodestruciton; The side chain that prolongs; The sugar of carbon bond connection; Redox active agent; Amino thioic acid sulfoacid; The toxicity part; Through isotope-labeled part; The biophysics probe; The phosphorescence group; Chemiluminescent groups; The electron dense group; The magnetic group; Insert group; Chromophoric group; Energy transfer agent; Biologically active agent; Detectable label; And any combination of above-mentioned substance; And
L is optionally, and is the connexon that is selected from the group that is made up of following each group when existing: alkylidene group, be substituted alkylidene group, alkenylene, be substituted alkenylene ,-O-,-O-(alkylidene group or be substituted alkylidene group)-,-S-,-S-(alkylidene group or be substituted alkylidene group)-, wherein k be 1,2 or 3-S (O) k-,-S (O) k(alkylidene group or be substituted alkylidene group)-,-C (O)-,-C (O)-(alkylidene group or be substituted alkylidene group)-,-C (S)-,-C (S)-(alkylidene group or be substituted alkylidene group)-,-N (R ')-,-NR '-(alkylidene group or be substituted alkylidene group)-,-C (O) N (R ')-,-CON (R ')-(alkylidene group or be substituted alkylidene group)-,-CSN (R ')-,-CSN (R ')-(alkylidene group or be substituted alkylidene group)-,-N (R ') CO-(alkylidene group or be substituted alkylidene group)-,-N (R ') C (O) O-,-(alkylidene group or be substituted alkylidene group)-O-N=CR '-,-(alkylidene group or be substituted alkylidene group)-C (O) NR '-(alkylidene group or be substituted alkylidene group)-,-(alkylidene group or be substituted alkylidene group)-S (O) k-(alkylidene group or be substituted alkylidene group)-S-,-(alkylidene group or be substituted alkylidene group)-S-S-,-S (O) kN (R ')-,-N (R ') C (O) N (R ')-,-N (R ') C (S) N (R ')-,-N (R ') S (O) kN (R ')-,-N (R ')-N=,-C (R ')=N-,-C (R ')=N-N (R ')-,-C (R ')=N-N=,-C (R ') 2-N=N-and-C (R ') 2-N (R ')-N (R ')-, wherein R ' is H, alkyl independently of one another or is substituted alkyl;
Its restricted condition is not for when A and B exist, and R is not a methyl.
65. according to the described method of claim 64, the wherein said oxime amino acid that contains has structure corresponding to formula (XII):
Figure A2005800443280028C1
66. according to the described method of claim 64, the wherein said oxime amino acid that contains has structure corresponding to formula (XIII):
Figure A2005800443280028C2
67. according to the described method of claim 64, wherein X is a water-soluble polymers.
68. according to the described method of claim 64, wherein X is the derivative of polyoxyethylene glycol.
69. according to the described method of claim 64, wherein X is a cytotoxic compound.
70. according to the described method of claim 64, wherein X is a medicine.
71. according to the described method of claim 64, wherein X is second polypeptide.
72. according to the described method of claim 71, wherein said second polypeptide is the non-natural amino acid polypeptides that contains oxime.
73. according to the described method of claim 71, wherein said second polypeptide have with according to the identical amino acid structure of the described non-natural amino acid polypeptides of claim 62.
74. according to the described method of claim 62, it further comprises pharmaceutically acceptable supporting agent.
75. according to the described method of claim 62, the wherein said non-natural amino acid polypeptides that contains oxime is the modified non-natural amino acid polypeptides that contains oxime.
76. method that is used for detecting the existence of patient's polypeptide, described method comprises the homology non-natural amino acid polypeptides of throwing with significant quantity that comprises the alpha-non-natural amino acid that at least one contains oxime, the wherein said oxime amino acid that contains is to use translation system to incorporate in the interior specific site of described polypeptide, and described translation system comprises:
(i) polynucleotide of coding said polypeptide, wherein said polynucleotide comprise corresponding to incorporating the described amino acid whose selection codon of specifying the site in advance of oxime that contains into, and
(ii) comprise the described amino acid whose tRNA of oxime that contains, wherein said tRNA has specificity to described selection codon.
77. according to the described method of claim 76, wherein said translation system is the in vivo translation system that comprises the cell of the organism that is selected from following group: prokaryotic organism, eukaryote, Mammals, intestinal bacteria, pseudomonas kind, fungi, yeast, archeobacteria, eubacterium, plant, insect and protobiont.
