CN101365335A - 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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CN101365335A
CN101365335A CNA2006800499954A CN200680049995A CN101365335A CN 101365335 A CN101365335 A CN 101365335A CN A2006800499954 A CNA2006800499954 A CN A2006800499954A CN 200680049995 A CN200680049995 A CN 200680049995A CN 101365335 A CN101365335 A CN 101365335A
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alkylidene
alkyl
carbon
group
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苗振伟
刘俊杰
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Ambrx Inc
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Ambrx Inc
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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 heterocycle, aldol-based, dicarbonyl, and/or diamine 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 heterocycle, aldol-based, dicarbonyl, and/or diamine 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
Related application
The application's case is advocated the U.S. Provisional Application case the 60/755th of application on December 30th, 2005, the 60/755th of application on December 30th, No. 338 1, the 60/755th of No. 711 and on December 30th, 2005 application, No. 018 right, the mode that all described patents are all quoted in full is incorporated herein.
Technical field
This paper describes compound, composition, technology and the strategy of preparation, purifying, sign and use alpha-non-natural amino acid, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides.
Background technology
The ability that non-genomic amino acids coding (that is, " alpha-non-natural amino acid ") is incorporated in the protein allows introducing can provide naturally occurring functional group (such as the ε-NH of lysine 2, sulfydryl-SH of cysteine, the imino group of histidine etc.) the chemical functional group of valuable substitute.Known some chemical functional group is inertia to the functional group seen in the amino acid of 20 kinds of common gene codes, but fully and effectively with can be incorporated into functional group reactions on the alpha-non-natural amino acid to form stable keys.
Several different methods now can be used for selectivity and introduces and not see in the protein, all functional groups seen in the amino acid of 20 kinds of common gene codes are chemical inertness and can be used for and the reagent that comprises some functional group effectively and optionally reacts the chemical functional group who stablizes covalent bond to form.
Summary of the invention
This paper describes method, composition, technology and the strategy of preparation, purifying, sign and use alpha-non-natural amino acid, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides.On the one hand for making method, composition, technology and the strategy of alpha-non-natural amino acid and/or non-natural amino acid polypeptides derivatization.In one embodiment, described method, composition, technology and strategy relate to chemical derivatization; In other embodiments, relate to biologically-derivedization; In other embodiments, relate to the physics derivatization; In other embodiments, relate to the combination of derivatization.In other or extra embodiment, described derivatization tool regioselectivity.In other or extra embodiment, described derivatization tool regiospecificity.In other or extra embodiment, described derivatization is stoichiometry or near-stoichiometric in the reagent that contains alpha-non-natural amino acid and derivatization reagent.In other or extra embodiment, described derivatization is fast at ambient temperature.In other or extra embodiment, described derivatization is to take place in the aqueous solution.In other or extra embodiment, described derivatization is in generation under the pH value between about 2 and about 10.In other or extra embodiment, described derivatization is in generation under the pH value between about 3 and about 8.In other or extra embodiment, described derivatization is in generation under the pH value between about 2 and about 9.In other or extra embodiment, described derivatization is in generation under the pH value between about 4 and about 9.In other or extra embodiment, described derivatization is generation under about 4 pH value.In another embodiment, described derivatization is to take place under about 8 pH value.In other or extra embodiment, described derivatization is stoichiometry, near-stoichiometric or class stoichiometry in the reagent that contains alpha-non-natural amino acid and derivatization reagent.In other or extra embodiment, provide and incorporate required group stoichiometry, near-stoichiometric or class stoichiometry on the non-natural amino acid polypeptides method.In other or extra embodiment, provide permission to incorporate required group stoichiometry, near-stoichiometric or class stoichiometry on the non-natural amino acid polypeptides strategy, reactant mixture, synthesis condition.
On the one hand for make the alpha-non-natural amino acid of peptide and protein chemistry derivatization based on the reactivity of dicarbapentaborane, described dicarbapentaborane comprises 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 carboxylic acid and/or at least one thioester substrate, and wherein said dicarbapentaborane can be 1,2-dicarbapentaborane, 1,3-dicarbapentaborane or 1, the 4-dicarbapentaborane.Other or additional aspect are for making the alpha-non-natural amino acid of peptide and protein chemistry derivatization based on the reactivity of two amidos, described two amidos comprise diazanyl, amidino groups, imido grpup, 1,1-two amidos, 1,2-two amidos, 1,3-two amidos and 1,4-two amidos.In other or extra embodiment, at least one above-mentioned alpha-non-natural amino acid is incorporated in the polypeptide, that is described embodiment is a non-natural amino acid polypeptides.In other or extra embodiment, on the side chain of alpha-non-natural amino acid, make it functionalized, so that itself and derivatization molecular reaction generation key, comprise based on the key of heterocycle (comprising nitrogen heterocyclic ring) and/or based on the key of aldehyde alcohol.Other or extra embodiment be for can containing the non-natural amino acid polypeptides of the non-natural amino acid polypeptides of key with generation with the derivatization molecular reaction, and described key comprises based on the key of heterocycle (comprising nitrogen heterocyclic ring) and/or based on the key of aldehyde alcohol.In other or extra embodiment, alpha-non-natural amino acid is selected from the amino acid with dicarbapentaborane and/or two amine side chains.In other or extra embodiment, alpha-non-natural amino acid comprises through covering side chain, and it comprises through covering two amidos and/or through covering dicarbapentaborane.In other or extra embodiment, alpha-non-natural amino acid comprises the group that is selected from following each thing: ketone-amine (that is, containing the group of ketone and amine), ketone-alkynes (that is the group that, contains ketone and alkynes) and alkene-diketone (that is the group that, contains dicarbapentaborane and alkene).
In other or extra embodiment, alpha-non-natural amino acid comprises the dicarbapentaborane side chain, and wherein said carbonyl is selected from ketone, aldehyde, carboxylic acid or ester (comprising thioesters).Another embodiment contains the alpha-non-natural amino acid that can form the functional group of heterocycle (comprising nitrogen heterocyclic ring) after handling with suitable functionalized reagent.In another embodiment, the structure of alpha-non-natural amino acid is similar to natural amino acid, but contains in the above-mentioned functional group one.In another embodiment, alpha-non-natural amino acid is similar to phenyl alanine or tyrosine (aromatic amino acid); And in another embodiment, alpha-non-natural amino acid is similar to alanine and leucine (hydrophobic amino acid).In one embodiment, alpha-non-natural amino acid has and the distinct characteristic of natural amino acid.In one embodiment, described distinct characteristic is the chemical reactivity of side chain, in another embodiment, even this distinct chemical reactivity allows under the situation that the side chain of the naturally occurring amino acid unit in polypeptide do not react, also carry out above-mentioned reaction as the side chain of the alpha-non-natural amino acid of the unit of same polypeptide.In another embodiment, the side chain of alpha-non-natural amino acid has the chemical property with naturally occurring amino acid whose side chain quadrature.In another embodiment, the side chain of alpha-non-natural amino acid comprises and contains the electrophilic group part; In another embodiment, containing electrophilic group part and can carry out nucleophillic attack to produce heterocyclic derivatives protein on the side chain of alpha-non-natural amino acid comprises nitrogen heterocyclic ring derivatization protein.In arbitrary the foregoing description of this paragraph, alpha-non-natural amino acid can be used as independent molecule existence or can incorporate in the polypeptide of any length; If be the latter, polypeptide can in addition and have naturally occurring amino acid or alpha-non-natural amino acid so.
Be the molecule that replaces through diamines on the other hand, it is used for the derivatization non-natural amino acid polypeptides of preparation based on heterocycle (comprising nitrogen heterocyclic ring) key, and wherein said two amidos are selected from hydrazine, amidine, imines, 1,1-diamines, 1,2-diamines, 1,3-diamines and 1,4-diamine groups.Another embodiment is the molecule that replaces through diamines, and it is used for via at the derivatization molecule and contain and form heterocycle (comprising nitrogen heterocyclic ring) key between the non-natural amino acid polypeptides of dicarbapentaborane and make the non-natural amino acid polypeptides derivatization that contains dicarbapentaborane.In other embodiments, the above-mentioned non-natural amino acid polypeptides that contains dicarbapentaborane is the non-natural amino acid polypeptides that contains diketone.In other or extra embodiment, the alpha-non-natural amino acid that contains dicarbapentaborane comprises described carbonyl and is selected from ketone, aldehyde, carboxylic acid or the ester side chain of (comprising thioesters).In other or extra embodiment, the molecule that replaces through diamines comprises the group that is selected from required functional group.In other or extra embodiment, the molecule that replaces through diamines be polyethylene glycol (PEG) molecule through the diamines replacement.In another embodiment, the side chain of alpha-non-natural amino acid has the chemical property with naturally occurring amino acid quadrature, and it makes alpha-non-natural amino acid to react with the molecular selectivity that replaces through diamines.In another embodiment, the side chain of alpha-non-natural amino acid comprise with contain diamines molecular selectivity reaction contain the electrophilic group part; In another embodiment, containing electrophilic group part and can carry out nucleophillic attack to produce heterocyclic derivatives protein on the alpha-non-natural amino acid side chain comprises nitrogen heterocyclic ring derivatization protein.What the embodiment described in the paragraph was correlated with therewith is modified non-natural amino acid polypeptides on the other hand, and it is to be obtained by derivatization molecule and non-natural amino acid polypeptides reaction.Other embodiment comprises any other modification of modified non-natural amino acid polypeptides.
On the other hand for prepare the molecule through the dicarbapentaborane replacement of derivatization non-natural amino acid polypeptides based on heterocycle (comprising nitrogen heterocyclic ring) key.Another embodiment is the molecule that replaces through dicarbapentaborane, and it is used for making the non-natural amino acid polypeptides derivatization that contains diamines via forming heterocycle (comprising nitrogen heterocycle).Another embodiment is for forming the molecule through the dicarbapentaborane replacement of described heterocycle (comprising nitrogen heterocycle) with the non-natural amino acid polypeptides that contains diamines in the pH value scope between about 4 and about 8.Another embodiment is the molecule that replaces through dicarbapentaborane, and it is used for via at the derivatization molecule and contain and form heterocycle (comprising nitrogen heterocyclic ring) key between the non-natural amino acid polypeptides of diamines and make the non-natural amino acid polypeptides derivatization that contains diamines.In another embodiment, the molecule that replaces through dicarbapentaborane is the molecule that replaces through diketone, is the molecule that replaces through keto-aldehyde in others, is the molecule that replaces through ketone acid in others, be the molecule that replaces through ketone ester in others, comprise the molecule that replaces through the ketone thioesters.In other embodiments, the molecule that replaces through dicarbapentaborane comprises the group that is selected from required functional group.In other or extra embodiment, the molecule that replaces through aldehyde be polyethylene glycol (PEG) molecule through the aldehyde replacement.In another embodiment, the side chain of alpha-non-natural amino acid has the chemical property with naturally occurring amino acid quadrature, and it makes alpha-non-natural amino acid to react with the molecular selectivity through carbonyl substituted.In another embodiment, the side chain of alpha-non-natural amino acid comprises the part (for example, two amidos) of reacting with the molecular selectivity that contains dicarbapentaborane; In another embodiment, the nucleophilic part on the alpha-non-natural amino acid side chain can be carried out the attack of parent's electricity to produce heterocyclic derivatives protein, comprises nitrogen heterocyclic ring derivatization protein.What the embodiment described in the paragraph was correlated with therewith is modified non-natural amino acid polypeptides on the other hand, and it is to be obtained by derivatization molecule and non-natural amino acid polypeptides reaction.Other embodiment comprises any other modification of modified non-natural amino acid polypeptides.
Be single, double and multifunctional linkers on the other hand based on heterocycle (comprising nitrogen heterocyclic ring) and/or aldehyde alcohol key generation derivatization non-natural amino acid polypeptides.Embodiment is that the molecule that the non-natural amino acid polypeptides that can be used for containing dicarbapentaborane is connected with other molecule connects base (difunctionality and multifunctional).Another embodiment is that the molecule that the non-natural amino acid polypeptides that can be used for containing diamines is connected with other molecule connects base (difunctionality and multifunctional).In another embodiment, the non-natural amino acid polypeptides that contains dicarbapentaborane comprises ketone, aldehyde, carboxylic acid, ester or thioesters side chain.Contain among the embodiment of non-natural amino acid polypeptides of diamines in utilization, molecule connects base and contains carbonyl at an one end; In other embodiments, described carbonyl is selected from aldehyde radical, ester group, thioester substrate or ketone group.In other or extra embodiment, the connection base molecule that replaces through diamines connects basic molecule for the polyethylene glycol (PEG) that replaces through diamines.In other or extra embodiment, the connection base molecule that replaces through dicarbapentaborane connects basic molecule for the polyethylene glycol (PEG) that replaces through dicarbapentaborane.In other embodiments, phrase " other molecule " comprises (only for instance) protein, other polymer and little molecule.In other or extra embodiment, the molecule that contains diamines connects base and comprises identical or suitable group at all ends, thus with the non-natural amino acid polypeptides reaction that contains dicarbapentaborane after, products therefrom is the same multimerization that contains the non-natural amino acid polypeptides of dicarbapentaborane.In other embodiments, turn to same dimerization with poly.In other or extra embodiment, the molecule that contains dicarbapentaborane connects base and comprises identical or suitable group at all ends, thus with the non-natural amino acid polypeptides reaction that contains diamines after, products therefrom is the same multimerization that contains the non-natural amino acid polypeptides of diamines.In other embodiments, turn to same dimerization with poly.In another embodiment, the side chain of alpha-non-natural amino acid has the chemical property with naturally occurring amino acid quadrature, and it makes alpha-non-natural amino acid to react with the base molecular selectivity that is connected that replaces through diamines.In another embodiment, the side chain of alpha-non-natural amino acid has the chemical property with naturally occurring amino acid quadrature, and it makes alpha-non-natural amino acid to react with the base molecular selectivity that is connected that replaces through dicarbapentaborane.In another embodiment, the side chain of alpha-non-natural amino acid comprise with contain diamines be connected base molecular selectivity reaction contain the electrophilic group part; In another embodiment, containing electrophilic group part and can carry out nucleophillic attack by the connection base molecule that contains diamines to produce heterocyclic derivatives protein on the alpha-non-natural amino acid side chain comprises nitrogen heterocyclic ring derivatization protein.What the embodiment described in the paragraph was correlated with therewith is through connection (modified) non-natural amino acid polypeptides on the other hand, and it is to obtain by connecting the reaction of basic molecule and non-natural amino acid polypeptides.Other embodiment comprises any other modification of the non-natural amino acid polypeptides that connects (modified).
For making protein derivedization to produce heterocyclic derivatives protein, comprise nitrogen heterocyclic ring derivatization method of protein on the one hand via dicarbapentaborane and the reaction of diamine reactant thing.Comprise in this respect that the condensation based on the reactant that contains dicarbapentaborane and diamines makes protein derivedization to produce the method for heterocyclic derivatives protein adduct (comprising nitrogen heterocyclic ring derivatization protein adduct).Extra or other embodiment is for to utilize diamines functionalized poly (ethylene glycol) (PEG) molecule to make the method that contains diketone protein or contain keto-aldehyde protein or contain ketone acid protein or contain ketone ester protein or contain ketone thioesters protein derivedization.In extra or others, the molecule that replaces through diamines can comprise protein, other polymer and little molecule.
Be used to make the method for the molecule that replaces through diamines of the protein derivedization that replaces through dicarbapentaborane on the other hand for chemosynthesis.In one embodiment, the molecule that replaces through diamines can comprise peptide, other polymer (non-branch and branch) and little molecule.Embodiment is suitable for making the method for the molecule that replaces through diamines of the non-natural amino acid polypeptides derivatization that contains dicarbapentaborane for preparation, only for instance, the described non-natural amino acid polypeptides that contains dicarbapentaborane comprises and contains diketone, contains keto-aldehyde, contains ketone acid, contains ketone ester and/or contain the non-natural amino acid polypeptides of ketone thioesters.In other or extra embodiment, in vivo alpha-non-natural amino acid is incorporated on locus specificity ground into during the translated protein.In other or extra embodiment, the molecule that replaces through diamines allows to make the alpha-non-natural amino acid locus specificity derivatization that contains dicarbapentaborane via each carbonyl of nucleophillic attack, thereby form the heterocyclic derivatives polypeptide in the locus specificity mode, comprise nitrogen heterocyclic ring derivatization polypeptide.In other or extra embodiment, preparation provides the mode that obtains multiple locus specificity derivatization polypeptide through the method for the molecule that diamines replaces.Other or extra embodiment are for synthesizing the method for diamines functionalized poly (ethylene glycol) (PEG) molecule.
Be used to make the method for the molecule that replaces through dicarbapentaborane of the non-natural amino acid polypeptides derivatization that replaces through diamines on the other hand for chemosynthesis.In one embodiment, the molecule that replaces through dicarbapentaborane is the molecule that replaces through diketone, keto-aldehyde, ketone acid, ketone ester and/or ketone thioesters.In another embodiment, the molecule that replaces through dicarbapentaborane comprises protein, polymer (non-branch and branch) and little molecule.In other or extra embodiment, described method replenish during the translated protein in vivo can site specific incorporation of non-natural amino acids technology.Thereby other or extra embodiment are suitable for providing with the non-natural amino acid polypeptides reaction that contains diamines the method for the molecule that replaces through dicarbapentaborane of locus specificity derivatization non-natural amino acid polypeptides for preparation.Other or extra embodiment are for synthesizing the method for polyethylene glycol (PEG) molecule that replaces through dicarbapentaborane.
Contain the method that diamines difunctionality connection base makes the non-natural amino acid polypeptides chemical derivatization that replaces through dicarbapentaborane for using on the other hand.Thereby an embodiment is for connecting the method that connection base that replaces through diamines and the protein that replaces through dicarbapentaborane produce heterocycle (comprising nitrogen heterocyclic ring) key via condensation reaction.In other or extra embodiment, the alpha-non-natural amino acid that replaces through dicarbapentaborane be the alpha-non-natural amino acid through diketone, keto-aldehyde, ketone acid, ketone ester and/or the replacement of ketone thioesters.In other or extra embodiment, use the difunctionality that contains diamines to connect base and make non-natural amino acid polypeptides locus specificity ground derivatization and/or accurately control three-dimensional structure simultaneously.In one embodiment, using described method that molecule is connected base (single, double and multifunctional) is connected with the non-natural amino acid polypeptides that contains dicarbapentaborane (for example comprise and contain diketone, keto-aldehyde, ketone acid, ketone ester and/or ketone thioesters), wherein at least one connects basic end and contains two amidos, and it can be connected with the non-natural amino acid polypeptides that contains dicarbapentaborane via heterocycle (comprising nitrogen heterocyclic ring) key.In other or extra embodiment, use these to connect the non-natural amino acid polypeptides that base will contain dicarbapentaborane and be connected with other molecule, described other molecule comprises for example protein, other polymer (branch and non-branch) and little molecule.
In certain embodiments, non-natural amino acid polypeptides is connected with water-soluble polymer.In certain embodiments, water-soluble polymer comprises polyalkylene glycol moiety.In certain embodiments, peg molecule is the double functional copolymer.In certain embodiments, the double functional copolymer is connected with second polypeptide.In certain embodiments, second polypeptide is identical with first polypeptide, and in other embodiments, second polypeptide is homopolypeptide not.In certain embodiments, non-natural amino acid polypeptides comprises at least two amino acid that are connected with the water-soluble polymer that comprises polyalkylene glycol moiety.
In certain embodiments, non-natural amino acid polypeptides comprises increases replacement, interpolation or the disappearance of non-natural amino acid polypeptides to the compatibility 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 that increases the non-natural amino acid polypeptides that is produced in the host cell.In certain embodiments, non-natural amino acid polypeptides comprises replacement, interpolation or the disappearance of the amino acid polypeptide adjusting protease resistant, serum half-life, immunogenicity and/or the expression that replace, add or lack with respect to not having.
In certain embodiments, non-natural amino acid polypeptides is activator, partial agonist, antagonist, partial antagonist or inverse agonist.In certain embodiments, activator, partial agonist, antagonist, partial antagonist or inverse agonist comprise the alpha-non-natural amino acid that is connected with water-soluble polymer.In certain embodiments, water-soluble polymer comprises polyalkylene glycol moiety.In certain embodiments, the polypeptide that comprises the alpha-non-natural amino acid that is connected with water-soluble polymer can prevent corresponding receptor dimerizationization.In certain embodiments, the polypeptides for modulating polypeptide and the combination that combines collocation thing, part or acceptor that comprise the alpha-non-natural amino acid that is connected with water-soluble polymer.In certain embodiments, polypeptides for modulating one or more polypeptide characteristic or activity of comprising the alpha-non-natural amino acid that is connected with water-soluble polymer.
In certain embodiments, select codon to be selected from: amber codon, ochre codon, opal codon, unique codon, rare codon, non-natural codon, five base codons and four base codons by the molecular group of following password.
This paper also describes the method for preparing the non-natural amino acid polypeptides that is connected with water-soluble polymer.In certain embodiments, described method comprises to make and comprises contacting with comprising with the water-soluble polymer of the part of described alpha-non-natural amino acid reaction through isolated polypeptide of alpha-non-natural amino acid.In certain embodiments, the alpha-non-natural amino acid of being incorporated into is to other water-soluble polymer tool reactivity to any anergy in 20 kinds of common amino acids.In certain embodiments, water-soluble polymer comprises polyalkylene glycol moiety.The molecular weight of polymer can be in broad range, include but not limited between about 100Da and about 100,000Da or higher between.The molecular weight of polymer can between the 000Da, include but not limited to about 100 between about 100Da and about 100,000Da, about 95,000Da, about 90,000Da, about 85,000Da, about 80,000Da, about 75,000Da, about 70,000Da, about 65,000Da, about 60,000Da, about 55,000Da, about 50,000Da, about 45,000Da, about 40,000Da, about 35,000Da, about 30,000Da, about 25,000Da, about 20,000Da, about 15,000Da, about 10,000Da, about 9,000Da, about 8,000Da, about 7,000Da, about 6,000Da, about 5,000Da, about 4,000Da, about 3,000Da, about 2,000Da, about 1,000Da, about 900Da, about 800Da, about 700Da, about 600Da, about 500Da, about 400Da, about 300Da, about 200Da and about 100Da.In certain embodiments, the molecular weight of polymer is between about 100Da and about 50, between the 000Da.In certain embodiments, the molecular weight of polymer is between about 100Da and about 40, between the 000Da.In certain embodiments, the molecular weight of polymer is between about 1, and 000Da and about 40 is between the 000Da.In certain embodiments, the molecular weight of polymer is between about 2, and 000Da is to about 50, between the 000Da.In certain embodiments, the molecular weight of polymer is between about 5, and 000Da and about 40 is between the 000Da.In certain embodiments, the molecular weight of polymer is between about 10, and 000Da and about 40 is between the 000Da.In certain embodiments, peg molecule is a branched polymers.The molecular weight of side chain PEG can be between about 1, and 000Da and about 100 between the 000Da, includes but not limited to about 100,000Da, about 95,000Da, about 90,000Da, about 85,000Da, about 80,000Da, about 75,000Da, about 70,000Da, about 65,000Da, about 60,000Da, about 55,000Da, about 50,000Da, about 45,000Da, about 40,000Da, about 35,000Da, about 30,000Da, about 25,000Da, about 20,000Da, about 15,000Da, about 10,000Da, about 9,000Da, about 8,000Da, about 7,000Da, about 6,000Da, about 5,000Da, about 4,000Da, about 3,000Da, about 2,000Da and about 1,000Da.In certain embodiments, the molecular weight of side chain PEG is between about 1, and 000Da and about 50 is between the 000Da.In certain embodiments, the molecular weight of side chain PEG is between about 1, and 000Da and about 40 is between the 000Da.In certain embodiments, the molecular weight of side chain PEG is between about 5, and 000Da and about 40 is between the 000Da.In certain embodiments, the molecular weight of side chain PEG is between about 5, and 000Da and about 20 is between the 000Da.In certain embodiments, the molecular weight of side chain PEG is between about 2, and 000Da is to about 50, between the 000Da.