78. according to the described method of claim 76, what the wherein said non-natural amino acid polypeptides that contains oxime comprised structure that at least one has formula (XI) contains oxime amino acid:
Figure A2005800443280029C1
Wherein:
A is optionally, and when exist, be low-grade alkylidene, be substituted low-grade alkylidene, rudimentary cycloalkylidene, be substituted rudimentary cycloalkylidene, lower alkenylene, be substituted lower alkenylene, alkynylene, the assorted alkyl in rudimentary Asia, be substituted inferior mix alkyl, rudimentary inferior Heterocyclylalkyl, be substituted rudimentary inferior Heterocyclylalkyl, arylidene, be substituted arylidene, inferior heteroaryl, be substituted inferior heteroaryl, alkarylene, be substituted alkarylene, inferior aralkyl or be substituted inferior aralkyl;
B is optionally, and is the connexon that is selected from the group that is made up of following each group when exist: low-grade alkylidene, be substituted low-grade alkylidene, lower alkenylene, be substituted lower alkenylene, alkyl is mixed in rudimentary Asia, be substituted rudimentary Asia mix alkyl ,-O-,-O-(alkylidene group or be substituted alkylidene group)-,-S-,-S-(alkylidene group or be substituted alkylidene group)-, wherein k be 1,2 or 3-S (O) k-,-S (O) k(alkylidene group or be substituted alkylidene group)-,-C (O)-,-C (O)-(alkylidene group or be substituted alkylidene group)-,-C (S)-,-C (S)-(alkylidene group or be substituted alkylidene group)-,-N (R ')-,-NR '-(alkylidene group or be substituted alkylidene group)-,-C (O) N (R ')-,-CON (R ')-(alkylidene group or be substituted alkylidene group)-,-CSN (R ')-,-CSN (R ')-(alkylidene group or be substituted alkylidene group)-,-N (R ') CO-(alkylidene group or be substituted alkylidene group)-,-N (R ') C (O) O-,-S (O) kN (R ')-,-N (R ') C (O) N (R ')-,-N (R ') C (S) N (R ')-,-N (R ') S (O) kN (R ')-,-N (R ')-N=,-C (R ')=N-,-C (R ')=N-N (R ')-,-C (R ')=N-N=,-C (R ') 2-N=N-and-C (R ') 2-N (R ')-N (R ')-, wherein R ' is H, alkyl independently of one another or is substituted alkyl;
R is H, alkyl, is substituted alkyl, cycloalkyl or is substituted cycloalkyl;
R 1Be H, amino protecting group, resin, amino acid, polypeptide or polynucleotide; And
R 2Be OH, ester protecting group, resin, amino acid, polypeptide or polynucleotide;
R 3With R 4Be H, halogen, low alkyl group or be substituted low alkyl group independently of one another, or R 3With R 4Or two R 3Group forms cycloalkyl or Heterocyclylalkyl according to circumstances;
R 5For H, alkyl, be substituted alkyl, thiazolinyl, be substituted thiazolinyl, alkynyl, be substituted alkynyl, alkoxyl group, be substituted alkoxyl group, alkyl alkoxy, be substituted alkyl alkoxy, polyoxyalkylene, be substituted polyoxyalkylene, aryl, be substituted aryl, heteroaryl, be substituted heteroaryl, alkaryl, be substituted alkaryl, aralkyl, be substituted aralkyl ,-(alkylidene group or be substituted alkylidene group)-ON (R ") 2,-(alkylidene group or be substituted alkylidene group)-C (O) S R " ,-(alkylidene group or be substituted alkylidene group)-S-S-(aryl or be substituted aryl) ,-C (O) R " ,-C (O) 2R " or-C (O) N (R ") 2, R wherein " independently of one another for hydrogen, alkyl, be substituted alkyl, thiazolinyl, be substituted thiazolinyl, alkoxyl group, be substituted alkoxyl group, aryl, be substituted aryl, heteroaryl, alkaryl, be substituted alkaryl, aralkyl or be substituted aralkyl;
Or R 5Be L-X, wherein