This paper also describes composition, and it comprises the polypeptide that comprises at least one alpha-non-natural amino acid as herein described and pharmaceutically acceptable supporting agent.In certain embodiments, alpha-non-natural amino acid is connected with water-soluble polymer.This paper also describes and comprises the pharmaceutically acceptable supporting agent and the medical composition of polypeptide, 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, by sugar moieties water-soluble polymer is connected with polypeptide.Prodrug, non-natural amino acid polypeptides and/or the modified non-natural amino acid polypeptides of alpha-non-natural amino acid are also described herein; The composition comprise described prodrug and pharmaceutically acceptable supporting agent is also described herein.The metabolite of alpha-non-natural amino acid, non-natural amino acid polypeptides and/or modified non-natural amino acid polypeptides is also described herein; Described metabolite can have the required activity of replenishing or working in coordination with the activity of strengthening alpha-non-natural amino acid, non-natural amino acid polypeptides and/or modified non-natural amino acid polypeptides.This paper also describes the purposes that alpha-non-natural amino acid as herein described, non-natural amino acid polypeptides and/or modified non-natural amino acid polypeptides are used for providing to organism (patient who comprises the described metabolite of needs) required metabolite.
This paper also describes and comprises the cell that coding comprises the polynucleotide of the polypeptide of selecting codon.In certain embodiments, described cell comprises quadrature RNA synzyme and/or quadrature tRNA so that alpha-non-natural amino acid is replaced into polypeptide.In certain embodiments, cell is in cell culture, and in other embodiments, cell is the multi-cell organism part of (comprising amphibian, reptile, birds and mammal).In any cell embodiment, other embodiment comprises that express polynucleotide is to produce non-natural amino acid polypeptides.Other embodiment produces the organism of non-natural amino acid polypeptides (comprising modified non-natural amino acid polypeptides) for utilizing alpha-non-natural amino acid as herein described.Other embodiment is the organism that contains alpha-non-natural amino acid as herein described, non-natural amino acid polypeptides and/or modified non-natural amino acid polypeptides.Described organism comprises unicellular and multi-cell organism, comprises amphibian, reptile, birds and mammal.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 is to produce by the ribosome translation.
This paper also describes the method that preparation comprises the polypeptide of alpha-non-natural amino acid.In certain embodiments, described method is included under the condition that allows express polypeptide, cultivates the cell of the polynucleotide, quadrature RNA synzyme and/or the quadrature tRNA that comprise one or more coding said polypeptides; With the described polypeptide of purifying from cell and/or medium.
This paper also describes the library of alpha-non-natural amino acid as herein described, or the library of non-natural amino acid polypeptides as herein described, or the library of modified non-natural amino acid polypeptides as herein described, or its combinatorial libraries.This paper also describes the array that contains at least one alpha-non-natural amino acid, at least one non-natural amino acid polypeptides and/or at least one modified alpha-non-natural amino acid.This paper also describes the array that comprises the polynucleotide of selecting codon that contains at least one coded polypeptide.Can use array as herein described to screen (polynucleotide by detecting coded polypeptide transcribe or by detecting the translation of polypeptide) at the generation of non-natural amino acid polypeptides in the organism.
This paper also describes at required screening active ingredients library as herein described, or uses array screening as herein described library as herein described, or at the method in other library of required screening active ingredients compound and/or polypeptide and/or polynucleotide.This paper also describes the purposes that is used to develop and find novel therapeutic agents and therapeutic agent itself from the described activity data of library screening.
This paper also describes the method for the treatment half life period, serum half-life or the circulation timei that increase polypeptide.In certain embodiments, described method comprises with at least one alpha-non-natural amino acid and replaces any or in the naturally occurring polypeptide with upper amino acid, and/or with polypeptide and water-soluble polymer coupling.
This paper also describes the method that needs the patient of described treatment with the treatment of the medical composition of effective dose, and described medical composition comprises the polypeptide that comprises alpha-non-natural amino acid and pharmaceutically acceptable supporting agent.In certain embodiments, with alpha-non-natural amino acid and water-soluble polymer coupling.
Other or alternate embodiment are for treating the method for illness, symptom or disease, described method comprises the non-natural amino acid polypeptides of throwing with the treatment effective dose, and described polypeptide comprises at least one alpha-non-natural amino acid that is selected from the group that is made up of following each thing: contain the heterocycle alpha-non-natural amino acid, contain the carbonyl alpha-non-natural amino acid, contain the dicarbapentaborane alpha-non-natural amino acid, contain the diamines alpha-non-natural amino acid, contain ketone alkynes alpha-non-natural amino acid or contain the ketoamine alpha-non-natural amino acid.In other embodiments, by biological synthesis method described alpha-non-natural amino acid is incorporated in the polypeptide as described herein.In other embodiments, by synthetic method described alpha-non-natural amino acid is incorporated in the polypeptide as described herein.In other or alternate embodiment, described non-natural amino acid polypeptides comprises at least one amino acid whose alpha-non-natural amino acid that is selected from formula I-LXVII.
Other or alternate embodiment are for treating the method for illness, symptom or disease, described method comprises the non-natural amino acid polypeptides of throwing with the treatment effective dose, and described polypeptide comprises at least one and contains heterocycle alpha-non-natural amino acid and gained and contain the heterocycle non-natural amino acid polypeptides and with respect to the naturally occurring amino acid polypeptide of homology the biological usability of polypeptide is increased.
Other or alternate embodiment are for treating the method for illness, symptom or disease, described method comprises the non-natural amino acid polypeptides of throwing with the treatment effective dose, and described polypeptide comprises at least one and contains heterocycle alpha-non-natural amino acid and gained and contain the heterocycle non-natural amino acid polypeptides and with respect to the naturally occurring amino acid polypeptide of homology the security features of polypeptide is increased.
Other or alternate embodiment are for treating the method for illness, symptom or disease, described method comprises the non-natural amino acid polypeptides of throwing with the treatment effective dose, and described polypeptide comprises at least one and contains the heterocycle alpha-non-natural amino acid and gained contains the heterocycle non-natural amino acid polypeptides makes polypeptide with respect to the naturally occurring amino acid polypeptide of homology water-soluble increase.
Other or alternate embodiment are for treating the method for illness, symptom or disease, described method comprises the non-natural amino acid polypeptides of throwing with the treatment effective dose, and described polypeptide comprises at least one and contains heterocycle alpha-non-natural amino acid and gained and contain the heterocycle non-natural amino acid polypeptides and with respect to the naturally occurring amino acid polypeptide of homology increased the treatment half life period of polypeptide.
Other or alternate embodiment are for treating the method for illness, symptom or disease, described method comprises the non-natural amino acid polypeptides of throwing with the treatment effective dose, and described polypeptide comprises at least one and contains heterocycle alpha-non-natural amino acid and gained and contain the heterocycle non-natural amino acid polypeptides and with respect to the naturally occurring amino acid polypeptide of homology the serum half-life of polypeptide is increased.
Other or alternate embodiment are for treating the method for illness, symptom or disease, described method comprises the non-natural amino acid polypeptides of throwing with the treatment effective dose, and described polypeptide comprises at least one and contains heterocycle alpha-non-natural amino acid and gained and contain the heterocycle non-natural amino acid polypeptides and with respect to the naturally occurring amino acid polypeptide of homology prolonged the circulation timei of polypeptide.
Other or alternate embodiment are for treating the method for illness, symptom or disease, described method comprises the non-natural amino acid polypeptides of throwing with the treatment effective dose, and described polypeptide comprises at least one and contains the heterocycle alpha-non-natural amino acid and gained contains the heterocycle non-natural amino acid polypeptides is regulated polypeptide with respect to the naturally occurring amino acid polypeptide of homology biologically active.
Other or alternate embodiment are for treating the method for illness, symptom or disease, described method comprises the non-natural amino acid polypeptides of throwing with the treatment effective dose, and described polypeptide comprises at least one and contains the heterocycle alpha-non-natural amino acid and gained contains the heterocycle non-natural amino acid polypeptides is regulated polypeptide with respect to the naturally occurring amino acid polypeptide of homology immunogenicity.
This paper also describes the method that detects the existence of polypeptide in patient's body, and it comprises throwing and polypeptide and pharmaceutically acceptable supporting agent.
Other or alternate embodiment are for detecting the method for the existence of polypeptide in patient's body, described method comprises throws and the polypeptide that comprises at least one alpha-non-natural amino acid, and described alpha-non-natural amino acid is selected from the group that is made up of following each thing: contain the heterocycle alpha-non-natural amino acid, contain the carbonyl alpha-non-natural amino acid, contain the dicarbapentaborane alpha-non-natural amino acid, contain the diamines alpha-non-natural amino acid, contain ketone alkynes alpha-non-natural amino acid or contain the ketoamine alpha-non-natural amino acid.In other embodiments, by biological synthesis method described alpha-non-natural amino acid is incorporated in the polypeptide as described herein.In other embodiments, by synthetic method described alpha-non-natural amino acid is incorporated in the polypeptide as described herein.In other or alternate embodiment, described non-natural amino acid polypeptides comprises at least one amino acid whose alpha-non-natural amino acid that is selected from formula I-LXVII.
Other or alternate embodiment are for detecting the method for the existence of polypeptide in patient's body, described method comprises throwing and comprises the polypeptide that at least one contains the heterocycle alpha-non-natural amino acid, and gained contains the heterocycle non-natural amino acid polypeptides makes polypeptide with respect to the naturally occurring amino acid polypeptide of homology biological usability increase.
Other or alternate embodiment are for detecting the method for the existence of polypeptide in patient's body, described method comprises throwing and comprises the polypeptide that at least one contains the heterocycle alpha-non-natural amino acid, and gained contains the heterocycle non-natural amino acid polypeptides makes polypeptide with respect to the naturally occurring amino acid polypeptide of homology security features increase.
Other or alternate embodiment are for detecting the method for the existence of polypeptide in patient's body, described method comprises throwing and comprises the polypeptide that at least one contains the heterocycle alpha-non-natural amino acid, and gained contains the heterocycle non-natural amino acid polypeptides makes polypeptide with respect to the naturally occurring amino acid polypeptide of homology water-soluble increase.
Other or alternate embodiment are for detecting the method for the existence of polypeptide in patient's body, described method comprises throwing and comprises the polypeptide that at least one contains the heterocycle alpha-non-natural amino acid, and gained contains the heterocycle non-natural amino acid polypeptides makes polypeptide with respect to the naturally occurring amino acid polypeptide of homology treatment half life period increase.
Other or alternate embodiment are for detecting the method for the existence of polypeptide in patient's body, described method comprises throwing and comprises the polypeptide that at least one contains the heterocycle alpha-non-natural amino acid, and gained contains the heterocycle non-natural amino acid polypeptides makes polypeptide with respect to the naturally occurring amino acid polypeptide of homology serum half-life increase.
Other or alternate embodiment are for detecting the method for the existence of polypeptide in patient's body, described method comprises throwing and comprises the polypeptide that at least one contains the heterocycle alpha-non-natural amino acid, and gained contains the heterocycle non-natural amino acid polypeptides makes polypeptide with respect to the naturally occurring amino acid polypeptide of homology prolongation circulation timei.
Other or alternate embodiment are for detecting the method for the existence of polypeptide in patient's body, described method comprises throwing and comprises the polypeptide that at least one contains the heterocycle alpha-non-natural amino acid, and gained contains the heterocycle non-natural amino acid polypeptides is regulated polypeptide with respect to the naturally occurring amino acid polypeptide of homology biologically active.
Other or alternate embodiment are for detecting the method for the existence of polypeptide in patient's body, described method comprises throwing and comprises the polypeptide that at least one contains the heterocycle alpha-non-natural amino acid, and gained contains the heterocycle non-natural amino acid polypeptides is regulated polypeptide with respect to the naturally occurring amino acid polypeptide of homology immunogenicity.
Should be appreciated that method and composition as herein described is not limited to ad hoc approach as herein described, scheme, cell-line, constructs body and reagent, and therefore can change.Should also be clear that term as used herein just for the purpose of describing specific embodiment, and do not plan to limit the scope of method and composition as herein described, its claim of will only being enclosed limits.
Unless indication done clearly in addition in context, otherwise as in this paper and the claim of enclosing use, singulative " " and " described " comprise a plurality of reference substances.
Unless define in addition, otherwise employed all scientific and technical terminologies of this paper have with one of ordinary skill in the art of the present invention as herein described and usually understand identical implication.Although can will be used for practice of the present invention as herein described or test, now only method for optimizing, device and material are described with method as herein described, device and materials similar or suitable any method, device and material.
All open cases that this paper is mentioned and patent all are that the mode of quoting in full is incorporated herein to reach description and to disclose the purpose of constructing body and method that may be used in combination with current described invention described in for example described open case.Open case discussed in this article only provides the disclosure before the application's case date of application.This paper where face in office all should not be construed as admits that date that inventor of the present invention haves no right to make described disclosure owing to existing invention or any other reason in advance.
Term " based on the key of aldehyde alcohol " or " based on the key that mixes aldehyde alcohol " are meant that a kind of carbonyls and another kind can be identical or the acid or the condensation of base catalysis type of the enolate/enol of carbonyls that can be inequality, and it is used to produce beta-hydroxy carbonyls (aldehyde alcohol).
As used herein, term " affinity labeling " be meant reversible or irreversibly in conjunction with another molecule with it is modified, with its destruction or with the mark of its formation compound.For instance, affinity labeling comprises enzyme and its substrate, perhaps antibody and its antigen.
Term " alkoxyl ", " alkyl amino " and " alkylthio group " (or thioalkoxy group) are to use with its conventional sense, and are meant the alkyl that is connected with molecule via oxygen atom, amino or sulphur atom respectively.
Unless otherwise mentioned, otherwise term " alkyl " itself or be meant to have appointment amount of carbon atom (that is C, as the part of another molecule meaning 1-C 10The meaning is 1 to 10 carbon) straight or branched or cyclic hydrocarbon group or its combination, it can be saturated fully, single or polyunsaturated and can comprise divalence and the multivalence group.The example of saturated hydrocarbyl includes but not limited to such as following group: the homologue of methyl, ethyl, n-pro-pyl, isopropyl, normal-butyl, the tert-butyl group, isobutyl group, sec-butyl, cyclohexyl, (cyclohexyl) methyl, cyclopropyl methyl, for example n-pentyl, n-hexyl, n-heptyl, n-octyl and isomeric compound etc.Unsaturated alkyl is the alkyl with one or more pairs key or triple bond.The example of unsaturated alkyl includes but not limited to vinyl, 2-acrylic, cyclobutenyl, 2-isopentene group, 2-(butadienyl), 2,4-pentadienyl, 3-(1, the 4-pentadienyl), acetenyl, 1-and 3-propinyl, 3-butynyl and higher homologue and isomeric compound.Unless otherwise mentioned, otherwise the alkyl derivative that this paper defines in more detail also planned to comprise in term " alkyl ", such as " assorted alkyl ", " alkylhalide group " and " homology alkyl ".
Term " alkylidene " itself or be meant divalent group derived from alkane, for example (CH as the part of another molecule meaning 2-) n, wherein n can be 1 to about 24.Only for instance, described group includes but not limited to have 10 or the group of carbon atom still less, such as structure-CH 2CH 2-and-CH 2CH 2CH 2CH 2-." low-carbon alkyl " or " low-carbon (LC) alkylidene " is for having 8 or the short alkyl or the alkylidene of chain of carbon atom still less usually.Unless otherwise mentioned, otherwise term " alkylidene " plans also to comprise that this paper is described as the group of " inferior assorted alkyl ".
Term " amino acid " is meant naturally occurring amino acid and alpha-non-natural amino acid, and amino acid analogue and amino acid analog thing to work with mode like the naturally occurring amino acids.Natural amino acids coding is 20 kinds of common amino acids (alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenyl alanine, proline, serine, threonine, tryptophan, tyrosine and valine) and pyrroles's lysine and selenocysteine.Amino acid analogue is meant the compound with Essential Chemistry structure identical with naturally occurring amino acid, and only for instance, α carbon combines with hydrogen, carboxyl, amino and R group.Described analog can have modified R group (for example, nor-leucine) or can have modified peptide main chain, still keeps the Essential Chemistry structure identical with naturally occurring amino acid simultaneously.The limiting examples of amino acid analogue comprises homoserine, nor-leucine, methionine sulfoxide, methionine methyl sulfonium.
The one-letter symbol that amino acid can be recommended with its title, its known usually trigram symbol or IUPAC-IUB biochemical nomenclature commission (Biochemical Nomenclature Commission) is in this article mentioned.In addition, nucleotide can be mentioned by its single-letter code of accepting usually.
" amino terminal modification group " is meant any molecule that can be connected with terminal amido.For instance, described terminal amido can be in the end of polymer molecule, and wherein said polymer molecule includes but not limited to polypeptide, polynucleotide and polysaccharide.End modified group includes but not limited to various water-soluble polymers, peptide or protein.Only for instance, end modified group comprises polyethylene glycol or seralbumin.End modified group can be used for changing the treatment feature of polymer molecule, includes but not limited to increase the serum half-life of peptide.
" antibody fragment " meaning is any form of the antibody except that the total length form.Antibody fragment herein comprises as the antibody than small component that is present in the full length antibody, and engineered antibody.Antibody fragment includes but not limited to Fv, Fc, Fab and (Fab ') 2, strand Fv (scFv), bifunctional antibody, three function antibodies, four function antibodies, bi-functional hybrid antibody, CDR1, CDR2, CDR3, CDR combination, variable region, framework region, constant region, heavy chain, light chain and variable region, and (Maynard ﹠amp such as alternative support non-antibody molecule, bispecific antibody; Georgiou, 2000, Annu.Rev.Biomed.Eng.2:339-76; Hudson, 1998, Curr.Opin.Biotechnol.9:395-402).Another functional structure is strand Fv (scFv), its 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 (Mr25,000) protein keep the specificity of antigen and compatibility usually in single polypeptide and the convenient structure unit (building block) of big antigentic specificity molecule can be provided.Unless do clearly indication in addition, otherwise use in the statement of term " antibody " and the claim particularly including " antibody fragment ".
As used herein, term " aromatic series " or " aryl " are meant a kind of closed-loop structure, and it has at least one and has the ring of conjugated pi electron system and comprise isocyclic aryl and heterocyclic aryl (or " heteroaryl " or " heteroaromatic ").Carbocyclic ring or heterocyclic aromatic group can contain 5 to 20 annular atomses.Described term comprises that monocycle, covalently bound or condensed ring encircle (that is the right ring of shared adjacent carbon atom) group more.Aromatic group can be unsubstituted or be substituted.The limiting examples of " aromatic series " or " aryl " comprises phenyl, 1-naphthyl, 2-naphthyl, 4-xenyl, anthryl and phenanthryl.The substituting group of each is selected from the substituent group that accepts as herein described in above-mentioned aryl and the heteroaryl ring system.
In brief, term " aromatic series " or " aryl " are when comprising as hereinbefore defined aryl and heteroaryl ring when being used in combination with other term (including but not limited to aryloxy group, fragrant sulphur oxygen base, aralkyl).Therefore, term " aralkyl " or " alkaryl " plan comprise the group (including but not limited to benzyl, phenethyl, pyridylmethyl etc.) that aryl is connected with alkyl, described alkyl comprises the alkyl of carbon atom (including but not limited to methylene) hetero atom (oxygen atom only for instance) displacement.The example of described aryl includes but not limited to phenoxymethyl, 2-pyridine radicals oxygen ylmethyl, 3-(1-naphthoxy) propyl group etc.
As used herein, term " arlydene " is meant divalent aryl.The limiting examples of " arlydene " comprises phenylene, inferior pyridine radicals, inferior pyrimidine radicals and inferior thienyl.The substituting group of arlydene is selected from the substituent group that accepts as herein described.
" double functional copolymer " (being also referred to as " difunctionality connect base ") be meant comprise two can with the other parts specific reaction to form the covalently or non-covalently polymer of the functional group of key.Described part can include but not limited to natural or alpha-non-natural amino acid or contain side group on the peptide of described natural or alpha-non-natural amino acid.Only for instance, difunctionality connect base can have can with the functional group of radical reaction on first peptide and can with another functional group of radical reaction on second peptide, comprise that first peptide, difunctionality connect the concatenator of the base and second peptide thereby form.Known many programs that all cpds is connected with peptide be connected basic molecule.For example referring to No. the 188th, 256, 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, the mode that described patent is quoted in full is incorporated herein." polyfunctional poly compound " (being also referred to as " multifunctional linkers ") be meant comprise two or more can with the polymer of the functional group of other parts reactions.Described part can include but not limited to be used to form the natural or alpha-non-natural amino acid of key covalently or non-covalently or contain side group (including but not limited to amino acid side group) on the peptide of described natural or alpha-non-natural amino acid.Double functional copolymer or polyfunctional poly compound can have any Len req or molecular weight, and provide specific required interval or conformation between molecule that can be through selecting to combine with its institute with one or more molecules that are being connected to compound or the compound.
As used herein, term " biological usability " is meant that material or its active part are from pharmaceutical dosage form transmission and speed that becomes available at site of action or in systemic circulation and degree.The biological usability increase is meant the increase of speed and degree that material or its active part become available from the pharmaceutical dosage form transmission and at site of action or in systemic circulation.For instance, the increase of biological usability can be indicated by the increase of material or the concentration of its active part in blood when with other material or active part comparison.The limiting examples that is used for assessing the method that biological usability increases is provided in example 21-25.The method can be used for assessing the biological usability of any polypeptide.
Term " bioactive molecule ", " biologically-active moiety " or " bioactivator " look like when being used for herein and are meant any material that can influence biosystem, path, molecule or interactional any physics or the biochemical characteristic relevant with organism (including but not limited to virus, bacterium, phage, transposons, prion, insect, fungi, plant, animal and human's class).Specifically, as used herein, bioactive molecule includes but not limited to expect and is used to diagnose, cure, alleviate, treat or prevents the disease of human or other animal or otherwise strengthen the health of the mankind or animal or any material of mental health conditions.The example of bioactive molecule includes but not limited to peptide, protein, enzyme, small-molecule drug, hard medicine, soft medicine, carbohydrate, inorganic atoms or molecule, dyestuff, lipid, nucleosides, radionuclide, oligonucleotide, toxin, cell, virus, liposome, particulate and micella.The kind that is applicable to the bioactivator of method and composition as herein described includes but not limited to medicine, prodrug, radionuclide, developer, polymer, antibiotic, fungicide, antivirotic, antiphlogistic, antitumor agent, cardiovascular agents, antianxiety agent, hormone, growth factor, steroid dose, microbe-derived toxin etc.
" adjusting biologically active " meaning refer to increase or reduces reactivity, the change polypeptide of polypeptide selectivity, strengthen or weaken the substrate selective of polypeptide.Can be undertaken through changing bioactive analysis by the biologically active of non-natural polypeptide is compared with the biologically active of natural polypeptides.
As used herein, term " biomaterial " is meant the material of biogenetic derivation, includes but not limited to the material by bio-reactor and/or recombination method and technology acquisition.
As used herein, term " biophysics probe " is meant and can detects or monitor the probe that molecular structure changes.Described molecule includes but not limited to protein, and " biophysics probe " can be used for detecting or monitoring protein and other macromolecular interaction.But the example of biophysics probe includes but not limited to the group of spin labeling, fluorogen and photoactivation.
As used herein, term " passes through biological synthesis method " and is meant any method of utilizing translation system (cell or acellular), comprises using at least a following assembly: polynucleotide, codon, tRNA and ribosome.For instance, can use method and the technology described in this paper " in vivo generation comprises the polypeptide of alpha-non-natural amino acid " and the limiting examples 20 that alpha-non-natural amino acid " is incorporated into by biological synthesis method " in the non-natural amino acid polypeptides.In addition, the selection method that can " incorporate " the useful alpha-non-natural amino acid in the non-natural amino acid polypeptides by biological synthesis method into is described in the limiting examples 20.
As used herein, term " vitamin h analog " (perhaps being also referred to as " vitamin h analogies ") is any molecule except that vitamin h, and it combines with avidin and/or streptavidin with high-affinity.
As used herein, term " carbonyl " be meant contain be selected from by-C (O)-,-S (O)-,-S (O) 2-and-group of the part of the group of C (S)-composition, include but not limited to contain the group of 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.Described carbonyl comprises ketone, aldehyde, carboxylic acid, ester and thioesters.In addition, described group can be the part of straight chain, side chain or ring molecule.
Term " carboxyl terminal modification group " is meant any molecule that can be connected with terminal carboxyl group.For instance, described terminal carboxyl group can be in the end of polymer molecule, and wherein said polymer molecule includes but not limited to polypeptide, polynucleotide and polysaccharide.End modified group includes but not limited to various water-soluble polymers, peptide or protein.Only for instance, end modified group comprises polyethylene glycol or seralbumin.End modified group can be used for changing the treatment feature of polymer molecule, includes but not limited to increase the serum half-life of peptide.
As used herein, term " but group of chemical cracking " (being also referred to as " chemically unstable " group) is meant the group of when being exposed to acid, alkali, oxidant, reductant, chemical initiator or radiation initiators fracture or cracking.
As used herein, term " chemiluminescent groups " is meant under situation about not heating because of the luminous group of chemical reaction.Only for instance, luminol (luminol) (5-amino-2,3-dihydro-1,4-phthalazine diketone) with as hydrogen peroxide (H 2O 2) oxidant under the situation that has alkali and metallic catalyst, react with produce the excitation state product (3-aminophthalic acid ester, 3-APA).
As used herein, term " chromophore " is meant the molecule of the light that absorbs visible wavelength, UV wavelength or IR wavelength.
As used herein, term " co-factor " is meant vital atom of macromolecular effect or molecule.Co-factor includes but not limited to some other factors that inorganic ions, coenzyme, protein or enzymic activity are essential.Example comprises the metal ion of ferroheme, the magnesium in the chlorophyll and protein in the hemochrome.
As used herein, " altogether folding " is meant folding process again, reaction or the method for at least two molecules of use, and described molecule is interact with each other and cause launching or incorrect folding molecule changes into suitably folding molecule.Only for instance, " altogether folding " uses at least two polypeptide, and it is interact with each other and cause launching or incorrect folding polypeptide changes into natural suitably folding polypeptide.Described polypeptide can contain natural amino acid and/or at least one alpha-non-natural amino acid.
As used herein, " comparison window " is meant and is used for any one section of close position that the sequence with close position of equal number is compared with reference sequences after two sequence of the best comparison.Described close position include but not limited to by about 20 to about 600 sequential cells, comprise about 50 to about 200 sequential cells and about 100 to about 150 groups that sequential cells is formed.Only for instance, the polypeptide that described sequence comprises polypeptide and contains alpha-non-natural amino acid, wherein sequential cells includes but not limited to natural and alpha-non-natural amino acid.In addition, only for instance, described sequence comprises polynucleotide, and wherein nucleotide is corresponding sequential cells.Affiliated field is used for the comparison method of the sequence of comparison as everyone knows.Can be by local homology's algorithm of (including but not limited to) Smith and Waterman (1970) Adv.Appl.Math.2:482c; The autoploidy 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; These algorithms computer-implemented (GAP, BESTFIT, FASTA and TFASTA among the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI); Perhaps artificial comparison and range estimation (for example referring to people such as Ausubel, Current Protocols in Molecular Biology (1995 [)) supply the best comparison of sequence relatively.
For instance, the algorithm that can be used for measuring sequence homogeneity and sequence similarity percentage is BLAST and BLAST2.0 algorithm, and it is described in people (1990) J.Mol.Biol.215:403-410 such as people such as Altschul (1997) Nuc.Acids Res.25:3389-3402 and Altschul respectively.Carrying out software that BLAST analyzes, can to pass through U.S. biotechnology information centre (National Center for Biotechnology Information) public available.The sensitivity and the speed of BLAST algorithm parameter W, T and X decision comparison.BLASTN program (being used for nucleotide sequence) is used following default parameters: word length (W) is 11, and expected value (E) is 10, M=5, and N=-4 also compares two chains.For amino acid sequence, the BLASTP program is used following default parameters: word length be 3 and expected value (E) be 10, score matrix (referring to Henikoff and Henikoff (1992) Proc.Natl with BLOSUM62, Acad.Sci.USA 89:10915), comparison value (B) is 50, expected value (E) is 10, M=5, two chains of N=-4 and comparison.When carrying out the BLAST algorithm, close " low complex degree " screening sequence usually.
The BLAST algorithm also carries out the statistical analysis (for example referring to Karlin and Altschul (1993) Proc.Natl.Acad.Sci.USA 90:5873-5787) of similitude between two sequences.A kind of similarity measurement method that the BLAST algorithm is provided is minimum summation probability (P (N)), and it provides the indication to the probability that will take place accidentally between two nucleotide or the amino acid sequence to mate.For instance, if minimum summation probability thinks so that less than about 0.2 or less than about 0.01 or less than about 0.001 nucleic acid and reference sequences are similar in the comparison of test nucleic acid and reference nucleic acid.
Term " conservative modify variant " be applicable to natural and alpha-non-natural amino acid and natural and non-natural nucleotide sequence with and make up.With regard to specific nucleic acid sequence, " the conservative variant of modifying " is meant the natural and non-natural nucleic acid of the natural and alpha-non-natural amino acid sequence that coding is identical or substantially the same, perhaps do not encode natural and during the alpha-non-natural amino acid sequence, refer to substantially the same sequence yet when natural and non-natural nucleic acid.For instance, because the degeneracy of genetic code, a large amount of identical nucleic acid of function any appointment protein of will encoding.For instance, codon GCA, GCC, GCG and GCU coded amino acid alanine all.Therefore, in each position of codon appointment alanine, codon can become any corresponding codon under the situation that does not change coded polypeptide.Described nucleic acid variation is " silent variant (silent variation) ", and it is a kind of for conservative modification variation.Therefore, for instance, the natural or non-natural nucleotide sequence of herein each natural or the non-natural polypeptide of encoding is also described each possible silent variant of natural or non-natural nucleic acid.One of ordinary skill in the art will recognize, each codon in the natural or non-natural nucleic acid TGG of the AUG of unique password of methionine and unique password that is generally tryptophan (be generally except) can be modified to obtain the identical molecule of function.Therefore, each silent variant of the natural and non-natural nucleic acid of the natural and non-natural polypeptide of coding is intrinsic in each described sequence.
For amino acid sequence, change, add or lack single natural and alpha-non-natural amino acid or the medium and small percentage of encoded sequence natural and alpha-non-natural amino acid nucleic acid, peptide, polypeptide or protein sequence indivedual replacements, lack or be added to " conservative modify variation ", wherein said change cause amino acid whose disappearance, amino acid whose interpolation or chemically similarly amino acid to the replacement of natural and alpha-non-natural amino acid.Affiliated field provides the conservative replacement table of functionally similar natural amino acid as everyone knows.Described conservative modification variant is except that for the polymorphic variant and do not get rid of polymorphic variant, also is homologue and allelomorph between the kind of methods described herein and composition.
One of ordinary skill in the art are known to provide intimate amino acid whose conservative replacement table.Below eight groups contain the conservative each other each other amino acid that replaces separately:
1) alanine (A), glycine (G)
2) aspartic acid (D), glutamic acid (E);
3) asparagine (N), glutamine (Q);
4) arginine (R), lysine (K);
5) isoleucine (I), leucine (L), methionine (M), valine (V);
6) phenyl alanine (F), tyrosine (Y), tryptophan (W);
7) serine (S), threonine (T); With
8) cysteine (C), methionine (M),
(for example referring to Creighton, Proteins:Structures and Molecular Properties (W H Freeman ﹠amp; Co.; The 2nd edition (in December, 1993)).
Unless otherwise mentioned, otherwise term " cycloalkyl " and " Heterocyclylalkyl " itself or represent the annular form of " alkyl " and " alkyl of mixing " during with the combination of other term respectively.Therefore, cycloalkyl or Heterocyclylalkyl comprise saturated, part is unsaturated and complete undersaturated ring key.In addition, for Heterocyclylalkyl, hetero atom can occupy the position that heterocycle is connected with the remainder of molecule.Hetero atom can include but not limited to oxygen, nitrogen or sulphur.The example of cycloalkyl includes but not limited to cyclopenta, cyclohexyl, 1-cyclohexenyl group, 3-cyclohexenyl group, suberyl etc.The example of Heterocyclylalkyl includes but not limited to 1-(1,2,5,6-tetrahydro pyridyl), 1-piperidyl, 2-piperidyl, 3-piperidyl, 4-morpholinyl, morpholinyl, oxolane-2-base, oxolane-3-base, thiophane-2-base, thiophane-3-base, 1-piperazinyl, 2-piperazinyl etc.In addition, multiring structure contained in described term, includes but not limited to dicyclo and tricyclic structure.Similarly, term " inferior Heterocyclylalkyl " itself or be meant divalent group, and term " cycloalkylidene " itself or look like as the part of another molecule and to be meant divalent group derived from cycloalkyl derived from Heterocyclylalkyl as the part of another molecule meaning.
As used herein, term " cyclodextrin " is meant in ring formation process by at least 6 cyclic carbohydrates of forming to 8 glucose molecules.The Outboard Sections of ring contains water soluble group; Encircling the center for can hold micromolecular nonpolar relatively chamber.
As used herein, term " cytotoxicity " is meant the compound of harm cell.
As used herein, " denaturant " is meant any compound or the material that will cause that reversible polymer launches.Only for instance, " denaturant " can cause the protein reversible expansion.The intensity of denaturant will be by the characteristic and the concentration decision of specific denaturant.For instance, denaturant includes but not limited to chaotropic agent, cleaning agent, water miscibility organic solvent, phosphatide or its combination.The limiting examples of chaotropic agent includes but not limited to urea, guanidine and thiocyanic acid sodium.The limiting examples of cleaning agent (for example can include but not limited to strong cleaning agent (such as lauryl sodium sulfate or APEO); Tween or Triton cleaning agent); sodium lauroyl sarcosine (Sarkosyl); gentle nonionic detergent (for example; digitonin); gentle cationic detergent (such as; N-＞2; 3-(dioleoyl oxygen base)-propyl group-N; N; the N-trimethyl ammonium); gentle ion cleaning agent (for example, sodium taurocholate or deoxysodium cholate) or amphion cleaning agent (include but not limited to DMPT (amphion cleaning agent (Zwittergent)); 3-(3-courage amidopropyl) dimethylamino-1-propane sulphate (CHAPS) and 3-(3-courage amidopropyl) dimethylamino-2-hydroxyl-1-propane sulfonate (CHAPSO)).The limiting examples of water miscibility organic solvent includes but not limited to acetonitrile, low-carbon (LC) alkanol (C especially 2-C 4Alkanol is such as ethanol or isopropyl alcohol) or lower alkanes glycol (C 2-C 4The alkane glycol is such as ethylene glycol) can be used as denaturant.The limiting examples of phosphatide includes but not limited to naturally occurring phosphatide, such as phosphatidyl-ethanolamine, phosphatid ylcholine, phosphatidylserine and phosphatidylinositols, or synthetic phospholipid derivative or variant, such as two hexanoyl phosphatid ylcholine or Diheptanoylphosphatidylcholines.
As used herein, term " required functional group " is meant any group that is selected from following each thing: mark, dyestuff, polymer, water-soluble polymer, polyethyleneglycol derivative, photocrosslinking agent, cytotoxic compound, medicine, affinity labeling, photoaffinity labeling, reactive compounds, resin, second protein or polypeptide or polypeptide analog, antibody or antibody fragment, metal-chelator, co-factor, fatty acid, carbohydrate, polynucleotide, DNA, RNA, antisense polynucleotide, sugar, water-soluble dendron shaped polymer, cyclodextrin, biomaterial, nano particle, spin labeling, fluorogen, the containing metal part, radioactive segment, novel functional group, with the covalently or non-covalently interactional group of other molecule, the light cage covers part, but actinic radiation excitation portion, part, but photoisomerization part, vitamin h, the vitamin h analog, and the part of heavy atom arranged, but chemical cracking group, but photodestruciton group, the side chain that prolongs, carbon connects sugar, redox active agent, amino thio-acid, toxic moiety, isotope-labeled part, the biophysics probe, the phosphorescence group, chemiluminescent groups, the electron dense group, the magnetic group, insert group, chromophore, energy transfer agent, bioactivator (in the case, bioactivator can comprise the reagent with therapeutic activity, and non-natural amino acid polypeptides or modified alpha-non-natural amino acid can with the therapeutic agent that is connected serve as auxiliary therapeutical agent or as the mode that therapeutic agent is delivered to the desired area in the organism), detectable label, little molecule, inhibition ribonucleic acid, the radioactive nucleus thuja acid, the neutron capture agent, biotin derivative, quantum dot, the nanometer mediator, the radioactivity mediator, abzyme, active composite activating agent, virus, adjuvant, aglycon, anaphylactogen, angiostatin, antihormones, antioxidant, fit, guide RNA, saponarin, shuttle vector, big molecule, mimic epitope, acceptor, reverse micelle, with its any combination.
As used herein, term " diamines " is meant the group/molecule that comprises at least two amine functional groups, includes but not limited to diazanyl, amidino groups, imido grpup, 1,1-two amidos, 1,2-two amidos, 1,3-two amidos and 1,4-two amidos.In addition, described group can be the part of straight chain, side chain or ring molecule.
As used herein, term " detectable label " but be meant the mark that the operational analysis technology is observed, described analytical technology includes but not limited to fluorescence, chemiluminescence, electron spin resonnance, ultraviolet absorption spectrum, mass spectrum, nuclear magnetic resonnance, magnetic resonance and electrochemical method.
As used herein, term " dicarbapentaborane " be meant 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, include 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.Described dicarbapentaborane comprises diketone, keto-aldehyde, ketone acid, ketone ester and ketone thioesters.In addition, described group can be the part of straight chain, side chain or ring molecule.Two parts in the dicarbapentaborane can be identical or different, and can comprise at any place of two parts and will produce the substituting group of (only for instance) ester, ketone, aldehyde, thioesters or acid amides.
As used herein, term " medicine " be meant be used to prevent, diagnose, relax, any material of treatment or cure diseases or symptom.
As used herein, term " dyestuff " is meant and contains chromophoric solubility coloring material.
As used herein, term " effective dose " be meant throw and medicament or the sufficient quantity of compound, it will alleviate the disease of being treated or one or more symptoms of symptom to a certain extent.The result can be the alleviating and/or relax perhaps any other required change of biosystem of symptom, symptom or the cause of disease of disease.For instance, throw and medicament or compound include but not limited to natural amino acid polypeptide, non-natural amino acid polypeptides, modified natural amino acid polypeptide or modified non-natural amino acid polypeptides.Can throw with the composition that contains described natural amino acid polypeptide, non-natural amino acid polypeptides, modified natural amino acid polypeptide or modified non-natural amino acid polypeptides for preventative, enhancing property and/or therapeutic treatment.Suitable " effectively " amount in any individual cases can be used such as the technology of dose escalation study and determine.
As used herein, term " electron dense group " is meant the group of scattered electron when shining with electron beam.Described group includes but not limited to ammonium molybdate, basic bismuth nitrate, cadmium iodide (99%), carbohydrazide, six Ferric Chloride Hydrateds, hexa (98.5%), anhydrous indium chloride, lanthanum nitrate, three hydration lead acetates, three hydration lead citrates, plumbi nitras, periodic acid, phosphomolybdic acid, phosphotungstic acid, the potassium ferricyanide, potassium ferrocyanide, ammoniated ruthenium oxychloride, silver nitrate, protargin (silverproteinate) (Ag calibrating: 8.0-8.5%) " by force ", tetraphenylporphines silver (S-TPPS), sodium chloraurate, sodium tungstate, thallium nitrate, thiosemicarbazides (TSC), uranyl acetate, uranyl nitrate and vanadic sulfate.
As used herein, term " energy transfer agent " is meant can be provided energy or accept the molecule of energy from another molecule to another molecule.Only for instance, FRET (fluorescence resonance energy transfer) (FRET) is a dipole-dipole coupling process, transfer to unexcited acceptor molecule, fluorescence ground is with the longer wavelength emission energy that provided subsequently excited energy non-radiation type by described process fluorescence donor molecule.
Term " enhancing " meaning is increase or prolongs the effect or the duration of required effect.For instance, the effect of " enhancing " therapeutic agent is meant the effect of therapeutic agent effect during the treatment that increases or prolong disease, illness or symptom or the ability of duration.As used herein, " enhancing effective dose " is meant the amount of the effect of therapeutic agent in the treatment that is enough to strengthen disease, illness or symptom.When being used for the patient, the effective dose that is used for this purposes will be decided on the order of severity of disease, illness or symptom and the course of disease, previous therapy, patient's health status with to the reaction of medicine and the doctor in charge's judgement.
As used herein, term " eucaryote " is meant the organism that belongs to system generation eucaryon field, includes but not limited to animal (including but not limited to mammal, insect, reptile, birds etc.), infusorian, plant (including but not limited to monocotyledon, dicotyledon and algae), fungi, yeast, flagellate, microsporozoite and protist.
As used herein, term " fatty acid " " be meant carboxylic acid with about C6 or longer hydrocarbon side chain.
As used herein, term " fluorogen " is meant and is exciting back emission photon and fluorescigenic thus molecule.
As used herein, term " functional group ", " active part ", " activated group ", " leaving group ", " reactive site ", " chemically reactive group " and " chemical reactivity part " are meant the part or the unit of molecule generation chemical reaction.Described term in chemical field in synonym to a certain extent, and be used in this article indicate carry out certain function or active and can with the part of the molecule of other molecular reaction.
Term " halogen " comprises fluorine, chlorine, iodine and bromine.
As used herein, term " halogen acyl group " is meant the acyl group that contains the halogen part, includes but not limited to-C (O) CH 3,-C (O) CF 3,-C (O) CH 2OCH 3Deng.
As used herein, term " alkylhalide group " is meant the alkyl that contains the halogen part, includes but not limited to-CF 3With-CH 2CF 3Deng.
As used herein, term " assorted alkyl " is meant straight or branched or cyclic hydrocarbon group or its combination, it is made up of the hetero atom that alkyl and at least one are selected from the group that is made up of O, N, Si and S, and wherein nitrogen and sulphur atom can according to circumstances can be according to circumstances through quaternized through oxidation and nitrogen heteroatom.Hetero atom O, N can be positioned at any interior location place of assorted alkyl or the position that alkyl is connected with the molecule remainder with S and Si.Example includes but not limited to-CH 2-CH 2-OCH 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 3With-CH=CH-N (CH 3)-CH 3In addition, at the most two hetero atoms can be continuous, such as-CH 2-NH-OCH 3With-CH 2-O-Si (CH 3) 3
Term " based on the key of heterocycle " or " heterocyclic bond " are meant by dicarbapentaborane and the formed part of diamines radical reaction.The gained product is a heterocycle, comprises heteroaryl or Heterocyclylalkyl.The gained heterocyclic radical serves as alpha-non-natural amino acid or non-natural amino acid polypeptides and is connected with chemistry between another functional group.In one embodiment, heterocyclic bond comprises the nitrogen heterocyclic ring key, only for instance, comprises pyrazoles key, pyrroles's key, indoles key, benzodiazepine key and pyrazoline ketonic bond.
Similarly, term " inferior assorted alkyl " is meant the divalent group derived from assorted alkyl, for example (but being not limited to)-CH 2-CH 2-S-CH 2-CH 2-and-CH 2-S-CH 2-CH 2-NH-CH 2-.For the assorted alkyl in Asia, identical or different hetero atom also can occupy any or two chain ends (including but not limited to alkylidene oxygen base, alkylenedioxy group, alkylidene amino, alkylidene diaminourea, aminooxy group alkylidene etc.).In addition, for alkylidene and inferior assorted alkyl linking group, the direction that the chemical formula of linking group is write does not hint the orientation of linking group.For instance, formula-C (O) 2R '-expression-C (O) 2R '-and-R ' C (O) 2-.
As used herein, term " heteroaryl " or " heteroaromatic " are meant and contain the heteroatomic aryl that at least one is selected from N, O and S; Wherein nitrogen and sulphur atom can according to circumstances can be through quaternized through oxidation and nitrogen-atoms.Heteroaryl can be substituted or be unsubstituted.Heteroaryl can be connected with the remainder of molecule by hetero atom.The limiting examples of heteroaryl comprises the 1-pyrrole radicals, the 2-pyrrole radicals, the 3-pyrrole radicals, the 3-pyrazolyl, the 2-imidazole radicals, the 4-imidazole radicals, 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-pyridine radicals, the 3-pyridine radicals, the 4-pyridine radicals, the 2-pyrimidine radicals, the 4-pyrimidine radicals, 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.
As used herein, term " homology alkyl " is meant the alkyl into alkyl.
As used herein, term " unanimity " is meant that two or more sequences or subsequence are identical.In addition, as used herein, term " in fact consistent " is meant when using comparison algorithm or comparing on the zone of comparison window or specified measurement and when comparing maximum correspondence, two or more have the sequence of identical sequential cells percentage by manual comparison and range estimation.Only for instance, if about 60% unanimity of the sequential cells in the specific region, about 65% unanimity, about 70% unanimity, about 75% unanimity, about 80% unanimity, about 85% unanimity, about 90% consistent or about 95% unanimity, two or more sequences can " consistent in fact " so.Described percentage is used to describe " the uniformity percentage " of two or more sequences.The uniformity of sequence can be present at least about the long zone of 75-100 sequential cells, long zone or (when the not specifying) whole sequence of about 50 sequential cells.This definition also is meant the complementarity of cycle tests.Only for instance, when amino acid residue is identical, two or more peptide sequence unanimities; And if about 60% unanimity of the amino acid residue in the appointed area, about 65% unanimity, about 70% unanimity, about 75% unanimity, about 80% unanimity, about 85% unanimity, about 90% consistent or about 95% unanimity, two or more peptide sequences " consistent in fact " so.Uniformity can be present at least about the zone of 75 zones to about 100 amino acid longs, about 50 amino acid longs or the whole sequence of (when not specifying) peptide sequence.In addition, only for instance, when the nucleic acid residue is identical, two or more polynucleotide consensus sequences; And if about 60% unanimity of the nucleic acid residue in the appointed area, about 65% unanimity, about 70% unanimity, about 75% unanimity, about 80% unanimity, about 85% unanimity, about 90% consistent or about 95% unanimity, two or more polynucleotide sequences " consistent in fact " so.Uniformity can be present at least about 75 to the long zone of about 100 nucleic acid, the zone of about 50 nucleic acid length or the whole sequence of (when not specifying) polynucleotide sequence.