X is selected from the group that is made up of following each thing: mark; Dyestuff; Polymkeric substance; Water-soluble polymers; The derivative of polyoxyethylene glycol; Photocrosslinking agent; Cytotoxic compound; Medicine; Affinity marker; The photoaffinity mark; Reactive compounds; Resin; Second protein or polypeptide or polypeptide analog; Antibody or antibody fragment; Metal chelator; Cofactor; Lipid acid; Carbohydrate; Polynucleotide; DNA; RNA; Antisense polynucleotide; Carbohydrate, water-soluble branch-shape polymer, cyclodextrin, biomaterial; Nanoparticle; Spin labeling; Fluorophore, metallic part; Radioactive segment; Novel functional group; With the covalently or non-covalently interactional group of other molecules; The light cage covers part; But photoisomerization part; Biological rope; The vitamin H analogue; Be combined with the part of heavy atom; But the group of chemical cracking; But the group of photodestruciton; The side chain that prolongs; The sugar of carbon bond connection; Redox active agent; Amino thioic acid sulfoacid; The toxicity part; Through isotope-labeled part; The biophysics probe; The phosphorescence group; Chemiluminescent groups; The electron dense group; The magnetic group; Insert group; Chromophoric group; Energy transfer agent; Biologically active agent; Detectable label; And any combination of above-mentioned substance; And
L is optionally, and is the connexon that is selected from the group that is made up of following each group when existing: alkylidene group, be substituted alkylidene group, alkenylene, be substituted alkenylene ,-O-,-O-(alkylidene group or be substituted alkylidene group)-,-S-,-S-(alkylidene group or be substituted alkylidene group)-, wherein k be 1,2 or 3-S (O) k-,-S (O) k(alkylidene group or be substituted alkylidene group)-,-C (O)-,-C (O)-(alkylidene group or be substituted alkylidene group)-,-C (S)-,-C (S)-(alkylidene group or be substituted alkylidene group)-,-N (R ')-,-NR '-(alkylidene group or be substituted alkylidene group)-,-C (O) N (R ')-,-CON (R ')-(alkylidene group or be substituted alkylidene group)-,-CSN (R ')-,-CSN (R ')-(alkylidene group or be substituted alkylidene group)-,-N (R ') CO-(alkylidene group or be substituted alkylidene group)-,-N (R ') C (O) O-,-(alkylidene group or be substituted alkylidene group)-O-N=CR '-,-(alkylidene group or be substituted alkylidene group)-C (O) NR '-(alkylidene group or be substituted alkylidene group)-,-(alkylidene group or be substituted alkylidene group)-S (O) k-(alkylidene group or be substituted alkylidene group)-S-,-(alkylidene group or be substituted alkylidene group)-S-S-,-S (O) kN (R ')-,-N (R ') C (O) N (R ')-,-N (R ') C (S) N (R ')-;-N (R ') S (O) kN (R ')-,-N (R ')-N=,-C (R ')=N-,-C (R ')=N-N (R ')-,-C (R ')=N-N=,-C (R ') 2-N=N-and-C (R ') 2-N (R ')-N (R ')-, wherein R ' is H, alkyl independently of one another or is substituted alkyl;
Its restricted condition is not for when A and B exist, and R is not a methyl.
79. according to the described method of claim 78, the wherein said oxime amino acid that contains has structure corresponding to formula (XII):
80. according to the described method of claim 78, the wherein said oxime amino acid that contains has structure corresponding to formula (XIII):
Figure A2005800443280032C1
81. according to the described method of claim 78, wherein X is selected from the group that is made up of following each thing: mark; Dyestuff; Affinity marker; The photoaffinity mark; Spin labeling; Fluorophore; Radioactive segment; Be combined with the part of heavy atom; Through isotope-labeled part; The biophysics probe; The phosphorescence group; Chemiluminescent groups; The electron dense group; The magnetic group; Chromophoric group; Energy transfer agent; Detectable label; And any combination of above-mentioned substance.
82. according to the described method of claim 78, it is that modified biosynthesizing contains the oxime non-natural amino acid polypeptides that wherein said biosynthesizing contains the oxime non-natural amino acid polypeptides.