For sequence relatively, a common sequence is served as the reference sequences of comparing with cycle tests.When using sequence comparison algorithm, in cycle tests and reference sequences input calculator, specify the subsequence coordinate in case of necessity, and specified sequence algorithm routine parameter.The default program parameter can be used, perhaps alternate parameter can be specified.Sequence comparison algorithm calculates the sequence identity percentage of cycle tests with respect to reference sequences based on program parameter subsequently.
As used herein, term " immunogenicity " is meant the antibody response of throwing with medicine.Can use for anti-non-natural amino acid polypeptides antibody in the biological fluid quantitatively and qualitative test obtain immunogenicity to the therapeutic non-natural amino acid polypeptides.Described calibrating includes but not limited to radioimmunoassay (RIA), enzyme-linked immunosorbent calibrating (ELISA), electrochemiluminescent immunoassay calibrating (LIA) and fluorescence immunoassay calibrating (FIA).To the immunogenic analysis of therapeutic non-natural amino acid polypeptides relate to throw with the therapeutic non-natural amino acid polypeptides after antibody response compare with the antibody response behind the therapeutic natural amino acid polypeptide with throwing.
As used herein, term " insertion agent " (being also referred to as " insertion group ") is meant the molecule internal clearance that can insert molecule or the chemicals in the intermolecular gap between the molecule.Only for instance, insert agent or group and can be the molecule in the base of piling up that inserts the dna double spiral.
As used herein, term " through separate " is to instigate the component of being paid close attention to separate with the non-component of paying close attention to and the component of being paid close attention to is removed from the non-component of paying close attention to.Can be drying regime or partial desiccation state through separate substance, perhaps be the form of solution (including but not limited to the aqueous solution).Can be the homogeneous attitude through separation component, or can be the part of medical composition through separation component, described medical composition comprises extra pharmaceutically acceptable supporting agent and/or excipient.But operational analysis chemical technology (including but not limited to polyacrylamide gel electrophoresis or high performance liquid chromatography) is measured purity and homogenieity.In addition, be in the preparation during existing main matter when separating component paid close attention to and the component of being paid close attention to, this paper is described as component purified in fact.As used herein, that term " purified " can refer to is at least 85% pure, at least 90% pure, at least 95% pure, at least 99% pure or purer component of being paid close attention to.Only for instance, when nucleic acid or protein do not have at least some under its native state with the cellular component of its association or nucleic acid or protein compression have been reduced to and are higher than it in vivo or during the degree of the concentration of in vitro making, described nucleic acid or protein be " through separation ".Equally, for instance, when from when open reading frame that gene side joint and coding remove the extragenic protein of being paid close attention to separates, gene is separated.
As used herein, term " mark " is meant to incorporate in the compound and be easy to and detects, and its physical distribution can be after testing and/or the material of monitoring whereby.
As used herein, term " key " is meant bond or the chemical part that forms by connecting basic functional group and the chemical reaction between another molecule.Described bond can include but not limited to covalent bond and non-covalent bond, and described chemical part can include but not limited to ester, carbonic ester, imine phosphate acid esters, hydrazone, acetal, ortho esters, peptide bond and oligonucleotides key.The key of the hydrolysis-stable meaning is meant that described key is stable in fact and (including but not limited under physiological conditions) under the useful pH value in one period long period that (possible even indefinite duration) do not react with water in water.The hydrolytically unstable or the degradable key meaning are that described key can be degraded in the water or the aqueous solution (comprising for example blood).Unstable or the degradable key meaning of enzymatic is that described key can be by one or more enzyme degradeds.Only for instance, PEG and related polymer can be in main polymer chain or one or more functional end-groups of main polymer chain and polymer molecule between be connected and comprise degradable linkage in the base.Described degradable linkage includes but not limited to the ester bond that formed by the pure radical reaction on PEG carboxylic acid or active PEG carboxylic acid and the bioactivator, wherein said ester group usually under physiological conditions hydrolysis with release bioactive agent.Other hydrolysis degradable linkage includes but not limited to carbonic acid ester bond; Imine linkage by amine and aldehyde reaction generation; By the phosphoric acid ester bond of alcohol with phosphate reaction formation; Hydrazone key as the product of hydrazides and aldehyde; As the acetal bonds of aldehyde with the product of alcohol; As the original acid ester key of formates with the product of alcohol; The peptide bond that forms by the carboxyl of terminal amido of (including but not limited to) polymer (such as PEG) and peptide; And the oligonucleotides key that forms by 5 ' hydroxyl of the phosphoramidite (phosphoramidite) of (including but not limited to) polymer ends base and oligonucleotides.
As used herein, term " medium " is meant and is used to grow and gathers cell and/or described cell is expressed and/or any medium of the product of secretion.Described " medium " includes but not limited to solution, solid, semisolid or rigid support thing, it can support or hold any host cell, for example comprises bacterial host cell, yeast host cell, insect host cell, plant host cell, eucaryote host cell, mammalian host cell, Chinese hamster ovary celI, prokaryotes host cell, Escherichia coli (E.coli) or pseudomonad (Pseudomonas) host cell and cellular content.Described " medium " include but not limited to grow host cell, medium of secrete polypeptide comprises the medium before or after the propagation step.Described " medium " also includes but not limited to contain the buffer solution of host cell lysate (for example, the polypeptide that produces in the cell) or reagent and host cell through dissolving or break to discharge polypeptide.
As used herein, term " metabolite " is meant 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 described 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 " are meant 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 described 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 " is meant that organism changes the general designation of the process of predetermined substance.Described process includes but not limited to hydrolysis and enzymatic reaction.The out of Memory of relevant metabolism can be from The PharmacologicalBasis of Therapeutics, and the 9th edition, McGraw-Hill (1996) obtains.Only for instance, the metabolite of natural amino acid polypeptide, non-natural amino acid polypeptides, modified natural amino acid polypeptide or modified non-natural amino acid polypeptides can be differentiated in the following manner: the host is thrown with natural amino acid polypeptide, non-natural amino acid polypeptides, modified natural amino acid polypeptide or modified non-natural amino acid polypeptides and analyzes tissue samples from the host; Perhaps cultivate natural amino acid polypeptide, non-natural amino acid polypeptides, modified natural amino acid polypeptide or modified non-natural amino acid polypeptides and liver cell in vitro and analyze the gained compound.
As used herein, term " metal-chelator " is meant the molecule that forms metal complex with metal ion.For instance, described molecule can form two or more coordinate bonds and can form ring structure with central metallic ions.
As used herein, term " containing metal part " is meant the group that contains metal ion, atom or particle.Described part include 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, the term part of heavy atom " and have " is meant and has usually the group of the ion of the atom heavier than carbon.Described ion or atom include but not limited to silicon, tungsten, gold, lead and uranium.
As used herein, term " modified " is meant the change of existence to natural amino acid, alpha-non-natural amino acid, natural amino acid polypeptide or non-natural amino acid polypeptides.Can pass through the synthetic back of natural amino acid, alpha-non-natural amino acid, natural amino acid polypeptide or non-natural amino acid polypeptides and modify, perhaps common translation modification or the posttranslational modification by natural amino acid, alpha-non-natural amino acid, natural amino acid polypeptide or non-natural amino acid polypeptides obtains described change or modification.Form " (the modified) " meaning refers to that natural amino acid, alpha-non-natural amino acid, natural amino acid polypeptide or the non-natural amino acid polypeptides discussed are modified according to circumstances, that is to say that 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 " is meant that the circulating half-life of modified bioactive molecule changes with respect to its plus or minus without modified forms.For instance, modified bioactive molecule includes but not limited to natural amino acid, alpha-non-natural amino acid, natural amino acid polypeptide or non-natural amino acid polypeptides.For instance, obtain blood sample during by each time point behind throwing and bioactive molecule or modified bioactive molecule, and the concentration of measuring molecule described in each sample is measured serum half-life.The correlation of serum-concentration and time allows to calculate serum half-life.For instance, through regulating the serum half-life that serum half-life can be increase, it can be facilitated improved dosage regimen or avoid toxic action.Described increase in the serum can be at least about 2 times, at least about 3 times, at least about 5 times or at least about 10 times.The limiting examples of the method that the assessment serum half-life increases is provided in the example 33.The method can be used for assessing the serum half-life of any polypeptide.
As used herein, term " the treatment half life period through regulating " is meant that the half life period of the modified bioactive molecule of treatment effective dose changes with respect to its plus or minus without modified forms.For instance, modified bioactive molecule includes but not limited to natural amino acid, alpha-non-natural amino acid, natural amino acid polypeptide or non-natural amino acid polypeptides.The pharmacokinetics and/or the pharmacodynamic profiles of molecule are measured the treatment half life period during for instance, by each time point of measurement dispensing back.The treatment half life period that increases can be facilitated useful especially dosage regimen, useful especially accumulated dose or be avoided undesirable effect.For instance, can be by increasing effect, increase or reducing the combining of modified molecule and its target, increase or reduce another parameter or without the mechanism of action or the increase of decorating molecule or reduce enzyme (protease only for instance) degraded of molecule is caused the increase for the treatment of the half life period.The limiting examples of the method that the assessment treatment half life period increases is provided in the example 33.The method can be used for assessing the treatment half life period of any polypeptide.
As used herein, term " nano particle " is meant that particle diameter is between the particle of about 500nm between about 1nm.
As used herein, term " near-stoichiometric " is meant that the ratio of the molal quantity of the compound that participates in chemical reaction is about 0.75 to about 1.5.
As used herein, term " non-eucaryote " is meant non-eucaryon organism.For instance, non-eucaryon organism can belong to fungal systems the territory takes place, and includes but not limited to Escherichia coli (Escherichia coli), extreme thermophilic bacteria (Thermus thermophilics) or bacillus stearothermophilus (Bacillus stearothermophilus), Pseudomonas fluorescence (Pseudomonas fluoresceins), pseudomonas aeruginosa (Pseudomonas aeruginosa), pseudomonas putida (Pseudomonas putida); Or ancient fungus strain system generation territory, include but not limited to Methanococcus jannaschii (Methanococcus jannaschii), thermophilic autotrophy methagen (Methanobacteriumthermoautotrophicum), super hyperthermophilic archaeon strain (Archaeoglobus fulgidus), strong thermophilic coccus (Pyrococcusfuriosus), hole is got over fireball bacterium (Pyrococcus horikoshii), thermophilic spring is given birth to archeobacteria (Aeuropyrum pernix) or Halophiles (Halobacterium) (having a liking for richly endowed bacterium of salt (Haloferax volcanii) and Halobacterium NRC-1 (Halobacterium species NRC-1) such as Wo Shi).
" alpha-non-natural amino acid " is meant the amino acid that is not a kind of or pyrroles's lysine or selenocysteine in 20 kinds of common amino acids.Can be " non-naturally encoded amino acid ", " alpha-non-natural amino acid (unnatural amino acid) ", " amino acid of non-natural existence " with other term that term " alpha-non-natural amino acid (non-natural amino acid) " synonym uses with and variously be connected and the form that is not connected with hyphen with hyphen.Term " alpha-non-natural amino acid " includes but not limited to the natural existence by modifying natural amino acids coding (including but not limited to 20 kinds of common amino acids or pyrroles's lysine and selenocysteine), but itself does not incorporate amino acid in the polypeptide chain of growth into by the translation compound.And non-naturally encoded naturally occurring amino acid whose example includes but not limited to N-acetyl-glucosamine base-L-serine, N-acetyl-glucosamine base-L-threonine and O-phosphotyrosine.In addition, term " alpha-non-natural amino acid " includes but not limited to naturally not exist and can obtain or can be by modifying the amino acid that alpha-non-natural amino acid obtains by synthesis mode.
As used herein, deoxyribonucleotide, dezyribonucleoside, ribonucleotide or the ribonucleotide that term " nucleic acid " is meant sub-thread or bifilar form with and polymer.Only for instance, described nucleic acid and nucleic acid polymers include but not limited to the analog of (i) natural nucleotide, and it has the binding characteristic similar to reference nucleic acid and with the mode metabolism similar to naturally occurring nucleotide; (ii) oligonucleotide analogs includes but not limited to PNA (peptidyl nucleic acid), is used for the DNA analog (thiophosphate, phosphoramidate etc.) of antisense technology; (iii) its conservative modification variant (including but not limited to that degenerate codon replaces) and complementary series and the clearly sequence of description.For instance, can one or more be selected by producing the 3rd of (or all) codons realize that through mixing sequence that base and/or deoxyinosine residue replace degenerate codon replaces (people such as Batzer, Nucleic Acid Res. 19:5081 (1991); People such as Ohtsuka, J.Biol.Chem.260:2605-2608 (1985); With people such as Rossolini, Mol.Cell.Probes 8:91-98 (1994)).
As used herein, term " oxidant " is meant can be from removing the compound or the material of electronics through the compound of oxidation.For instance, oxidant comprises but is not limited to through oxidized glutathione, cystine, cystamine, through the oxidation dithiothreitol (DTT), through oxidation erythrite and oxygen.Various oxidants are applicable in the method and composition as herein described.
As used herein, term " pharmaceutically acceptable " be meant the biologically active that can not eliminate compound or characteristic and nontoxic relatively (promptly, material can be thrown with individual and can not cause undesirable biological agent or with harmful mode and any component interaction that contains the composition of described material) material, include but not limited to salt, supporting agent or thinner.
As used herein, term " photoaffinity labeling " is meant to have when when exposure and mark that it is had the mark of group of molecule formation key of compatibility.Only for instance, described key can be covalent bond or non-covalent bond.
As used herein, term " the light cage covers part " is meant when with the illumination of certain wavelength covalently or non-covalently the group in conjunction with other lewis' acid.
As used herein, term " but photodestruciton group " is meant the group of fracture when exposure.
As used herein, term " photocrosslinking agent " is meant the compound that comprises two or more functional groups, and described functional group can react when exposure and form covalently or non-covalently key with two or more monomers or polymerizable molecular.
As used herein, term " but photoisomerization part " is meant the group that is become another kind of isomeric form when with optical illumination by a kind of isomeric form.
As used herein, term " poly alkylene glycol " is meant linearity or branch polyhydroxyl polyether polyalcohol.Described poly alkylene glycol includes but not limited to polyethylene glycol, polypropylene glycol, polytetramethylene glycol and its derivative.Other exemplary embodiments is for example listed in the commercial supplier catalogue, such as Shearwater Corporation catalogue " Polyethylene Glycol andDerivatives for Biomedical Applications " (2001).Only for instance, described polyhydroxyl polyether polyalcohol has between about 0.1kDa to the mean molecule quantity between about 100kDa.For instance, described polyhydroxyl polyether polyalcohol includes but not limited between about 100Da and about 100,000Da or higher between.The molecular weight of polymer can between the 000Da, include but not limited to about 100 between about 100Da and about 100,000Da, about 95,000Da, about 90,000Da, about 85,000Da, about 80,000Da, about 75,000Da, about 70,000Da, about 65,000Da, about 60,000Da, about 55,000Da, about 50,000Da, about 45,000Da, about 40,000Da, about 35,000Da, about 30,000Da, about 25,000Da, about 20,000Da, about 15,000Da, about 10,000Da, about 9,000Da, about 8,000Da, about 7,000Da, about 6,000Da, about 5,000Da, about 4,000Da, about 3,000Da, about 2,000Da, about 1,000Da, about 900Da, about 800Da, about 700Da, about 600Da, about 500Da, about 400Da, about 300Da, about 200Da and about 100Da.In certain embodiments, the molecular weight of polymer is between about 100Da and about 50, between the 000Da.In certain embodiments, the molecular weight of polymer is between about 100Da and about 40, between the 000Da.In certain embodiments, the molecular weight of polymer is between about 1, and 000Da and about 40 is between the 000Da.In certain embodiments, the molecular weight of polymer is between about 2, and 000Da is to about 50, between the 000Da.In certain embodiments, the molecular weight of polymer is between about 5, and 000Da and about 40 is between the 000Da.In certain embodiments, the molecular weight of polymer is between about 10, and 000Da and about 40 is between the 000Da.In certain embodiments, poly-(ethylene glycol) molecule is a branched polymers.The molecular weight of side chain PEG can be between about 1, and 000Da and about 100 between the 000Da, includes but not limited to about 100,000Da, about 95,000Da, about 90,000Da, about 85,000Da, about 80,000Da, about 75,000Da, about 70,000Da, about 65,000Da, about 60,000Da, about 55,000Da, about 50,000Da, about 45,000Da, about 40,000Da, about 35,000Da, about 30,000Da, about 25,000Da, about 20,000Da, about 15,000Da, about 10,000Da, about 9,000Da, about 8,000Da, about 7,000Da, about 6,000Da, about 5,000Da, about 4,000Da, about 3,000Da, about 2,000Da and about 1,000Da.In certain embodiments, the molecular weight of side chain PEG is between about 1, and 000Da and about 50 is between the 000Da.In certain embodiments, the molecular weight of side chain PEG is between about 1, and 000Da and about 40 is between the 000Da.In certain embodiments, the molecular weight of side chain PEG is between about 5, and 000Da and about 40 is between the 000Da.In certain embodiments, the molecular weight of side chain PEG is between about 5, and 000Da and about 20 is between the 000Da.In certain embodiments, the molecular weight of side chain PEG is between about 2, and 000Da is to about 50, between the 000Da.
As used herein, term " polymer " " be meant by repeating the molecule that subunit constitutes.Described molecule includes but not limited to polypeptide, polynucleotide or polysaccharide or poly alkylene glycol.
Term " polypeptide ", " peptide " and " protein " are used interchangeably in this article to refer to the amino acid residue polymer.That is to say, be equally applicable to the description of peptide and the description of protein, and also be applicable to the description of polypeptide at the description of the description of peptide and protein at the description of polypeptide.Described term is applicable to that naturally occurring amino acid polymer and one or more amino acid residues are the amino acid polymer of alpha-non-natural amino acid.In addition, described " polypeptide ", " peptide " and " protein " comprise the amino acid chain of any length, comprise full length protein, and wherein amino acid residue is to connect by the covalency peptide bond.
Term " posttranslational modification " be meant after natural or alpha-non-natural amino acid have been incorporated polypeptide chain into by translation, taken place to described amino acid whose any modification.Described modification include but not limited to common translation in vivo modify, altogether translation in vitro modify (such as, in cell free translation system), in vitro modify after in vivo modifying and translate after the translation.
As used herein, term " prodrug " or " pharmaceutically acceptable prodrug " are meant the medicament that or in vitro changes into parent drug in vivo, wherein it can not eliminate biologically active or characteristic and nontoxic relatively (that is, the material throwing can be able to not caused undesirable biological agent or any component interaction to be harmful to mode and to contain the composition of described material with individuality) of medicine.Prodrug is generally prodrug, and it is after throwing and individuality and absorption subsequently, and transforming (such as by the metabolic pathway conversion) via particular procedure is activity or the material that has more activity.Some prodrugs have to be present in to be made it have than low activity and/or gives medicine dissolution or the chemical group of certain other characteristic on the prodrug.With chemical group from prodrug cracking and/or modify after, produce active medicine.Prodrug changes into active medicine by enzymatic or non-enzymatic reaction in vivo.Prodrug can provide the plysiochemical characteristic of improvement, such as the transfer characteristic of dissolubility, enhancing preferably (such as, selectively targeted specific cells, tissue, organ or part) and the pharmacotherapy of improvement be worth.The benefit of described prodrug include but not limited to (i) compare with parent drug be easy to the dispensing; (ii) prodrug can and be a biological utilisation by oral administration, and parent drug is not all right; (iii) compare with parent drug, prodrug also can have improved dissolution in medical composition.Prodrug comprises non-activity or the active active medicine derivative that reduces on the pharmacology.Prodrug can through be designed to by handle medicinal property (such as, plysiochemical, biological medicine or pharmacokinetic properties) regulate the medicine that arrives required site of action or the amount of bioactive molecule.The limiting examples of prodrug is a non-natural amino acid polypeptides, it is to throw and to promote that leap is wherein water-soluble the disadvantageous cell membrane of mobility is transmitted with the form of ester (" prodrug "), the rear flank becomes carboxylic acid through the metabolism hydrolysis, promptly active entity subsequently in the wherein water-soluble useful cell but enter.Prodrug can be designed to reversible medicaments derivative and strengthen the dressing agent of medicine to the conveying of site-specific sex organization to be used as.
As used herein, term " prevention effective dose " is meant the amount of the composition that contains at least one non-natural amino acid polypeptides or at least one modified non-natural amino acid polypeptides that prophylactically is applied to the patient, and it will alleviate one or more symptoms of disease, symptom or the illness of being treated to a certain extent.In described prophylactic applications, described amount is decided by patient's health status, body weight etc.It will be appreciated by those skilled in the art that by normal experiment (including but not limited to the dosage escalation clinical testing) and can determine described prevention effective dose.
As used herein, term " through protection " is meant to exist and prevents " protecting group " or the part that chemical reactivity functional group reacts under some reaction condition.Protecting group will be looked the type of the chemically reactive group of being protected and be changed.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) reactive group is a mercaptan, protecting group can be adjacent pyridyl disulfide so; And if (iii) chemically reactive group be carboxylic acid (such as, butyric acid or propionic acid) or hydroxyl, so protecting group can be benzyl or alkyl (such as, methyl, ethyl or the tert-butyl group).
Only for instance, end-blocking/protecting group can be selected from:
Figure A200680049995D00461
?
Figure A200680049995D00462
Pi-allyl
Figure A200680049995D00463
?
Figure A200680049995D00464
?
Figure A200680049995D00465
The tert-butyl group
Figure A200680049995D00466
(C 6H 5) 3C—
Figure A200680049995D00467
。
The trityl acetyl group
In addition, protecting group includes but not limited to the photo-labile group, such as Nvoc and other known protecting group of MeNvoc and affiliated field.Other protecting group is described in Greene and Wuts, Protective Groups in OrganicSynthesis, the 3rd edition, John Wiley ﹠amp; Sons, New York, NY, in 1999, described document is that the mode of quoting in full is incorporated herein.
As used herein, term " radioactive segment " be meant its nuclear from put out nuclear radiation (such as, α, β or γ particle) group; Wherein the α particle is a helion, and the β particle is an electronics, and the γ particle is a high-energy photon.
As used herein, term " reactive compounds " be meant under proper condition can with the compound of another atom, molecule or compound reaction.
Term " recombinant host cell " (being also referred to as " host cell ") is meant the cell that comprises exogenous polynucleotide, wherein is used for the method that exogenous polynucleotide inserts cell is included but not limited to other method of generation recombinant host cell known in direct picked-up, transduction, f pairing or the affiliated field.Only for instance, described exogenous polynucleotide can be the nonconformity carrier, includes but not limited to plasmid; Maybe can be incorporated in the host genome.
As used herein, term " redox active agent " is meant oxidation or reduces the molecule of another molecule that redox active agent becomes reduction or oxidation state thus.The example of redox active agent includes but not limited to ferrocene, quinone, Ru 2+/3+Complex compound, Co 2+/3+Complex compound and Os 2+/3+Complex compound.
As used herein, term " reductant " is meant the compound or the material that electronics can be added in the compound that is reduced.For instance, reductant includes but not limited to dithiothreitol (DTT) (DTT), 2 mercapto ethanol, dithioerythritol, cysteine, cysteamine (2-aminoothyl mercaptan) and through reduced glutathione.Described reductant can be used for (only for instance) to be remained on sulfydryl to go back in ortho states and the original molecule or intermolecular disulfide bond.
As used herein, " folding again " described suitably folding or deployed condition changed into any process, reaction or method natural or suitably folding conformation.Only for instance, with regard to disulfide bond, the folding again polypeptide that will contain disulfide bond changes into natural or suitably folding conformation by suitably not folding or deployed condition.The described polypeptide that contains disulfide bond can be natural amino acid polypeptide or non-natural amino acid polypeptides.
As used herein, term " resin " is meant the soluble polymer beads of HMW.Only for instance, described bead can be used as the synthetic supporter of solid-phase peptide or connect the position of molecule before purifying.
As used herein, term " sugar " is meant a series of carbohydrate, includes but not limited to sugar, monose, oligosaccharides and polysaccharide.
As used herein, term " safety " or " security features " are meant and may throw side effect with relevant (relating to the number of times of throwing with medicine) with medicine.For instance, think to throw to have the excellent safety feature with the medicine that repeatedly and only produces slight side effect or do not have side effects.The limiting examples of the method for assessment security features is provided in the example 26.The method can be used for assessing the security features of any polypeptide.
As used herein, phrase " with ... selective cross " or " with ... specific hybrid " be meant that when specific nucleotide sequence was present in the complex mixture that includes but not limited to full cell or library DNA or RNA, molecule combined, forms duplex or hybridization with described sequence under stringent hybridization condition.
As used herein, term " spin labeling " is meant the molecule that contains the atom that represents the not sharing electron spin that can detect by ESR spectrum (that is, stable paramagnetism group) or one group of atom and can be connected with another molecule.Described spin labeling molecule includes but not limited to nitroxyl and nitroxide (nitroxide) and can be the single spin mark or two spin labeling.
As used herein, term " stoichiometry " is meant that the ratio of the molal quantity of the compound that participates in chemical reaction is about 0.9 to about 1.1.
As used herein, term " class stoichiometry " is meant when changing reaction condition or has the chemical reaction that becomes stoichiometry or near-stoichiometric under the situation of additive.The change of described reaction condition includes but not limited to the increase of temperature or the change of pH value.Described additive includes but not limited to accelerator.
Phrase " stringent hybridization condition " is meant the hybridization of sequence under low ionic strength and hot conditions of DNA, RNA, PNA or other nucleic acid mimics or its combination.For instance, under stringent condition, probe will with the hybridization of target sequence in its complex mixture (including but not limited to full cell or library DNA or RNA) at nucleic acid, but not with other sequence hybridization in complex mixture.Stringent condition is sequence dependent and will be with varying environment different.For instance, than long sequence specific hybrid under higher temperature.Stringent hybridization condition includes but not limited to (i) heat fusion joint (T that the bit sequencing is listed as under specify ion intensity and pH value m) low about 5-10 ℃; (ii) arrive about 1.0M for about 0.01M in about pH7.0 salinity under about pH8.3, and be at least about 30 ℃ for temperature the short probe (including but not limited to about 10), and be at least about 60 ℃ for temperature the long probe (including but not limited to) greater than 50 nucleotide to about 50 nucleotide; (iii) add destabilizing agent, include but not limited to formamide; (iv) 50% formamide, 5 * SSC and 1% SDS cultivate down at 42 ℃; Or 5 * SSC, about 1% SDS, cultivate down at 65 ℃, and in 0.2 * SSC and about 0.1% SDS, washing under 65 ℃, reach the time between about 5 minutes to about 120 minutes.Only for instance, the detection of selectivity or specific hybrid includes but not limited to double at least the positive signal of background.The extensive guidance of related nucleic acid hybridization sees Tijssen, Laboratory Techniques in Biochemistry and MolecularBiology--Hybridization with Nucleic Probes is in " Overview of principles of hybridization andthe strategy of nucleic acid assays " (1993).
As used herein, term " individuality " is meant the animal into treatment, observation or experimental subjects.Only for instance, individuality can be (but being not limited to) mammal, includes but not limited to the mankind.
As used herein, term " purified in fact " be meant the component paid close attention to can be in fact or essentially no before purifying, follow usually the component paid close attention to or with other component 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 about 1% (with dry weight basis) pollution components, 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, can from host cell, be purified into natural amino acid polypeptide or non-natural amino acid polypeptides from n cell or under the situation of reorganization generation natural amino acid polypeptide or non-natural amino acid polypeptides.For instance, when the preparation of natural amino acid polypeptide or non-natural amino acid polypeptides 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 about 1% (with dry weight basis) pollutant, described preparation can be " purified in fact ".For instance, when producing natural amino acid polypeptide or non-natural amino acid polypeptides by host cell reorganization, natural amino acid polypeptide or non-natural amino acid polypeptides can account for dry cell weight about 30%, about 25%, about 20%, about 15%, about 10%, about 5%, about 4%, about 3%, about 2% about 1% or lower ratio exist.For instance, when producing natural amino acid polypeptide or non-natural amino acid polypeptides by the host cell reorganization, natural amino acid polypeptide or non-natural amino acid polypeptides can account for the about 5g/L of dry cell weight, 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 lower amount is present in the medium.For instance, such as by proper method (including but not limited to SDS/PAGE analysis, RP-HPLC, SEC and Capillary Electrophoresis) mensuration, 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 higher purity level.
Term " substituting group " (being also referred to as " noiseless substituting group ") is meant the group that can be used for another group on the displacer molecule.Described group includes but not limited to halogen, C 1-C 10Alkyl, C 2-C 10Thiazolinyl, C 2-C 10Alkynyl, C 1-C 10Alkoxyl, C 5-C 12Aralkyl, C 3-C 12Cycloalkyl, C 4-C 12Cycloalkenyl group, phenyl, be substituted phenyl, toluyl, 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, 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 each m is 1 to 8) ,-C (O) NR 2,-C (S) NR 2,-SO 2NR 2,-NRC (O) NR 2,-NRC (S) NR 2, its salt etc.Each R group in the previous list includes but not limited to H, alkyl or is substituted alkyl, aryl or is substituted aryl or alkaryl.When the explanation of the conventional chemical formula by writing from left to right substituting group, it contains by writing structure from right to left resulting at chemically identical substituting group equally, for example, and-CH 2O-and-OCH 2-identical.
Only for instance, the substituting group of alkyl and assorted alkyl (comprising the group that is called alkylidene, thiazolinyl, inferior assorted alkyl, assorted thiazolinyl, alkynyl, cycloalkyl, Heterocyclylalkyl, cycloalkenyl group and heterocycloalkenyl) includes 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 2The assorted alkyl that each R group in the previous list includes but not limited to hydrogen, is substituted or is unsubstituted, the aryl that is substituted or is unsubstituted (include but not limited to replace aryl), alkyl, alkoxyl or the thioalkoxy group or the aralkyl that are substituted or are unsubstituted through 1-3 halogen.When two R groups were connected to identical nitrogen-atoms, it can be combined to form 5,6 or 7 yuan of rings with nitrogen-atoms.For instance ,-NR 2Plan includes but not limited to 1-pyrrolidinyl and 4-morpholinyl.
For instance, the substituting group of aryl and heteroaryl include 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 ,-NR-C (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 and fluorine (C 1-C 4) alkyl, its quantity be zero on the aromatic ring system the sum of open valence state; And wherein each the R group in the previous list includes but not limited to hydrogen, alkyl, assorted alkyl, aryl and heteroaryl.
As used herein, term " treatment effective dose " is meant the amount of the patient's who throws and suffered from disease, symptom or illness the composition that contains at least one non-natural amino acid polypeptides and/or at least one modified non-natural amino acid polypeptides, and it is enough to cure or contain or alleviate to a certain extent to small part one or more symptoms of disease, illness or the symptom of being treated.The effectiveness of described composition is decided on the condition that includes but not limited to following factor: the order of severity of disease, illness or symptom and the course of disease, previous therapy, patient's health status and to the reaction of medicine and the doctor in charge's judgement.Only for instance, the treatment effective dose can be determined by normal experiment (including but not limited to the dosage escalation clinical testing).
As used herein, term " thioalkoxy group " is meant the sulfur-bearing alkyl that is connected with molecule via oxygen atom.
Term " heat fusion joint " or T m50% temperature (under specify ion intensity, pH value and nucleic acid concentration) of hybridizing with the probe of target complementation and target sequence during for balance.
As used herein, term " toxic moiety " is meant and can works the mischief or dead compound.
As used herein, term " treatment " comprises mitigation, alleviates or improves the symptom of disease or symptom; Prevent other symptom; Improve or the potential metabolic disease of prevention symptom because of; Suppress disease or symptom, the development of disease or symptom is stagnated; Alleviate disease or symptom; Cause disappearing of disease or symptom; Alleviation is resembled by the disease that disease or symptom cause; Perhaps stop the symptom of disease or symptom.Term " treatment " includes but not limited to preventative and/or therapeutic treatment.
As used herein, term " water-soluble polymer " is meant any polymer that dissolves in the water-based solvent.Described water-soluble polymer includes but not limited to polyethylene glycol, polyethylene glycol propionic aldehyde, its single C 1-C 10Alkoxyl or aryloxy group derivative (are described in United States Patent (USP) the 5th, 252, in No. 714, it is incorporated herein by reference), mono methoxy-polyethylene glycol, polyvinylpyrrolidone, polyvinyl alcohol, polyaminoacid, the divinyl ether maleic anhydride, N-(2-hydroxypropyl)-Methacrylamide, glucan, glucan derivative (comprising dextran sulfate), polypropylene glycol, poly(propylene oxide)/ethylene oxide copolymer, polyoxy ethylization polyalcohol, heparin, heparin fragment, polysaccharide, oligosaccharides, glycan, cellulose and cellulose derivatives (including but not limited to methylcellulose and carboxymethyl cellulose), seralbumin, starch and starch derivatives, polypeptide, poly alkylene glycol and its derivative, the copolymer of poly alkylene glycol and its derivative, polyvinyl ethyl ether and alpha-beta-poly-[(2-ethoxy)-DL-asparagine etc., or its mixture.For instance, described water-soluble polymer and natural amino acid polypeptide or non-natural amino acid polypeptides coupling can cause changing, and include but not limited to water-soluble increase, serum half-life increases or through regulating, the treatment half life period is with respect to not modified form increase or through regulating, biological usability increases, biologically active is through regulating, prolong circulation timei, immunogenicity is through regulating, physics association feature (including but not limited to assemble and polymer formation) is through regulating, receptors bind changes, the combination that combines the collocation thing with one or more changes and receptor dimerizationization or multimerization change.In addition, described water-soluble polymer can have or can not have the biologically active of himself.
Unless otherwise mentioned, otherwise affiliated art scope in mass spectrum, NMR, HPLC, protein chemistry, biochemistry, recombinant DNA technology and pharmacological conventional method all can use.
The compound that this paper provided (including but not limited to alpha-non-natural amino acid, non-natural amino acid polypeptides, modified non-natural amino acid polypeptides and the reagent of making above-claimed cpd) comprises isotope-labeled compound, the person is identical described in each chemical formula that it is provided with this paper and the structure, but one or more atoms are through atomic mass or mass number and common atomic mass or the different atomic substitutions of mass number of natural world.Can incorporate the isotope that isotopic example in the The compounds of this invention comprises hydrogen, carbon, nitrogen, oxygen, fluorine and chlorine into, respectively such as 2H, 3H, 13C, 14C, 15N, 18O, 17O, 35S, 18F, 36Cl.Some isotope-labeled compound as herein described (for example, and have such as 3H and 14The radioisotopic compound of C) can be used in the distribution calibrating of medicine and/or matrix organization.In addition, use isotope (such as deuterium, promptly 2H) replace and to provide some treatment advantage because of higher metabolic stability (for example, the dosage demand of the in vivo half life period of increase or reduction).
Some compound herein (include but not limited to alpha-non-natural amino acid, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides, and the reagent of making above-claimed cpd) has asymmetric carbon atom and therefore can exist with enantiomter or diastereomeric form.Can by known method (for example, chromatography and/or fractional crystallization) non-enantiomer mixture be divided into its indivedual diastereoisomers according to physico chemistry difference.Can by with suitable optically active compound (for example, alcohol) reaction changes into non-enantiomer mixture with enantiomeric mixture, separate diastereoisomer and indivedual diastereoisomers are transformed the corresponding pure enantiomter of (for example, hydrolysis) one-tenth, thereby separate enantiomter.Think that all described isomer (comprising diastereoisomer, enantiomter and its mixture) all are the part of composition as herein described.
In extra or other embodiment, compound as herein described (include but not limited to alpha-non-natural amino acid, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides, and the reagent of making above-claimed cpd) is used with the form of prodrug.In extra or other embodiment, compound as herein described (includes but not limited to alpha-non-natural amino acid, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides, and the reagent of making above-claimed cpd) throw with the organism that needs is arranged after metabolism to produce metabolite, described metabolite is used to produce required effect subsequently, comprises required therapeutic action.The active metabolite that other or extra embodiment are the non-natural amino acid polypeptides of alpha-non-natural amino acid and " modified or not modified ".
Method as herein described and composite comprise N-oxide, crystal form (being also referred to as polymorph) or the pharmaceutically acceptable salt that uses alpha-non-natural amino acid, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides.In certain embodiments, alpha-non-natural amino acid, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides can tautomeric forms exist.In the scope of alpha-non-natural amino acid, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides that all dynamic isomers all are included in this paper and are provided.In addition, alpha-non-natural amino acid as herein described, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides not the solvation form and with pharmaceutically acceptable solvent (such as, water, ethanol etc.) the solvation form that forms exists.Think that the solvation form of alpha-non-natural amino acid, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides that this paper provided also discloses in this article to some extent.
Compounds more as herein described (include but not limited to alpha-non-natural amino acid, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides, and the reagent of making above-claimed cpd) can some tautomeric forms exist.Think that all described tautomeric forms all are the part of composition as herein described.And, for example think that all enols-ketone group form of herein any compound (include but not limited to alpha-non-natural amino acid, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides, and the reagent of making above-claimed cpd) is the part of composition as herein described.
Compounds more as herein described (include but not limited to alpha-non-natural amino acid, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides, and the reagent of making above-claimed cpd) be acidity and can with pharmaceutically acceptable salt forming cation.Compounds more as herein described (include but not limited to alpha-non-natural amino acid, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides, and the reagent of making above-claimed cpd) can be alkalescence and therefore can form salt with pharmaceutically acceptable anion.All described salt (comprising disalt) all in the scope of composition as herein described and its can prepare by conventional method.For instance, can prepare salt by making acid in aqueous medium, non-aqueous media or part aqueous medium, the contact with alkaline entity.By using at least a salt that reclaims in the following technology: filter, precipitate subsequent filtration, evaporating solvent or freeze-drying under the situation of the aqueous solution with non-solvent.
The acid proton that exists in the parent alpha-non-natural amino acid through metal ion (for example, alkali metal ion, alkaline-earth metal ions or aluminium ion) displacement or during with the organic base coordination, can form the pharmaceutically acceptable salt of non-natural amino acid polypeptides disclosed herein.In addition, can use the salt form of the non-natural amino acid polypeptides that the salt preparation of raw material or intermediate disclosed.Non-natural amino acid polypeptides as herein described that can be by making free alkali form and pharmaceutically acceptable inorganic or organic acid reaction are prepared into pharmaceutically acceptable acid-addition salts (it is one type a pharmaceutically acceptable salt) with non-natural amino acid polypeptides as herein described.Perhaps, non-natural amino acid polypeptides as herein described that can be by making free acid form and pharmaceutically acceptable inorganic or organic base react non-natural amino acid polypeptides as herein described are prepared into pharmaceutically acceptable base addition salts (it is one type a pharmaceutically acceptable salt).
The type of pharmaceutically acceptable salt includes but not limited to: the acid-addition salts that (1) and inorganic acid form, all example hydrochloric acids of described inorganic acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid etc.; Or the acid-addition salts that forms with organic acid, described organic acid is such as acetate, propionic acid, caproic acid, the pentamethylene propionic acid, glycolic, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3-(4-hydroxy benzoyl) benzoic acid, cinnamic acid, mandelic acid, Loprazolam, ethane sulfonic acid, 1, the 2-ethane disulfonic acid, the 2-hydroxyethanesulfonic acid, benzene sulfonic acid, the 2-naphthalene sulfonic acids, 4-methyl bicycle-[2.2.2] oct-2-ene-1-formic acid, glucoheptonic acid, 4,4 '-di-2-ethylhexylphosphine oxide-(3-hydroxyl-2-subunit-1-formic acid), the 3-phenylpropionic acid, trimethylace tonitric, butylacetic acid, lauryl sulfate, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid etc.; (2) acid proton that in parent compound, exists formed salt during through metal ion (for example, alkali metal ion, alkaline-earth metal ions or aluminium ion) displacement or with the organic base coordination.Acceptable organic base comprises monoethanolamine, diethanol amine, triethanolamine, tromethamine, N-methylglucosamine etc.Acceptable inorganic base comprises aluminium hydroxide, slaked lime, potassium hydroxide, sodium carbonate, sodium hydroxide etc.
Can make the corresponding equilibrium ion that ins all sorts of ways and analyze and differentiate the pharmaceutically acceptable salt of non-natural amino acid polypeptides, described method includes 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 described in the use-case 87-91 and method are tested the therapeutic activity of the pharmaceutically acceptable salt of described non-natural amino acid polypeptides.
Should be appreciated that, when mentioning salt, comprise its solvent addition form or crystal form, especially solvate or polymorph.Solvate contains the solvent of stoichiometry or non-stoichiometric amount, and normally utilizes pharmaceutically acceptable solvent (such as water, ethanol etc.) to form in crystallization process.When solvent forms hydrate during for water, or when solvent formation alcoholates when be pure.Polymorph comprises having the different crystal filling arrangement that same compound element is formed.Polymorph has different X-ray diffraction figure, infrared spectrum, fusing point, density, hardness, crystalline form, optics and electrology characteristic, stability and dissolubility usually.Can cause the monocrystalline form to be preponderated such as the various factors of recrystallisation solvent, crystalline rate and storage temperature again.
Can use multiple technologies to realize the polymorph of pharmaceutically acceptable salt of non-natural amino acid polypeptides and/or the screening and the sign of solvate, described technology includes but not limited to that heat analysiss, x x ray diffraction, spectroscopy, steam adsorb and microscopy.Heat analysis method is at heat chemistry degraded or thermal physical process (including but not limited to polymorphic transformation), and described method is used to analyze relation, gravimetry loss between each polymorph form, finds glass transformation temperature or is used for the research of excipient tolerability.Described method includes but not limited to differential scanning calorimetry (DSC), modulation system differential scanning calorimetry (MDCS), thermogravimetric analysis (TGA) and the infrared coupling analysis of thermogravimetric (TG/IR).X-ray diffraction method includes but not limited to monocrystalline and powder diffractometer and synchrotron radiation source (synchrotron source).Employed various spectral technique includes but not limited to Raman spectrum (Raman), FTIR, UVIS and NMR (liquid and solid-state).Various microscopy technology include but not limited to petrographic microscope, utilize the scanning electron microscopy (SEM) of energy dispersion X-ray analysis (EDX), utilize the environmental scanning electron microscope of EDX to learn (in gas or steam atmosphere), IR microscopy and Raman microscope.
Description of drawings
Can wherein utilize the principle of the inventive method, composition, device and equipment by obtain better understanding with reference to the embodiment of following statement illustrative embodiment and its alterations for the feature and the advantage of the inventive method and composition:
Fig. 1 provides the non-limiting schematic diagram of relation of some aspect of method as herein described, composition, strategy and technology.
Fig. 2 provides the illustrative limiting examples of the type of the alpha-non-natural amino acid that contains diamines as herein described.
Fig. 3 provides the illustrative limiting examples of the type of the alpha-non-natural amino acid that contains dicarbapentaborane as herein described.
Fig. 4 provides the illustrative limiting examples of the type of the alpha-non-natural amino acid that contains ketone alkynes as herein described.
Fig. 5 is provided for preparing the illustrative limiting examples of the synthetic method of alpha-non-natural amino acid as herein described.
Fig. 6 is provided for preparing the illustrative limiting examples of the synthetic method of alpha-non-natural amino acid as herein described.
Fig. 7 is provided for preparing the illustrative limiting examples of the synthetic method of alpha-non-natural amino acid as herein described.
Fig. 8 is provided for preparing the illustrative limiting examples of the synthetic method of alpha-non-natural amino acid as herein described.
Thereby Fig. 9 provides to utilize and contains the non-natural amino acid polypeptides that the posttranslational modification of dicarbapentaborane reagent contains diamines and form the modified illustrative limiting examples that contains the heterocycle non-natural amino acid polypeptides.
Thereby Figure 10 provides to utilize and contains the non-natural amino acid polypeptides that the posttranslational modification of dicarbapentaborane reagent contains diamines and form the modified illustrative limiting examples that contains the heterocycle non-natural amino acid polypeptides.
Figure 11 A) provides the illustrative limiting examples of the formation of heterocyclic bond as herein described.
Figure 11 B) provide as herein described contain alpha-non-natural amino acid through covering dicarbapentaborane and go to protect after the illustrative limiting examples of formation of heterocyclic bond.
Thereby Figure 12 provides to utilize and contains the non-natural amino acid polypeptides that the posttranslational modification of diamines reagent contains dicarbapentaborane and form the modified illustrative limiting examples that contains the heterocycle non-natural amino acid polypeptides.
Thereby Figure 13 provides to utilize and contains the non-natural amino acid polypeptides that the posttranslational modification of diamines reagent contains dicarbapentaborane and form the modified illustrative limiting examples that contains the heterocycle non-natural amino acid polypeptides.
Figure 14 provides the illustrative limiting examples of the protein modification that uses composition as herein described, method, technology and strategy.
Figure 15 provides the illustrative limiting examples of the protein modification that uses composition as herein described, method, technology and strategy.
Figure 16 provides the illustrative limiting examples of the protein modification that uses composition as herein described, method, technology and strategy.
Figure 17 provides the illustrative limiting examples of the protein Pegylation that uses composition as herein described, method, technology and strategy.
Figure 18 provides the synthetic illustrative limiting examples that contains PEG reagent, and described reagent can be used for modifying non-natural amino acid polypeptides contains PEG with formation the non-natural amino acid polypeptides through the heterocycle connection.
Figure 19 provides the synthetic illustrative limiting examples that contains PEG reagent, and described reagent can be used for modifying non-natural amino acid polypeptides contains PEG with formation the non-natural amino acid polypeptides through the heterocycle connection.