83. the method for the separating property of a facilitation polypeptide, it comprises that utilization comprises that at least one contains the homology non-natural amino acid polypeptides of oxime alpha-non-natural amino acid, the wherein said oxime amino acid that contains is to use translation system to incorporate in the interior specific site of described polypeptide, and described translation system comprises:
(i) polynucleotide of coding said polypeptide, wherein said polynucleotide comprise corresponding to incorporating the described amino acid whose selection codon of specifying the site in advance of oxime that contains into, and
(ii) comprise the described amino acid whose tRNA of oxime that contains, wherein said tRNA has specificity to described selection codon.
84. 3 method according to Claim 8, wherein said translation system is the in vivo translation system that comprises the cell of the organism that is selected from following group: prokaryotic organism, eukaryote, Mammals, intestinal bacteria, fungi, pseudomonas kind, yeast, archeobacteria, eubacterium, plant, insect and protobiont.
85. what 3 method according to Claim 8, the wherein said non-natural amino acid polypeptides that contains oxime comprised structure that at least one has formula (XI) contains oxime amino acid:
Figure A2005800443280032C2
Wherein:
A is optionally, and when exist, be low-grade alkylidene, be substituted low-grade alkylidene, rudimentary cycloalkylidene, be substituted rudimentary cycloalkylidene, lower alkenylene, be substituted lower alkenylene, alkynylene, the assorted alkyl in rudimentary Asia, be substituted inferior mix alkyl, rudimentary inferior Heterocyclylalkyl, be substituted rudimentary inferior Heterocyclylalkyl, arylidene, be substituted arylidene, inferior heteroaryl, be substituted inferior heteroaryl, alkarylene, be substituted alkarylene, inferior aralkyl or be substituted inferior aralkyl;
B is optionally, and is the connexon that is selected from the group that is made up of following each group when exist: low-grade alkylidene, be substituted low-grade alkylidene, lower alkenylene, be substituted lower alkenylene, alkyl is mixed in rudimentary Asia, be substituted rudimentary Asia mix alkyl ,-O-,-O-(alkylidene group or be substituted alkylidene group)-,-S-,-S-(alkylidene group or be substituted alkylidene group)-, wherein k be 1,2 or 3-S (O) k-,-S (O) k(alkylidene group or be substituted alkylidene group)-,-C (O)-,-C (O)-(alkylidene group or be substituted alkylidene group)-,-C (S)-,-C (S)-(alkylidene group or be substituted alkylidene group)-,-N (R ')-,-NR '-(alkylidene group or be substituted alkylidene group)-,-C (O) N (R ')-,-CON (R ')-(alkylidene group or be substituted alkylidene group)-,-CSN (R ')-,-CSN (R ')-(alkylidene group or be substituted alkylidene group)-,-N (R ') CO-(alkylidene group or be substituted alkylidene group)-,-N (R ') C (O) O-,-S (O) kN (R ')-,-N (R ') C (O) N (R ')-,-N (R ') C (S) N (R ')-,-N (R ') S (O) kN (R ')-,-N (R ')-N=,-C (R ')=N-,-C (R ')=N-N (R ')-,-C (R ')=N-N=,-C (R ') 2-N=N-and-C (R ') 2-N (R ')-N (R ')-, wherein R ' is H, alkyl independently of one another or is substituted alkyl;
R is H, alkyl, is substituted alkyl, cycloalkyl or is substituted cycloalkyl;
R 1Be H, amino protecting group, resin, amino acid, polypeptide or polynucleotide; And
R 2Be OH, ester protecting group, resin, amino acid, polypeptide or polynucleotide;
R 3With R 4Be H, halogen, low alkyl group or be substituted low alkyl group independently of one another, or R 3With R 4Or two R 3Group forms cycloalkyl or Heterocyclylalkyl according to circumstances;
R 5For H, alkyl, be substituted alkyl, thiazolinyl, be substituted thiazolinyl, alkynyl, be substituted alkynyl, alkoxyl group, be substituted alkoxyl group, alkyl alkoxy, be substituted alkyl alkoxy, polyoxyalkylene, be substituted polyoxyalkylene, aryl, be substituted aryl, heteroaryl, be substituted heteroaryl, alkaryl, be substituted alkaryl, aralkyl, be substituted aralkyl ,-(alkylidene group or be substituted alkylidene group)-ON (R ") 2,-(alkylidene group or be substituted alkylidene group)-C (O) SR " ,-(alkylidene group or be substituted alkylidene group)-S-S-(aryl or be substituted aryl) ,-C (O) R " ,-C (O) 2R " or-C (O) N (R ") 2, R wherein " independently of one another for hydrogen, alkyl, be substituted alkyl, thiazolinyl, be substituted thiazolinyl, alkoxyl group, be substituted alkoxyl group, aryl, be substituted aryl, heteroaryl, alkaryl, be substituted alkaryl, aralkyl or be substituted aralkyl;