Figure 20 provides the synthetic illustrative limiting examples that contains difunctionality PEG reagent, and described reagent can be used for modifying non-natural amino acid polypeptides contains PEG with formation the non-natural amino acid polypeptides through the heterocycle connection.
The synthetic illustrative limiting examples that Figure 21 provides difunctionality to connect base, described connection base can be used for modifying non-natural amino acid polypeptides to form the non-natural amino acid polypeptides that connects through heterocycle.
Figure 22 provides the synthetic illustrative limiting examples that contains trifunctional PEG reagent, and described reagent can be used for modifying non-natural amino acid polypeptides contains PEG with formation the non-natural amino acid polypeptides through the heterocycle connection.
Figure 23 provides by using composition as herein described, method, technology and strategy that non-natural amino acid polypeptides is connected the non-limiting diagram of the illustrative that makes the protein Pegylation with the PEG group.
The non-limiting diagram of illustrative that Figure 24 provides difunctionality to connect the purposes of base, it uses composition as herein described, method, technology and strategy to be connected the base modification and to be connected non-natural amino acid polypeptides via PEG.
The non-limiting diagram of illustrative that Figure 25 provides difunctionality to connect the purposes of base, it uses composition as herein described, method, technology and strategy via being connected the base modification and being connected non-natural amino acid polypeptides.
The non-limiting diagram of illustrative that Figure 26 provides trifunctional to connect the purposes of base, it uses composition as herein described, method, technology and strategy to be connected the base modification via PEG and is connected non-natural amino acid polypeptides and makes the basic Pegylation of described connection.
The non-limiting diagram of illustrative that Figure 27 provides difunctionality to connect the purposes of base, it uses composition as herein described, method, technology and strategy to modify non-natural amino acid polypeptides and described polypeptide is connected with the PEG group.
Figure 28 provides the synthetic non-limiting diagram of illustrative that contains pyrazole compound.
Figure 29 provides the non-limiting diagram of illustrative of using synthetic non-natural amino acid polypeptides of composition as herein described, method, technology and strategy and PEG group.
Embodiment
I. foreword
Recently reported the brand new technical in the protein science, it provides and has overcome the prospect of modifying relevant numerous limitations with the protein loci specificity.Specifically, novel assembly has been added to prokaryotes Escherichia coli (Escherichia coli, E.coli) (for example referring to people such as L.Wang, (2001), Science 292:498-500) and eucaryote S. cervisiae (Sacchromyces cerevisia, S.cerevisiae) in the protein biosynthesis machine of (for example people such as J.Chin, Science 301:964-7 (2003)), described machine can in vivo be incorporated alpha-non-natural amino acid in the protein into.Made to respond amber codon TAG in this way multiple new amino acid with novel chemistry, physics or biological nature effectively and is with high fidelity incorporated in the protein of Escherichia coli and yeast, but described new amino acid comprises photoaffinity labeling and photoisomerization amino acid, ketone group amino acid and glycosylation amino acid.For example referring to people such as J.W.Chin, (2002), Journal ofthe American Chemical Society 124:9026-9027 (mode of quoting is in full incorporated into); J.W.Chin ， ﹠amp; P.G. Schultz, (2002), ChemBioChem 3 (11): 1135-1137 (mode of quoting is in full incorporated into); People such as J.W.Chin, (2002), PNAS United States of America 99 (71): 11020-11024 (mode of quoting is in full incorporated into); With L. Wang ， ﹠amp; P.G.Schultz, (2002), Chem.Comm., 1-11 (mode of quoting is in full incorporated into).These researchs confirm, might selectivity and introduce routinely and do not see in the protein, all functional groups seen in the amino acid of 20 kinds of common gene codes are chemical inertness and can be used for effectively and optionally reacting the chemical functional group who stablizes covalent bond to form.
II. summarize
Fig. 1 is the limiting examples of composition as herein described, method, technology and strategy.On the one hand, this paper describes and to be used for producing and use comprises at least one alpha-non-natural amino acid or has the instrument (method, composition, technology) of polypeptide of the modified alpha-non-natural amino acid of dicarbapentaborane, diamines, ketone alkynes, ketoamine or heterocycle (comprising nitrogen heterocycle).Dicarbapentaborane includes but not limited to diketone, keto-aldehyde, ketone acid, ketone ester and ketone thioesters, and two amidos include but not limited to hydrazine, amidine, imines, 1,1-two amidos, 1,2-two amidos, 1,3-two amidos and 1,4-two amidos.Described alpha-non-natural amino acid can contain other functional group, includes but not limited to required functional group.It should be noted that above-mentioned various functional group do not plan to hint that the member of a functional group can not classify as the member of another functional group.In fact, looking particular condition will exist overlapping.Only for instance, water-soluble polymer and polyethyleneglycol derivative are overlapping on scope, yet, described overlapping and not exclusively and therefore two functional groups all quote hereinbefore.
As shown in Figure 1, on the one hand for using the method for method as herein described, composition and the choice of technology and design polypeptide to be finished.Can polypeptide from the beginning novel in design, comprise that (only for instance) is as the part (designing, synthesize, characterize and/or test multiple polypeptides under the described situation) of high-throughput screening method or be based upon on researcher's the basis of interest.Also can come polypeptide novel in design according to the structure of known or the polypeptide that part characterizes.Only for instance, growth hormone gene superfamily (vide infra) has become the theme of scientific circles' further investigation; Can come polypeptide novel in design according to one or more members' of this gene superfamily structure.Selecting to replace and/or modify the amino acid whose principle of which (which) will be in describing separately herein.Also will describe the selection of using which kind of modification herein, and described selection can be used for satisfying experimenter or terminal use's needs.Described needs can include but not limited to control the therapeutic efficiency of polypeptide; Improve the security features of polypeptide; Regulate pharmacokinetics, pharmacology and/or the pharmacodynamics of polypeptide, increase water-soluble, biological usability, increase serum half-life, increase the treatment half life period, regulate immunogenicity, regulate biologically active or prolong circulation timei such as (only for instance).In addition, described modification comprises that (only for instance) provides other functional group to polypeptide; Label, mark or detectable signal are incorporated in the polypeptide; Make the stalling characteristic facility of polypeptide; Any combination with above-mentioned modification.
This paper also describes to be modified into and contains diamines, dicarbapentaborane, ketone alkynes, ketoamine or heterocycle (comprising nitrogen heterocycle) or can be modified to contain the alpha-non-natural amino acid of diamines, dicarbapentaborane, ketone alkynes, ketoamine or heterocycle (comprising nitrogen heterocycle).Dicarbapentaborane can include but not limited to diketone, keto-aldehyde, ketone acid, ketone ester and ketone thioesters, and diamines can include but not limited to hydrazine, amidine, imines, 1,1-two amidos, 1,2-two amidos, 1,3-two amidos and 1,4-two amidos.The method that comprises generation, purifying, sign and the described alpha-non-natural amino acid of use in this respect.On the other hand, this paper describes and to incorporate at least one described alpha-non-natural amino acid in the polypeptide method, strategy and technology.Also comprise the described method that contains the polypeptide of at least one described alpha-non-natural amino acid of generation, purifying, sign and use in this respect.Also comprise generation, purifying, sign and use the oligonucleotides composition and the method for (comprising DNA and RNA) in this respect, described oligonucleotides can be used for producing the polypeptide that contains at least one alpha-non-natural amino acid to small part.The composition and the method that also comprise generation, purifying, sign and use cell in this respect, described cell can be expressed the described oligonucleotides that can be used for producing to small part the polypeptide that contains at least one alpha-non-natural amino acid.
Therefore, this paper provides and describes the polypeptide that comprises at least one alpha-non-natural amino acid or have the modified alpha-non-natural amino acid of diamines, dicarbapentaborane, ketone alkynes, ketoamine or heterocycle (comprising nitrogen heterocycle).The alpha-non-natural amino acid of modifying through dicarbapentaborane can include but not limited to diketone, keto-aldehyde, ketone acid, ketone ester and ketone thioesters, and can include but not limited to hydrazine, amidine, imines, 1 through diamine modified alpha-non-natural amino acid, 1-two amidos, 1,2-two amidos, 1,3-two amidos and 1,4-two amidos.In certain embodiments, the polypeptide that has at least one alpha-non-natural amino acid or contain the modified alpha-non-natural amino acid of diamines, dicarbapentaborane, ketone alkynes, ketoamine or heterocycle (comprising nitrogen heterocycle) comprises at least one common translation or posttranslational modification in certain position of polypeptide.In described embodiment, the alpha-non-natural amino acid of modifying through dicarbapentaborane can further include but not limited to diketone, keto-aldehyde, ketone acid, ketone ester and ketone thioesters, and can further include but not limited to hydrazine, amidine, imines, 1 through diamine modified alpha-non-natural amino acid, 1-two amidos, 1,2-two amidos, 1,3-two amidos and 1,4-two amidos.In certain embodiments; translation or posttranslational modification are (for example to take place via the cell machine altogether; glycosylation, acetylization, acidylate, lipid-modified, palmitoylation, palmitate addition, phosphorylation, the modification of glycolipid key etc.); in many cases; described common translation or posttranslational modification based on the cell machine is that naturally occurring amino acid sites place takes place on polypeptide; yet; in certain embodiments, be that alpha-non-natural amino acid site on polypeptide takes place based on the common translation of cell machine or posttranslational modification.
In other embodiments, posttranslational modification does not utilize the cellular machine device, but utilize chemical method as herein described or other to be applicable to that the method for specific reactivity group (includes but not limited to contain dicarbapentaborane by molecule (including but not limited to required functional group) that will comprise second reactive group and the alpha-non-natural amino acid that at least one comprises first reactive group, diketone, keto-aldehyde, ketone acid, ketone ester, the ketone thioesters, ketone alkynes, ketoamine, diamines, hydrazine, amidine, imines, 1, the 1-diamines, 1, the 2-diamines, 1, the 3-diamines, 1, the alpha-non-natural amino acid of 4-diamines or heterocycle (comprising nitrogen heterocyclic ring) functional group) connects functional group is provided.In certain embodiments, translation or posttranslational modification are in vivo carrying out in eukaryotic or non-eukaryotic altogether.In certain embodiments, translation or posttranslational modification are in vitro carried out under the situation of not utilizing the cellular machine device altogether.Also comprise the described method that contains the polypeptide of at least one described alpha-non-natural amino acid through posttranslational modification of generation, purifying, sign and use in this respect.
Also comprise in the scope of method as herein described, composition, strategy and technology and can (contain dicarbapentaborane, diketone, keto-aldehyde, ketone acid, ketone ester, ketone thioesters, ketone alkynes, ketoamine, diamines, hydrazine, amidine, imines, 1 with alpha-non-natural amino acid as the part of polypeptide; 1-diamines, 1; 2-diamines, 1; 3-diamines, 1,4-diamines or its are through the protection form) thus reaction produces any the reagent in the above-mentioned posttranslational modification.In general, gained will contain at least one heterocycle (comprising nitrogen heterocyclic ring) or aldehyde alcohol base through the alpha-non-natural amino acid of posttranslational modification; Modified heterocycle of gained or aldehyde alcohol base alpha-non-natural amino acid can experience follow-up modification reaction.Comprise also that in this respect generation, purifying, sign and use can carry out the described compositions and methods of any described posttranslational modification to described alpha-non-natural amino acid.
In certain embodiments, polypeptide comprises common translation or the posttranslational modification that at least one is undertaken by a kind of host cell in vivo, and wherein said posttranslational modification is not to be undertaken by another host cell type usually.In certain embodiments, polypeptide comprises at least one by common translation or posttranslational modification that eukaryotic carries out in vivo, and wherein said translation altogether or posttranslational modification are not to be undertaken by non-eukaryotic usually.The example of described altogether translation or posttranslational modification includes but not limited to glycosylation, acetylization, acidylate, lipid-modified, palmitoylation, palmitate addition, phosphorylation, the modification of glycolipid key etc.In one embodiment, altogether translation or posttranslational modification comprise by GlcNAc-asparagine key oligosaccharides to be connected with asparagine and (include but not limited to that oligosaccharides comprises (GlcNAc-Man) 2The situation of-Man-GlcNAc-GlcNAc etc.).In another embodiment, altogether translation or posttranslational modification comprise by GalNAc-serine, GalNAc-threonine, GlcNAc-serine or GlcNAc-threonine key oligosaccharides (including but not limited to Gal-GalNAc, Gal-GlcNAc etc.) are connected with serine or threonine.The example of secretory signal sequence include but not limited to prokaryotic secretion signal sequence, eucaryon secretory signal sequence, directed toward bacteria express 5 '-eucaryon secretory signal sequence, novel secretory signal sequence, transelminase secretory signal sequence, Omp A secretory signal sequence and the phage secretory signal sequence optimized.The example of secretory signal sequence includes but not limited to STII (prokaryotes), Fd GIII and M13 (phage), Bgl2 (yeast) and derives from the burst bla of transposons.In certain embodiments, protein or polypeptide can comprise secretion or positioning sequence, epi-position label, FLAG label, polyhistidyl label, GST fusion etc.Also comprise the described method that contains the polypeptide of at least one described translation altogether or posttranslational modification of generation, purifying, sign and use in this respect.In other embodiments, the glycosylation non-natural amino acid polypeptides is to produce with nonglycosylated form.Can produce the described non-glycosylated form of glycosylation alpha-non-natural amino acid by several different methods, described method comprise from through separate or purified in fact or not purified glycosylation non-natural amino acid polypeptides remove the oligosaccharides group with chemistry or enzymatic mode; In not making the glycosylated host of described non-natural amino acid polypeptides, produce alpha-non-natural amino acid (described host comprises through engineered or do not make glycosylated prokaryotes of described polypeptide or eucaryote through sudden change); Glycosylation inhibitor is introduced in the cell culture medium, and wherein said non-natural amino acid polypeptides is by the glycosylated eucaryote of described polypeptide is produced; Or the combination of any described method.This paper also describes the described non-glycosylated form of common glycosylated non-natural amino acid polypeptides (the glycosylation meaning is meant when producing glycosylated polypeptide usually) under making the glycosylated condition of naturally occurring polypeptide.Certainly, the described non-glycosylated form of common glycosylated non-natural amino acid polypeptides can be not purified form, purified in fact form or through unpack format.
Non-natural amino acid polypeptides can contain at least one, contain dicarbapentaborane, diketone, keto-aldehyde, ketone acid, ketone ester, ketone thioesters, ketone alkynes, ketoamine, diamines, hydrazine, amidine, imines, 1 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; 1-diamines, 1; 2-diamines, 1; 3-diamines, 1,4-diamines, heterocycle (comprising nitrogen heterocycle), aldehyde alcohol base or its alpha-non-natural amino acid through the protection form.Alpha-non-natural amino acid can be identical or different, for example can be in protein 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20 or more different loci place comprise 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20 or how different alpha-non-natural amino acids.In certain embodiments, existing at least one (but being less than all) specific amino acids replaces through alpha-non-natural amino acid in the protein of natural existence form.
The method and composition that this paper provided and described comprises polypeptide; it comprises at least one and contains dicarbapentaborane, diketone, keto-aldehyde, ketone acid, ketone ester, ketone thioesters, ketone alkynes, ketoamine, diamines, hydrazine, amidine, imines, 1; 1-diamines, 1; 2-diamines, 1; 3-diamines, 1,4-diamines, heterocycle (comprising nitrogen heterocycle), aldehyde alcohol base or its are through protection or through the alpha-non-natural amino acid of the form of covering.Will at least one alpha-non-natural amino acid introduce can allow in polypeptide to use and link chemistry, it relates to the specificity chemical reaction of (including but not limited to) and one or more alpha-non-natural amino acids, and not with the 20 seed amino acids reaction of common existence.After incorporating into, also can be by utilizing described herein or being applicable to that existing particular functional group in the natural amino acids coding or substituent chemical method modify the amino acid side chain that non-natural exists.
Alpha-non-natural amino acid method and composition as herein described provide have multiple functional group, the material of substituting group or part with include but not limited to the concatenator of other material of required functional group.
In certain embodiments, alpha-non-natural amino acid as herein described, non-natural amino acid polypeptides, connection base and reagent (compound that comprises formula I-LXVII) can be stablized in the aqueous solution down in the acid condition of appropriateness (including but not limited to that about pH2 is to about pH8).In other embodiments, described compound can be stablized 1 month under the condition of appropriateness acidity at least.In other embodiments, described compound can be stablized at least 2 weeks approximately under the condition of appropriateness acidity.In other embodiments, described compound can be stablized under the condition of appropriateness acidity at least 5 days approximately.
Composition as herein described, method, technology and strategy on the other hand for research or use the method for any above-mentioned modified or not modified non-natural amino acid polypeptides.Only for instance, comprise treatment, diagnosis in this respect, based on calibrating, industry, cosmetics, botany, environment, energy generation, the consumer goods and/or military use, it will benefit from the polypeptide that comprises modified or not modified non-natural amino acid polypeptides or protein.
III. the position of alpha-non-natural amino acid in the polypeptide
Method and composition as herein described comprises to be incorporated one or more alpha-non-natural amino acids in the polypeptide into.One or more alpha-non-natural amino acids can be incorporated into the specific location that one or more do not destroy polypeptide active.This can replace realization by carrying out " guarding ", includes but not limited to replace 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 in the active unwanted position.
Can use multiple biochemistry and structural approach to select the required site that replaces for alpha-non-natural amino acid in the polypeptide.Any position of polypeptide chain all is applicable to selection incorporating alpha-non-natural amino acid into, and selects to be based upon on the basis of appropriate design or by selecting at random to realize any or to be not the purpose of special needs.The selection in required site can have the non-natural amino acid polypeptides (it can or keep not modified through further modification) of any desirable characteristics or activity based on generation, includes but not limited to activator, super agonist, partial agonist, inverse agonist, antagonist, receptors bind conditioning agent, receptor activity modulators, the conditioning agent with the combination that combines the collocation thing, combination collocation thing active regulator, combination collocation thing conformation conditioning agent; Dimer or polymer form; Specific activity or characteristic do not have change mutually with natural molecule; Perhaps control any physics of polypeptide or chemical property (such as, dissolubility, gathering or stability).For instance, can use the method that includes but not limited to point mutation analysis, alanine scanning or homologue scan method to differentiate and be polypeptide biologically active desired position in the polypeptide.Except that differentiating the good candidate that replaces for alpha-non-natural amino acid for being to the visual required activity of looking for for polypeptide of the residue the vital residue of biologically active by the method that includes but not limited to alanine or homologue scanning mutagenesis.Perhaps, through differentiating the good candidate that replaces for alpha-non-natural amino acid for being to the also visual required activity of looking for for polypeptide in the vital site of biologically active.Another alternative method will be for utilizing alpha-non-natural amino acid to replace continuously simply and observing influence to polypeptide active in the indivedual positions on polypeptide chain.Any way, technology or method that selection is used for alpha-non-natural amino acid is replaced the position of any polypeptide all are applicable in method as herein described, technology and the composition.
Also can check the natural structure that has a mutant that contains disappearance and the active protein zone of polypeptide with the replacement of determining to tolerate probably alpha-non-natural amino acid.In that remove possibly can't tolerate behind the residue of replacement of alpha-non-natural amino acid, the influence that is substituted in each rest position place that can use the method inspection of the part that includes but not limited to related polypeptide and any association or protein-bonded three-dimensional structure to be recommended.The X-ray crystallography of many polypeptide and NMR structure all can be from Protein Data Bank (Protein Data Bank, PDB; Www.rcsb.org) obtain, PDB is that the central database and can be used for that contains the three-dimensional structure data of protein and nucleic acid molecule is differentiated the amino acid position that can replace through alpha-non-natural amino acid.In addition, if can't obtain three-dimensional structure data, can set up the model of research polypeptide secondary structure and tertiary structure so.Therefore, can easily obtain the identity of the amino acid position that can replace through alpha-non-natural amino acid.
The exemplary site of incorporating alpha-non-natural amino acid into includes but not limited to: be not included in potential receptors bind zone or be used for site with the zone that combines albumen or part combination; Can be exposed to the site of solvent wholly or in part; Have minimum with contiguous residue or do not have the site of interaction of hydrogen bond; But bottom line is exposed to the site of contiguous reactive residue; And/or can as the flexible zone of height of estimating by specific polypeptide and its association acceptor, part or protein-bonded three-dimensional crystalline structure in the site.
Multiple alpha-non-natural amino acid can replace the ad-hoc location in the polypeptide or incorporate in the ad-hoc location in the polypeptide.For instance, select for the specific alpha-non-natural amino acid of incorporating into preferred conservative the replacement according to the inspection of polypeptide and its association part, acceptor and/or protein-bonded three-dimensional crystalline structure.
In one embodiment, method as herein described comprises to be incorporated alpha-non-natural amino acid in the polypeptide into, and wherein said alpha-non-natural amino acid comprises first reactive group; With described polypeptide is contacted with the molecule that comprises second reactive group (including but not limited to required functional group).In certain embodiments, first reactive group is that carbonyl or the dicarbapentaborane part and second reactive group are two amine moieties, forms heterocyclic bond thus.In certain embodiments, first reactive group is that two amine moieties and second reactive group are carbonyl or dicarbapentaborane part, forms heterocyclic bond thus.
In some cases, one or more alpha-non-natural amino acids are replaced or incorporate into polypeptide in other interpolation, replacement or disappearance combination to influence other chemistry, physics, pharmacology and/or biological nature.In some cases, described other adds, replaces 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, part and/or protein-bonded affinity.In some cases, described other interpolation, replacement or disappearance can increase the dissolubility (including but not limited to when expressing) of polypeptide in Escherichia coli or other host cell.In certain embodiments, for increasing the deliquescent purpose of polypeptide, after in Escherichia coli or other recombinant host cell, expressing, select other site except that another site of incorporating alpha-non-natural amino acid into to replace for natural coding or alpha-non-natural amino acid.In certain embodiments, polypeptide comprises another interpolation, replacement or disappearance, and it is regulated to the association part, in conjunction with the affinity of albumen and/or acceptor; Regulate (including but not limited to increases or reduce) receptor dimerizationization; Make receptor dimer stable; Regulate circulating half-life; Adjustment release or biological usability; Be beneficial to purifying; Perhaps improve or change specific dosing way.Similarly, non-natural amino acid polypeptides can comprise chemistry or enzymatic lysis sequence, protease cracking sequence, reactive group, antibody binding domain (including but not limited to FLAG or poly histidine) or contain the sequence (including but not limited to FLAG, poly histidine, GST etc.) of other compatibility, perhaps connect molecule (including but not limited to vitamin h), described connection molecule improves and detects that (including but not limited to GFP), transhipment, prodrug release or activation, size by tissue or cell membrane reduce, other characteristic of purifying or polypeptide.
IV. as the somatotropin supergene family of sample
Method as herein described, composition, strategy and technology are not limited to particular type, kind or the family of polypeptide or protein.In fact, almost any polypeptide all can be through design or modified to comprise at least one modified or not modified alpha-non-natural amino acid as herein described.Only for instance, polypeptide can with the therapeutic protein homology that is selected from the group that forms by following each thing: α-1 antitrypsin, angiostatin, AHF, antibody, antibody fragment, apolipoprotein (apolipoprotein), apoprotein (apoprotein), atrionatriuretic factor, atrial natriuretic polypeptins, atrial natriuretic peptide, the C-X-C chemotactic factor (CF), 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 part, cell factor, the CC chemotactic factor (CF), monocyte chemoattractant protein-1, monocyte chemoattractant protein-2, MCP-3, monocyte inflammatory protein-1 α, monocyte inflammatory protein-i β, RANTES, 1309, R83915, R91733, HCC1, T58847, D31065, T64262, CD40, the CD40 part, the c-kit part, collagen, group's stimulating factor (CSF), complement factor 5a, complement inhibitor, complement receptor 1, cell factor, epithelium neutrophilic granulocyte activation peptide-78, MIP-16, MCP-1, epidermal growth factor (EGF), epithelium neutrophilic granulocyte activation peptide, hematopoietin (EPO), toxin comes off, factors IX, factor VII, Factor IX, factor X, fibroblast growth factor (FGF), fibrinogen, fiber adhesion albumen, four-helix bundle albumen, G-CSF, glp-1, GM-CSF, glucocerebrosidase, gonadotropin, growth factor, growth factor receptors, grf, Hedgelog protein, haemoglobin, hepatocyte growth factor (hGF), hirudin, human growth hormone (hGH), human serum albumins, ICAM-1, the ICAM-1 acceptor, LFA-1, the LFA-1 acceptor, insulin, insulin-like growth factor (IGF), IGF-I, IGF-II, interferon (IFN), IFN-α, IFN-β, IFN-γ, any interferoid molecule or IFN family member, interleukins (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, neurotrophic factor (neurturin), neutrophil inhibitory factor (nif) (NIF), tumour inhibitor M (oncostatinM), BMP, oncoprotein, paracitonin, parathyroid hormone, PD-ECSF, PDGF, peptide hormone, pleiotropin, a-protein, protein G, pth, the pyrogenicity exotoxin A, pyrogenicity exotoxin B, PEC, pyy, relaxins, feritin, SCF, the atom synthetic proteins, soluble complement acceptor I, solubility I-CAM1, soluble interleukin receptor, soluble TNF acceptor, somatomedin (somatomedin), growth hormone release inhibiting hormone (somatostatin), somatotropin (somatotropin), streptokinase, super antigen (superantigen), staphylococcal enterotoxin (staphylococcal enterotoxin), SEA, SEB, SEC1, SEC2, SEC3, SED, SEE, steroid hormone receptor, superoxide dismutase, the toxic shock syndrome toxin, Thymosin alpha 1, tissue type plasminogen activator, TGF (TGF), TNF, tumor necrosis factor, 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, progesterone receptor, the stosterone acceptor, aldosterone receptor, ldl receptor and cortisone (being called " required polypeptide " hereinafter).
Therefore, provide following description by example for illustration purposes and only, and should not be construed as the scope of restriction method as herein described, composition, strategy and technology about somatotropin (GH) supergene family.In addition, mention the example of the described generic term of GH polypeptide plan use in the application's case as any member of GH supergene family.Therefore, should be appreciated that this paper about GH polypeptide or described modification of protein and the chemical any member who can be applicable to the GH supergene family equally, comprises the member who lists especially herein.
Following protein comprises by the protein of the coded by said gene of somatotropin (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.,
Figure A200680049995D0063144448QIETU
BY THE
Figure A200680049995D0063144501QIETU
Figure A200680049995D0063144517QIETU
Figure A200680049995D0063144526QIETU
(1996)): somatotropin, galactin, galactagogin, hematopoietin (EPO), TPO (TPO), interleukin 2 (IL-2), IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-11, IL-12 (p35 subunit), IL-13, IL-15, tumour inhibitor M, the cilium neurotrophic factor, leukaemia inhibitory factor, interferon-alpha, interferon-, the ε interferon, IFN-, omega interferon, the τ interferon, granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage group stimulating factor (GM-CSF), macrophage group stimulating factor (M-CSF) and heart nutrient-1 (cardiotrophin-1) (" GH supergene family ").Expection will be differentiated other member of this gene family by gene clone and order-checking in the future.The member of GH supergene family has similar secondary and tertiary structure, although it has limited amino acid or consensus dna sequence usually.Total architectural feature makes that the newcomer and the alpha-non-natural amino acid method and composition as herein described that are easy to sldh gene family are suitable equally.
Measured the structure of the various kinds of cell factor by X-ray diffraction and NMR research, described cell factor comprises 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 Science154:1779-1782 (1991) such as K.; 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 (, Nature 363:172-176 (1993)) Milburn, although and lack significant primary sequence autoploidy, described structure is still showed surprising conservative of GH structure.According to modeling and other research, think the IFN member of family (people such as Lee, J.Interferon.Cytokine Res.15:341 (1995) for this reason; 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)).A large amount of other cell factors and growth factor (comprise cilium neurotrophic factor (CNTF), leukaemia inhibitory factor (LIF), TPO (TPO), tumour inhibitor M, macrophage group stimulating factor (M-CSF), IL-3, IL-6, IL-7, IL-9, IL-12, IL-13, IL-15 and granulocyte colony stimulating factor (G-CSF), and IFN, such as interferon-alpha, interferon-, omega interferon, the τ interferon, ε interferon and IFN-) all belong to this family and (summarize in Mott and Campbell CurrentOpinion in Structural Biology 5:114-121 (1995); Silvennoinen and Ihle (1996) SIGNALLINGBY THE HEMATOPOIETIC
Figure A200680049995D0064144251QIETU
Figure A200680049995D0064144309QIETU
In).Think that now all above-mentioned cell factors and growth factor constitute a big gene family.
Except that total similar secondary and tertiary structure, the member of this family also has a specific character, promptly its must the oligomerization cell surface receptor with signal transduction path in the activating cell.Some GH family members (including but not limited to GH and EPO) are in conjunction with the acceptor of single type and make it form homodimer.Other family member (including but not limited to IL-2, IL-4 and IL-6) is in conjunction with the acceptor of more than one types and make acceptor form heterodimer or more senior aggregation (people such as Davis, (1993) Science 260:1805-1808; People such as Paonessa, 1995) EMBO is J.14:1942-1951; Mott and Campbell, Current Opinion in Structural Biology 5:114-121 (1995)).Mutagenesis research shows, the same with GH, these other cell factors and growth factor contain a plurality of (common two) receptor binding site, and successively in conjunction with its homoreceptor (Mott and Campbell, Current Opinion in Structural Biology 5:114-121 (1995); People such as Matthews, (1996) Proc.Natl.Acad.Sci.USA 93:9471-9476).The same with GH, these other family members' major receptors binding site mainly appears in four α spirals and the A-B ring.Specific amino acids in the helical bundle of participation receptors bind is different between the family member.Structurally relevant with the interactional most of cell surface receptor of GH supergene family member and comprise another big multigene family.For example referring to United States Patent (USP) the 6th, 608, No. 183, its mode of quoting in full is incorporated herein.
The common conclusions that obtains from relevant each member's of GH supergene family mutation research is: the ring that connects the α spiral tends to receptors bind irrelevant usually.Specifically, lack the B-C ring and seem unimportant for most of (if being not whole) family member's receptors bind.For this reason, in GH supergene family member, the B-C ring can replace through alpha-non-natural amino acid as described herein.A-B ring, C-D ring (with the interferoid/IL-10 member's of GH superfamily D-E ring) also can replace through alpha-non-natural amino acid.Near spiral A and also tend to irrelevant and can be the site of introducing alpha-non-natural amino acid away from the amino acid of last spiral with receptors bind.In certain embodiments, alpha-non-natural amino acid is substituted any position in ring structure, includes but not limited to preceding 1,2,3,4,5,6,7 or the amino acids more of A-B, B-C, C-D or D-E ring.In certain embodiments, alpha-non-natural amino acid is substituted in last 1,2,3,4,5,6,7 or more in the amino acids of A-B, B-C, C-D or D-E ring.
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-) contains that N is connected and/or O connects sugar.Glycosylation site in the protein almost only appears in the ring zone and does not appear in the α helical bundle.Because the ring zone is common and receptors bind is irrelevant, and, alpha-non-natural amino acid is replaced the useful site of introducing protein so it can be because it is the covalently bound site of glycosyl.These amino acid connect the amino acid surface exposure that is connected glycosylation site with O owing to comprise N in the protein, so can be the site that alpha-non-natural amino acid replaces.Therefore, the huge glycosyl of native protein tolerable is connected to protein in these site, and glycosylation site tends to away from receptor binding site.
May other member of GH gene family will be found in future.Area of computer aided secondary that can be by institute's predicted protein matter sequence and tertiary structure analysis and the newcomer who differentiates the GH supergene family through design with the selection technology of differentiating the molecule that combines with specific target.The member of GH supergene family has four or five amphiphilic spirals that connect by non-helical amino acid (ring zone) usually.Protein can contain the hydrophobic signal sequence to promote emiocytosis at its N end.Described newfound GH supergene family member is also included within the method and composition as herein described.
V. alpha-non-natural amino acid
The alpha-non-natural amino acid that is used for method and composition as herein described has at least one in following four characteristics: at least one functional group on (1) alpha-non-natural amino acid side chain has at least a and amino acid (that is alanine, 20 kinds of common gene codes, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenyl alanine, proline, serine, threonine, tryptophan, tyrosine and valine) the chemical reactivity quadrature or at least with the feature that is present in the naturally occurring amino acid whose chemical reactivity quadrature in the polypeptide that comprises alpha-non-natural amino acid and/or active and/or reactive; (2) alpha-non-natural amino acid of being introduced is inertia at the amino acid to 20 kinds of common gene codes chemically in fact; (3) alpha-non-natural amino acid stably can be incorporated in the polypeptide, preferably be had the stability suitable or stable under typical physiological condition, and preferred described in addition incorporating into can be via in vivo system's generation with naturally occurring amino acid; And (4) alpha-non-natural amino acid comprises dicarbapentaborane, diketo, ketaldonyl, the ketone acid base, the ketone ester base, the ketone thioester substrate, the ketone alkynyl, the ketoamine base, diamines, the aldehyde alcohol base, two amidos, diazanyl, amidino groups, imido grpup, 1,1-two amidos, 1,2-two amidos, 1,3-two amidos, 1,4-two amidos, heterocycle (comprising nitrogen heterocycle), perhaps can be by preferably (certain in the biological nature of not destroying the polypeptide that comprises alpha-non-natural amino acid with reagent, except that the destruction of described biological nature is the situation of purpose of modification/conversion) condition under reaction and change into dicarbapentaborane, diketo, ketaldonyl, the ketone acid base, the ketone ester base, the ketone thioester substrate, the ketone alkynyl, the ketoamine base, diamines, the aldehyde alcohol base, two amidos, diazanyl, amidino groups, imido grpup, 1,1-two amidos, 1,2-two amidos, 1,3-two amidos, 1,4-two amidos, the functional group of heterocycle (comprising nitrogen heterocycle), perhaps preferred wherein said conversion can be under aqueous conditions between under the pH value between about 4 and about 10 or between between about 3 and about 8 or between taking place between about 2 to about 9 or under the pH value between about 4 and about 9, or the reactive site on the preferred wherein alpha-non-natural amino acid is close potential point.The amino acid whose illustrative limiting examples that satisfies this four specific character of alpha-non-natural amino acid that can be used for composition as herein described and method is provided among Fig. 2-4.Any amount of alpha-non-natural amino acid can be introduced in the polypeptide.Alpha-non-natural amino acid also can comprise through protection or dicarbapentaborane, heterocycle (comprising nitrogen heterocycle), ketone alkynes, ketoamine, aldehyde alcohol base, two amidos through covering, perhaps make through blocking group go protection or make through cover can change into after group goes to cover dicarbapentaborane, heterocycle (comprising nitrogen heterocycle), ketone alkynes, ketoamine, aldehyde alcohol base or two amidos through protection or through covering group.
But can be used for the amino acid that alpha-non-natural amino acid in the method and composition as herein described includes but not limited to comprise the photoactivated cross-linking agent, the amino acid of spin labeling, fluorescence amino acid, metal is in conjunction with 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 molecule, but the light cage covers and/or photoisomerization amino acid, the amino acid that comprises vitamin h or vitamin h analog, glycosylation amino acid (such as, sugar-substituted serine), the amino acid of other carbohydrate modification, ketone group containing amino acid, the amino acid that comprises polyethylene glycol or other polyethers, the amino acid that heavy atom replaces, but but chemical cracking and/or photodestruciton amino acid, compare the amino acid (include but not limited to polyethers or long chain hydrocarbon, include but not limited to) of side chain with prolongation greater than about 5 or greater than about 10 carbon with natural amino acid, carbon containing connects the amino acid of sugar, redox active amino acids, the amino acid and the amino acid that comprises one or more toxic moieties that contain amino thio-acid.
In certain embodiments, alpha-non-natural amino acid comprises sugar moieties.Described amino acid whose example comprises N-acetyl group-L-glucose amido-L-serine, N-acetyl group-L-galactose amido-L-serine, N-acetyl group-L-glucose amido-L-threonine, N-acetyl group-L-glucose amido-altheine and O-mannose amido-L-serine.Described amino acid whose example also comprises naturally occurring N key between amino acid and the sugar or the O key example through the uncommon covalent bond of occurring in nature (including but not limited to alkene, oxime, thioesters, acid amides, heterocycle (comprising nitrogen heterocyclic ring), dicarbapentaborane etc.) displacement.Described amino acid whose example also comprises uncommon sugar in the naturally occurring protein, such as 2-deoxidation-glucose, 2-deoxy-galactose etc.
Via alpha-non-natural amino acid being incorporated in the polypeptide and incorporate multiple advantage and the manipulation that chemical part in the described polypeptide provides described polypeptide into.For instance, Du Te alpha-non-natural amino acid (including but not limited to have the amino acid of benzophenone and nitrine aryl (including but not limited to the phenylazide side chain)) for example allows in vivo and photo-crosslinking protein effectively in vitro.The example of photoreactivity alpha-non-natural amino acid includes but not limited to azido-phenyl alanine with to benzoyl-phenyl alanine.Subsequently can be arbitrarily crosslinked by the polypeptide that excites the interim contrast that photoreactive group is provided to make to have the photoreactivity alpha-non-natural amino acid.In a limiting examples, the methyl of alpha-non-natural amino acid can be through tagging (including but not limited to) methyl substituted as partial structurtes and dynamics probe (including but not limited to use nuclear magnetic resonnance and vibrational spectrum).
A. the structure of alpha-non-natural amino acid and synthetic: two amidos, class two amidos, through covering two amidos and through protecting two amidos
Amino acid with nucleophilic reactivity group allows especially to connect via electrophilic addition reaction the various reactions of molecule.Described nucleophilic reactivity group comprises that two amidos (comprise diazanyl, amidino groups, imido grpup, 1; 1-two amidos, 1; 2-two amidos, 1; 3-two amidos and 1,4-two amidos), class two amidos (it has with reactive like the diamines base class and structurally be similar to two amidos), through covering two amidos (it can easily change into two amidos) or through protecting two amidos (it has after going protection and be reactive like the diamines base class).Described amino acid comprises the amino acid of (I) structure that has formula:
Figure A200680049995D00671
Wherein:
A is optional, and when existing, it is the low-carbon (LC) alkylidene, be substituted low-carbon (LC) alkylidene, low-carbon (LC) cycloalkylidene, be substituted the low-carbon (LC) cycloalkylidene, the low-carbon (LC) alkenylene, be substituted low-carbon (LC) alkenylene, alkynylene, the inferior assorted alkyl of low-carbon (LC), be substituted inferior assorted alkyl, the inferior Heterocyclylalkyl of low-carbon (LC), be substituted low-carbon (LC) inferior Heterocyclylalkyl, arlydene, be substituted arlydene, inferior heteroaryl, be substituted inferior heteroaryl, alkarylene, be substituted alkarylene, inferior aralkyl or be substituted inferior aralkyl;
B is optional, and when existing, its at one end with contain the connection base that two amine moieties are connected, described connection base is selected from the group that is made up of following each group: the low-carbon (LC) alkylidene, be substituted low-carbon (LC) alkylidene, low-carbon (LC) alkenylene, be substituted the low-carbon (LC) alkenylene, the inferior assorted alkyl of low-carbon (LC), be substituted the low-carbon (LC) Asia mix alkyl ,-O-(alkylidene or be substituted alkylidene)-,-S-(alkylidene or be substituted alkylidene)-,-C (O) R " ,-C (O) R "-,-S (O) k(alkylidene or be substituted alkylidene)-(wherein k is 1,2 or 3) ,-C (O)-(alkylidene or be substituted alkylidene)-,-C (S)-(alkylidene or be substituted alkylidene)-,-NR " (alkylidene or be substituted alkylidene)-,-CON (R ")-(alkylidene or be substituted alkylidene)-,-CSN (R ")-(alkylidene or be substituted alkylidene)-and-N (R ") CO-(alkylidene or be substituted alkylidene)-, each R wherein is " independently for H, alkyl or be substituted alkyl;
J is
Figure A200680049995D00672
Or
Figure A200680049995D00673
Wherein:
R 8And R 9Be independently selected from H, alkyl, be substituted alkyl, cycloalkyl, be substituted cycloalkyl or amine protecting group;
T 1Be bond, the C that is substituted according to circumstances 1-C 4Alkylidene, the C that is substituted according to circumstances 1-C 4Alkenylene or the assorted alkyl that is substituted according to circumstances;
T 2Be the C that is substituted according to circumstances 1-C 4Alkylidene, the C that is substituted according to circumstances 1-C 4Alkenylene, the assorted alkyl that is substituted according to circumstances, the aryl that is substituted according to circumstances or the heteroaryl that is substituted according to circumstances;
Wherein each optional substituting group is independently selected from low-carbon alkyl, is substituted low-carbon alkyl, low-carbon naphthenic, is substituted low-carbon naphthenic, low-carbon (LC) thiazolinyl, is substituted the low-carbon (LC) thiazolinyl, alkynyl, the assorted alkyl of low-carbon (LC), is substituted assorted alkyl, low-carbon (LC) Heterocyclylalkyl, is substituted the low-carbon (LC) Heterocyclylalkyl, aryl, is substituted aryl, heteroaryl, is substituted heteroaryl, alkaryl, is substituted alkaryl, aralkyl or is substituted 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;
R 3And R 4Be H, halogen, low-carbon alkyl or be substituted low-carbon alkyl, perhaps R independently of one another 3And R 4Or two R 3Group forms cycloalkyl or Heterocyclylalkyl according to circumstances;
Or-the A-B-J-R group form together comprise at least one two amido, through protecting two amidos or dicyclo or tricyclic naphthenes base or Heterocyclylalkyl through covering two amidos;
Or-the B-J-R group form together comprise at least one two amido, through protecting two amidos or dicyclo or tricyclic naphthenes base or cyclophane base or Heterocyclylalkyl through covering two amidos;
Or-the J-R group form together comprise at least one two amido, through protecting two amidos or monocycle or bicyclic cycloalkyl or Heterocyclylalkyl through covering two amidos;
Wherein-last at least one amido of A-B-J-R is the amine through protection according to circumstances.
On the one hand for comprising the compound of structure 1 or 2:
Wherein:
A is optional, and when existing, it is the low-carbon (LC) alkylidene, be substituted low-carbon (LC) alkylidene, low-carbon (LC) cycloalkylidene, be substituted the low-carbon (LC) cycloalkylidene, the low-carbon (LC) alkenylene, be substituted low-carbon (LC) alkenylene, alkynylene, the inferior assorted alkyl of low-carbon (LC), be substituted inferior assorted alkyl, the inferior Heterocyclylalkyl of low-carbon (LC), be substituted low-carbon (LC) inferior Heterocyclylalkyl, arlydene, be substituted arlydene, inferior heteroaryl, be substituted inferior heteroaryl, alkarylene, be substituted alkarylene, inferior aralkyl or be substituted inferior aralkyl;
B is optional, and when existing, its at one end with contain the connection base that two amine moieties are connected, described connection base is selected from the group that is made up of following each group: the low-carbon (LC) alkylidene, be substituted low-carbon (LC) alkylidene, low-carbon (LC) alkenylene, be substituted the low-carbon (LC) alkenylene, the inferior assorted alkyl of low-carbon (LC), be substituted the low-carbon (LC) Asia mix alkyl ,-O-(alkylidene or be substituted alkylidene)-,-S-(alkylidene or be substituted alkylidene)-,-C (O) R " ,-S (O) k(alkylidene or be substituted alkylidene)-(wherein k is 1,2 or 3) ,-C (O)-(alkylidene or be substituted alkylidene)-,-C (S)-(alkylidene or be substituted alkylidene)-,-NR " (alkylidene or be substituted alkylidene)-,-CON (R ")-(alkylidene or be substituted alkylidene)-,-CSN (R ")-(alkylidene or be substituted alkylidene)-and-N (R ") CO-(alkylidene or be substituted alkylidene)-, each R wherein is " independently for H, alkyl or be substituted alkyl;
T 1Be bond or CH 2And T 2Be CH;
Wherein each optional substituting group is independently selected from low-carbon alkyl, is substituted low-carbon alkyl, low-carbon naphthenic, is substituted low-carbon naphthenic, low-carbon (LC) thiazolinyl, is substituted the low-carbon (LC) thiazolinyl, alkynyl, the assorted alkyl of low-carbon (LC), is substituted assorted alkyl, low-carbon (LC) Heterocyclylalkyl, is substituted the low-carbon (LC) Heterocyclylalkyl, aryl, is substituted aryl, heteroaryl, is substituted heteroaryl, alkaryl, is substituted alkaryl, aralkyl or is substituted aralkyl;
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 3And R 4Be H, halogen, low-carbon alkyl or be substituted low-carbon alkyl, perhaps R independently of one another 3And R 4Or two R 3Group forms cycloalkyl or Heterocyclylalkyl according to circumstances;
Or-A-B-contain two amine moieties form together comprise at least one two amido, through protecting two amidos or bicyclic cycloalkyl through covering two amidos or Heterocyclylalkyl;
Or-B-contain two amine moiety groups form together comprise at least one two amido, through protecting two amidos or dicyclo or tricyclic naphthenes base or cyclophane base or Heterocyclylalkyl through covering two amidos;
Wherein-A-B-contains on two amine moieties at least one amido according to circumstances for through protection amine;
Or its active metabolite, salt or pharmaceutically acceptable prodrug or solvate.