Or R 5Be L-X, wherein
X is selected from the group that is made up of following each thing: mark; Dyestuff; Polymkeric substance; Water-soluble polymers; The derivative of polyoxyethylene glycol; Photocrosslinking agent; Cytotoxic compound; Medicine; Affinity marker; The photoaffinity mark; Reactive compounds; Resin; Second protein or polypeptide or polypeptide analog; Antibody or antibody fragment; Metal chelator; Cofactor; Lipid acid; Carbohydrate; Polynucleotide; DNA; RNA; Antisense polynucleotide; Carbohydrate, water-soluble branch-shape polymer, cyclodextrin, biomaterial; Nanoparticle; Spin labeling; Fluorophore, metallic part; Radioactive segment; Novel functional group; With the covalently or non-covalently interactional group of other molecules; The light cage covers part; But photoisomerization part; Vitamin H; The vitamin H analogue; Be combined with the part of heavy atom; But the group of chemical cracking; But the group of photodestruciton; The side chain that prolongs; The sugar of carbon bond connection; Redox active agent; Amino thioic acid sulfoacid; The toxicity part; Through isotope-labeled part; The biophysics probe; The phosphorescence group; Chemiluminescent groups; The electron dense group; The magnetic group; Insert group; Chromophoric group; Energy transfer agent; Biologically active agent; Detectable label; And any combination of above-mentioned substance; And
L is optionally, and is the connexon that is selected from the group that is made up of following each group when existing: alkylidene group, be substituted alkylidene group, alkenylene, be substituted alkenylene ,-O-,-O-(alkylidene group or be substituted alkylidene group)-,-S-,-S-(alkylidene group or be substituted alkylidene group)-, wherein k be 1,2 or 3-S (O) k-,-S (O) k(alkylidene group or be substituted alkylidene group)-,-C (O)-,-C (O)-(alkylidene group or be substituted alkylidene group)-,-C (S)-,-C (S)-(alkylidene group or be substituted alkylidene group)-,-N (R ')-,-NR '-(alkylidene group or be substituted alkylidene group)-,-C (O) N (R ')-,-CON (R ')-(alkylidene group or be substituted alkylidene group)-,-CSN (R ')-,-CSN (R ')-(alkylidene group or be substituted alkylidene group)-,-N (R ') CO-(alkylidene group or be substituted alkylidene group)-,-N (R ') C (O) O-,-(alkylidene group or be substituted alkylidene group)-O-N=CR '-,-(alkylidene group or be substituted alkylidene group)-C (O) NR '-(alkylidene group or be substituted alkylidene group)-,-(alkylidene group or be substituted alkylidene group)-S (O) k-(alkylidene group or be substituted alkylidene group)-S-,-(alkylidene group or be substituted alkylidene group)-S-S-,-S (O) kN (R ')-,-N (R ') C (O) N (R ')-,-N (R ') C (S) N (R ')-,-N (R ') S (O) kN (R ')-,-N (R ')-N=,-C (R ')=N-,-C (R ')=N-N (R ')-,-C (R ')=N-N=,-C (R ') 2-N=N-and-C (R ') 2-N (R ')-N (R ')-, wherein R ' is H, alkyl independently of one another or is substituted alkyl;
Its restricted condition is not for when A and B exist, and R is not a methyl.
86. 3 described methods according to Claim 8, the wherein said oxime amino acid that contains has structure corresponding to formula (XII):
Figure A2005800443280034C1
87. 6 described methods according to Claim 8, the wherein said oxime amino acid that contains has structure corresponding to formula (XIII):
Figure A2005800443280035C1
88. 6 described methods according to Claim 8, wherein X is a water-soluble polymers.
89. 6 described methods according to Claim 8, wherein X is the derivative of polyoxyethylene glycol.
90. according to the described method of claim 78, the wherein said non-natural amino acid polypeptides that contains oxime is the modified non-natural amino acid polypeptides that contains oxime.
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