An embodiment is the compound of structure 1 or 2, and wherein A is the low-carbon (LC) alkylidene that is substituted or is unsubstituted, or the arlydene that is selected from the group that is made up of phenylene, inferior pyridine radicals, inferior pyrimidine radicals or inferior thienyl that is unsubstituted or is substituted.Another embodiment is the compound of structure 1 or 2, wherein B be the low-carbon (LC) alkylidene, be substituted the low-carbon (LC) alkylidene ,-O-(alkylidene or be substituted alkylidene)-,-C (O)-(alkylidene or be substituted alkylidene)-,-CON (R ")-(alkylidene or be substituted alkylidene)-,-S (alkylidene or be substituted alkylidene)-,-S (O) (alkylidene or be substituted alkylidene)-or-S (O) 2(alkylidene or be substituted alkylidene)-.Another embodiment is the compound of structure 1 or 2, and wherein B is-O (CH 2)-,-NHCH 2-,-C (O)-(CH 2)-,-CONH-(CH 2)-,-SCH 2-,-S (=O) CH 2-or-S (O) 2CH 2-.Another embodiment is the compound of structure 1 or 2, wherein R 1Be H, tertbutyloxycarbonyl (Boc), 9-fluorenyl methoxy carbonyl (Fmoc), N-acetyl group, tetrafluoro acetyl group (TFA) or benzene methoxycarbonyl group (Cbz).Compound according to claim 1, wherein R 1Be resin, amino acid, polypeptide or polynucleotide.Another embodiment is the compound of structure 1 or 2, wherein R 2Be OH, O-methyl, O-ethyl or the O-tert-butyl group.Another embodiment is the compound of structure 1 or 2, wherein R 2Be resin, amino acid, polypeptide or polynucleotide.Another embodiment is the compound of structure 1 or 2, wherein R 2Be polynucleotide.Another embodiment is the compound of structure 1 or 2, wherein R 2Be ribonucleic acid (RNA).Other embodiment is the compound of structure 1 or 2, wherein R 2Be tRNA.Another embodiment is the compound of structure 1 or 2, wherein tRNA identification selection codon specifically.Another embodiment is the compound of structure 1 or 2, wherein selects codon to be selected from by the molecular group of following password: amber codon, ochre codon, opal codon, unique codon, rare codon, non-natural codon, five base codons and four base codons.Other embodiment is the compound of structure 1 or 2, wherein R 2For suppressing tRNA.
The amino acid whose following limiting examples that comprises (I) structure that has formula:
Figure A200680049995D00701
Figure A200680049995D00702
With
Figure A200680049995D00703
Described alpha-non-natural amino acid also can be salt form, maybe can incorporate in non-natural amino acid polypeptides, polymer, polysaccharide or the polynucleotide and/or according to circumstances through posttranslational modification.
In certain embodiments, formula (I) compound can be stablized 1 month in the aqueous solution under the acid condition of gentleness at least.In certain embodiments, formula (I) compound can stablize at least 2 weeks under the acid condition of gentleness.In certain embodiments, formula (I) compound can be stablized 5 days under the acid condition of gentleness at least.In certain embodiments, described acid condition is about 2 to about 8 pH value.
In some embodiment of formula (I) compound, B is the low-carbon (LC) alkylidene, be substituted the low-carbon (LC) alkylidene ,-O-(alkylidene or be substituted alkylidene)-, C (R ')=NN (R ')-,-N (R ') CO-, C (O)-,-C (R ')=N-,-C (O)-(alkylidene or be substituted alkylidene)-, CON (R ') (alkylidene or be substituted alkylidene)-,-S (alkylidene or be substituted alkylidene)-,-S (O) (alkylidene or be substituted alkylidene)-or-S (O) 2(alkylidene or be substituted alkylidene)-.In some embodiment of formula (I) compound, B is-O (CH 2)-,-CH=N-, CH=NNH-,-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-acetyl group, tetrafluoro acetyl group (TFA) or benzene methoxycarbonyl group (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-tert-butyl group.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 ribonucleic acid (RNA).In some embodiment of formula (I) compound, R 2Be tRNA.In some embodiment of formula (I) compound, described tRNA is the identification selection codon specifically.In some embodiment of formula (I) compound, select codon to be selected from: amber codon, ochre codon, opal codon, unique codon, rare codon, non-natural codon, five base codons and four base codons by the molecular group of following password.In some embodiment of formula (I) compound, R 2For suppressing tRNA.
In addition, the amino acid with formula (I) structure comprises the amino acid of (II) structure that has formula:
Figure A200680049995D00711
Each R wherein aBe independently selected from the group that forms by following each group: H, halogen, alkyl, 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 each R ' is H, alkyl independently, is substituted alkyl, cycloalkyl or is substituted cycloalkyl.
Other or extra embodiment are and structure 3 or 4 corresponding compounds:
Figure A200680049995D00721
Each R wherein aBe independently selected from the group that forms by following each group: H, halogen, alkyl, be substituted alkyl ,-N (R ") 2,-C (O) N (R ") 2,-OR " and-S (O) kR ", wherein k is 1,2 or 3, wherein each R " is independently for H, alkyl or be substituted alkyl.
Also comprise amino acid whose following limiting examples with formula (II) structure:
Figure A200680049995D00722
Figure A200680049995D00723
With
Figure A200680049995D00724
Described alpha-non-natural amino acid also can be salt form, maybe can incorporate in non-natural amino acid polypeptides, polymer, polysaccharide or the polynucleotide and/or according to circumstances through posttranslational modification.
Amino acid with formula (I) structure also can be have formula (III) structure through the protection form:
Figure A200680049995D00731
Wherein, Prot is an amine protecting group, includes but not limited to:
Figure A200680049995D00733
Or
Figure A200680049995D00734
In certain embodiments, at least one amido of group J can be through protection; Or in other embodiments, two amidos are all through protection.
What in addition, have formula (III) structure comprises the amino acid of (IV) structure that has formula through protection amino acid:
Figure A200680049995D00735
Have formula (IV) structure through protecting amino acid whose limiting examples to comprise:
Figure A200680049995D00736
With
Figure A200680049995D00741
Described alpha-non-natural amino acid also can be salt form, maybe can incorporate in non-natural amino acid polypeptides, polymer, polysaccharide or the polynucleotide and/or according to circumstances through posttranslational modification.
Another embodiment is and has at least one to have the polypeptide of the compound of structure 1 or 2.
Another embodiment is a polypeptide, wherein said polypeptide for the protein of the therapeutic protein homology that is selected from the group that forms by required polypeptide.
Other limiting examples that contains the diamines alpha-non-natural amino acid is showed among Fig. 2.Contain and amino acid whose non-limiting exemplary synthesizing of diamines be described in herein and be provided among Fig. 7 and Fig. 8.
B. the structure of alpha-non-natural amino acid and synthetic: dicarbapentaborane, class dicarbapentaborane, through covering dicarbapentaborane and through the protection dicarbapentaborane
Amino acid with electrophilic reaction group allows especially to connect via nucleophilic addition the various reactions of molecule.Described electrophilic reaction group comprises dicarbapentaborane (comprising diketo, ketaldonyl, ketone acid base, ketone ester base and ketone thioester substrate), class dicarbapentaborane (it has similarly reactive and structurally be similar to dicarbapentaborane with dicarbapentaborane), through covering dicarbapentaborane (it can easily change into dicarbapentaborane) or through protection dicarbapentaborane (its going protection then have similarly reactive) with dicarbapentaborane.Described amino acid comprises the amino acid of (V) structure that has formula:
Figure A200680049995D00742
Wherein:
A is optional, and when existing, it is the low-carbon (LC) alkylidene, be substituted low-carbon (LC) alkylidene, low-carbon (LC) cycloalkylidene, be substituted the low-carbon (LC) cycloalkylidene, the low-carbon (LC) alkenylene, be substituted low-carbon (LC) alkenylene, alkynylene, the inferior assorted alkyl of low-carbon (LC), be substituted inferior assorted alkyl, the inferior Heterocyclylalkyl of low-carbon (LC), be substituted low-carbon (LC) inferior Heterocyclylalkyl, arlydene, be substituted arlydene, inferior heteroaryl, be substituted inferior heteroaryl, alkarylene, be substituted alkarylene, inferior aralkyl or be substituted inferior aralkyl;
B is optional, and when existing, its at one end with contain the connection base that two amine moieties are connected, described connection base is selected from the group that is made up of following each group: the low-carbon (LC) alkylidene, be substituted low-carbon (LC) alkylidene, low-carbon (LC) alkenylene, be substituted the low-carbon (LC) alkenylene, the inferior assorted alkyl of low-carbon (LC), be substituted the low-carbon (LC) Asia mix alkyl ,-O-(alkylidene or be substituted alkylidene)-,-S-(alkylidene or be substituted alkylidene)-,-C (O) R " ,-S (O) k(alkylidene or be substituted alkylidene)-(wherein k is 1,2 or 3) ,-C (O)-(alkylidene or be substituted alkylidene)-,-C (S)-(alkylidene or be substituted alkylidene)-,-NR " (alkylidene or be substituted alkylidene)-,-CON (R ")-(alkylidene or be substituted alkylidene)-,-CSN (R ")-(alkylidene or be substituted alkylidene)-and-N (R ") CO-(alkylidene or be substituted alkylidene)-, each R wherein is " independently for H, alkyl or be substituted alkyl;
K is
Figure A200680049995D00752
Or
Figure A200680049995D00753
Wherein
T 1Be bond, the C that is substituted according to circumstances 1-C 4Alkylidene, the C that is substituted according to circumstances 1-C 4Alkenylene or the assorted alkyl that is substituted according to circumstances;
Wherein each optional substituting group is independently selected from the low-carbon (LC) alkylidene, is substituted the low-carbon (LC) alkylidene, the low-carbon (LC) cycloalkylidene, is substituted low-carbon (LC) cycloalkylidene, low-carbon (LC) alkenylene, is substituted the low-carbon (LC) alkenylene, alkynylene, the inferior assorted alkyl of low-carbon (LC), is substituted inferior assorted alkyl, the inferior Heterocyclylalkyl of low-carbon (LC), is substituted low-carbon (LC) inferior Heterocyclylalkyl, arlydene, is substituted arlydene, inferior heteroaryl, is substituted inferior heteroaryl, alkarylene, is substituted alkarylene, inferior aralkyl or is substituted inferior aralkyl;
T 2Be selected from the group that forms by following each group: the low-carbon (LC) alkylidene, be substituted low-carbon (LC) alkylidene, low-carbon (LC) alkenylene, be substituted the low-carbon (LC) alkenylene, the inferior assorted alkyl of low-carbon (LC), be substituted the inferior assorted alkyl of low-carbon (LC) ,-O-,-O-(alkylidene or be substituted alkylidene)-,-S-,-S-(alkylidene or be substituted alkylidene)-,-S (O) k-(wherein k is 1,2 or 3) ,-S (O) k(alkylidene or be substituted alkylidene)-,-C (O)-,-C (O)-(alkylidene or be substituted alkylidene)-,-C (S)-,-C (S)-(alkylidene or be substituted alkylidene)-,-N (R ')-,-NR '-(alkylidene or be substituted alkylidene)-,-C (O) N (R ')-,-CON (R ')-(alkylidene or be substituted alkylidene)-,-CSN (R ')-,-CSN (R ')-(alkylidene or be substituted alkylidene)-,-N (R ') CO-(alkylidene or be substituted alkylidene)-,-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 each R ' is H, alkyl independently or is substituted alkyl;
T 3For
Figure A200680049995D00754
Or Each X wherein 1Be independently selected from the group that forms by following each group :-O-,-S-,-N (H)-,-N (R)-,-N (Ac)-and-N (OMe)-; X 2For-OR ,-OAc ,-SR ,-N (R) 2,-N (R) (Ac) ,-N (R) (OMe) or N 3, and wherein each R ' is H, alkyl independently or is substituted alkyl;
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;
Or-the A-B-K-R group form together comprise at least one carbonyl (comprising dicarbapentaborane), through protection carbonyl (comprise through protection dicarbapentaborane) or through covering dicyclo or the tricyclic naphthenes base or the Heterocyclylalkyl of carbonyl (comprising) through covering dicarbapentaborane;
Or-the K-R group form together comprise at least one carbonyl (comprising dicarbapentaborane), through protection carbonyl (comprise through protection dicarbapentaborane) or through covering monocycle or the bicyclic cycloalkyl or the Heterocyclylalkyl of carbonyl (comprising) through covering dicarbapentaborane.
In addition, the amino acid with formula (V) structure comprises the amino acid of (VI) structure that has formula:
Wherein:
M 1For bond ,-C (R 3) (R 4)-,-O-,-S-,-C (R 3) (R 4)-C (R 3) (R 4)-,-C (R 3) (R 4)-O-,-C (R 3) (R 4)-S-,-O-C (R 3) (R 4)-,-S-C (R 3) (R 4) ,-C (R 3)=C (R 3)-or-C (R 4)=C (R 4)-;
R 3And R 4Be independently selected from H, halogen, alkyl, be substituted alkyl, cycloalkyl or be substituted cycloalkyl,
Perhaps R 3And R 4Or two R 3Group or two R 4Group forms cycloalkyl or Heterocyclylalkyl according to circumstances.
Amino acid with formula (VI) structure comprises the amino acid of (VII) structure that has formula:
Wherein:
Each R aBe independently selected from the group that forms by following each group: H, halogen, alkyl, be substituted alkyl ,-N (R ') 2,-C (O) R ' ,-C (O) N (R ') 2,-OR ' and-S (O) kR ', wherein k is 1,2 or 3, and each R ' is H, alkyl independently or is substituted alkyl.
Amino acid with formula (VII) structure also comprises the amino acid with formula (VIII) and formula (IX) structure:
Figure A200680049995D00771
Comprising following non-limiting amino acid with formula (VIII) or formula (IX) structure:
Figure A200680049995D00772
With
Figure A200680049995D00773
Described alpha-non-natural amino acid also can be salt form, maybe can incorporate in non-natural amino acid polypeptides, polymer, polysaccharide or the polynucleotide and/or according to circumstances through posttranslational modification.
Other amino acid that contains dicarbapentaborane comprises the amino acid of (X) structure that has formula:
Wherein:
A is optional, and when existing, it is the low-carbon (LC) alkylidene, be substituted low-carbon (LC) alkylidene, low-carbon (LC) cycloalkylidene, be substituted the low-carbon (LC) cycloalkylidene, the low-carbon (LC) alkenylene, be substituted low-carbon (LC) alkenylene, alkynylene, the inferior assorted alkyl of low-carbon (LC), be substituted inferior assorted alkyl, the inferior Heterocyclylalkyl of low-carbon (LC), be substituted low-carbon (LC) inferior Heterocyclylalkyl, arlydene, be substituted arlydene, inferior heteroaryl, be substituted inferior heteroaryl, alkarylene, be substituted alkarylene, inferior aralkyl or be substituted inferior aralkyl;
B is optional, and when existing, its at one end with contain the connection base that two amine moieties are connected, described connection base is selected from the group that is made up of following each group: the low-carbon (LC) alkylidene, be substituted low-carbon (LC) alkylidene, low-carbon (LC) alkenylene, be substituted the low-carbon (LC) alkenylene, the inferior assorted alkyl of low-carbon (LC), be substituted the low-carbon (LC) Asia mix alkyl ,-O-(alkylidene or be substituted alkylidene)-,-S-(alkylidene or be substituted alkylidene)-,-C (O) R " ,-S (O) k(alkylidene or be substituted alkylidene)-(wherein k is 1,2 or 3) ,-C (O)-(alkylidene or be substituted alkylidene)-,-C (S)-(alkylidene or be substituted alkylidene)-,-NR " (alkylidene or be substituted alkylidene)-,-CON (R ")-(alkylidene or be substituted alkylidene)-,-CSN (R ")-(alkylidene or be substituted alkylidene)-and-N (R ") CO-(alkylidene or be substituted alkylidene)-, each R wherein is " independently for H, alkyl or be substituted alkyl;
M 2For
Figure A200680049995D00782
Figure A200680049995D00783
Or
Figure A200680049995D00784
Wherein (a) expression and the bond of B group, and (b) bond of expression and each carbonyl;
T 3For bond, C (R) (R), O or S;
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 3And R 4Be independently selected from H, halogen, alkyl, be substituted alkyl, cycloalkyl or be substituted cycloalkyl, perhaps R 3And R 4Or two R 3Group or two R 4Group forms cycloalkyl or Heterocyclylalkyl according to circumstances.
Amino acid with formula (X) structure comprises the amino acid of have formula (XI) and formula (XII) structure:
Figure A200680049995D00791
Wherein:
Each R aBe independently selected from the group that forms by following each group: H, halogen, alkyl, be substituted alkyl ,-N (R ') 2,-C (O) R ' ,-C (O) N (R ') 2,-OR ' and-S (O) kR ', wherein k is 1,2 or 3, and each R ' is H, alkyl independently or is substituted alkyl.
In addition, the amino acid with formula (XI) and formula (XII) structure comprises the amino acid of have formula (XIII) and formula (XIV) structure:
Figure A200680049995D00792
Also comprise following amino acid with formula (XIV) structure:
Figure A200680049995D00793
With
Figure A200680049995D00794
Described alpha-non-natural amino acid can be salt form, maybe can incorporate in non-natural amino acid polypeptides, polymer, polysaccharide or the polynucleotide and according to circumstances through posttranslational modification.
Other amino acid that contains dicarbapentaborane comprises the amino acid of (XV) structure that has formula:
Figure A200680049995D00801
Wherein:
B is optional, and when existing, it is the connection base that is selected from the group that is made up of following each group: the low-carbon (LC) alkylidene, be substituted low-carbon (LC) alkylidene, low-carbon (LC) alkenylene, be substituted the low-carbon (LC) alkenylene, the inferior assorted alkyl of low-carbon (LC), be substituted the inferior assorted alkyl of low-carbon (LC) ,-O-,-O-(alkylidene or be substituted alkylidene)-,-S-,-C (O) R " ,-S-(alkylidene or be substituted alkylidene)-,-S (O) k-(wherein k is 1,2 or 3) ,-S (O) k(alkylidene or be substituted alkylidene)-,-C (O)-,-NS (O) 2-,-OS (O) 2-,-C (O)-(alkylidene or be substituted alkylidene)-,-C (S)-,-C (S)-(alkylidene or be substituted alkylidene)-,-N (R ')-,-NR '-(alkylidene or be substituted alkylidene)-,-C (O) N (R ')-,-CON (R ')-(alkylidene or be substituted alkylidene)-,-CSN (R ')-,-CSN (R ')-(alkylidene or be substituted alkylidene)-,-N (R ') CO-,-N (R ') CO-(alkylidene or be substituted alkylidene)-,-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 ')-,-C (R ')=N-,-C (R ')=N-N (R ')-,-C (R ') 2-N=N-and-C (R ') 2-N (R ')-N (R ')-, wherein each R ' is H, alkyl independently or is substituted alkyl;
M 1For bond ,-C (R 3) (R 4)-,-O-,-S-,-C (R 3) (R 4)-C (R 3) (R 4)-,-C (R 3) (R 4)-O-,-C (R 3) (R 4)-S-,-O-C (R 3) (R 4)-,-S-C (R 3) (R 4) ,-C (R 3)=C (R 3)-or-C (R 4)=C (R 4)-;
T 3For bond, C (R) (R), O or S;
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 3And R 4Be independently selected from H, halogen, alkyl, be substituted alkyl, cycloalkyl or be substituted cycloalkyl, perhaps R 3And R 4Or two R 3Group or two R 4Group forms cycloalkyl or Heterocyclylalkyl according to circumstances;
Each R aBe independently selected from the group that forms by following each group: H, halogen, alkyl, be substituted alkyl ,-N (R ') 2,-C (O) R ' ,-C (O) N (R ') 2,-OR ' and-S (O) kR ', wherein k is 1,2 or 3, and each R ' is H, alkyl independently or is substituted alkyl; And n is 0 to 8.
Also comprise following amino acid with formula (XV) structure:
Figure A200680049995D00811
With
Figure A200680049995D00813
Described alpha-non-natural amino acid can be salt form, maybe can incorporate in non-natural amino acid polypeptides, polymer, polysaccharide or the polynucleotide and according to circumstances through posttranslational modification.
Has amino acid through the protection carbonyl
Also comprise and have at least one amino acid with formula (XVI) structure through the protection carbonyl:
Figure A200680049995D00814
Wherein:
A is optional, and when existing, it is the low-carbon (LC) alkylidene, be substituted low-carbon (LC) alkylidene, low-carbon (LC) cycloalkylidene, be substituted the low-carbon (LC) cycloalkylidene, the low-carbon (LC) alkenylene, be substituted low-carbon (LC) alkenylene, alkynylene, the inferior assorted alkyl of low-carbon (LC), be substituted inferior assorted alkyl, the inferior Heterocyclylalkyl of low-carbon (LC), be substituted low-carbon (LC) inferior Heterocyclylalkyl, arlydene, be substituted arlydene, inferior heteroaryl, be substituted inferior heteroaryl, alkarylene, be substituted alkarylene, inferior aralkyl or be substituted inferior aralkyl;
B is optional, and when existing, its at one end with contain the connection base that two amine moieties are connected, described connection base is selected from the group that is made up of following each group: the low-carbon (LC) alkylidene, be substituted low-carbon (LC) alkylidene, low-carbon (LC) alkenylene, be substituted the low-carbon (LC) alkenylene, the inferior assorted alkyl of low-carbon (LC), be substituted the low-carbon (LC) Asia mix alkyl ,-O-(alkylidene or be substituted alkylidene)-,-S-(alkylidene or be substituted alkylidene)-,-C (O) R " ,-S (O) k(alkylidene or be substituted alkylidene)-(wherein k is 1,2 or 3) ,-C (O)-(alkylidene or be substituted alkylidene)-,-C (S)-(alkylidene or be substituted alkylidene)-,-NR " (alkylidene or be substituted alkylidene)-,-CON (R ")-(alkylidene or be substituted alkylidene)-,-CSN (R ")-(alkylidene or be substituted alkylidene)-and-N (R ") CO-(alkylidene or be substituted alkylidene)-, each R wherein is " independently for H, alkyl or be substituted alkyl;
M 1For bond ,-C (R 3) (R 4)-,-O-,-S-,-C (R 3) (R 4)-C (R 3) (R 4)-,-C (R 3) (R 4)-O-,-C (R 3) (R 4)-S-,-O-C (R 3) (R 4)-,-S-C (R 3) (R 4) ,-C (R 3)=C (R 3)-or-C (R 4)=C (R 4)-;
T 3For bond, C (R) (R), O or S;
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 3And R 4Be independently selected from H, halogen, alkyl, be substituted alkyl, cycloalkyl or be substituted cycloalkyl, perhaps R 3And R 4Or two R 3Group or two R 4Group forms cycloalkyl or Heterocyclylalkyl according to circumstances; And
T 4For the carbonyl-protection base, include but not limited to
Figure A200680049995D00821
Figure A200680049995D00822
Or
Figure A200680049995D00823
Each X wherein 1Be independently selected from the group that forms by following group: O, S, NH, NR ', N-Ac and N-OMe; And X 2For O-R, O-Ac, SR, S-Ac, N (R ') (R '), N (R ') (Ac), N (R ') (OMe) or N 3
Amino acid with formula (XVI) structure comprises the amino acid of have formula (XVII), formula (XVIII) and formula (XIX) structure:
Figure A200680049995D00831
Wherein:
Each R aBe independently selected from the group that forms by following each group: H, halogen, alkyl, be substituted alkyl ,-N (R ') 2,-C (O) R ' ,-C (O) N (R ') 2,-OR ' and-S (O) kR ', wherein k is 1,2 or 3, and each R ' is H, alkyl independently or is substituted alkyl.
In addition, the amino acid of having that comprises have formula (XX), formula (XXI), formula (XXII), formula (XXIII), formula (XXIV) and formula (XXV) structure through the protection carbonyl:
Figure A200680049995D00832
Wherein:
X 1Be O, S, NH, NR ', N-Ac or N-OMe; And
Each R ' is H, alkyl independently or is substituted alkyl.
In addition, comprise the following amino acid that contains through the protection carbonyl:
Figure A200680049995D00841
Figure A200680049995D00842
With
Described alpha-non-natural amino acid can be salt form, maybe can incorporate in non-natural amino acid polypeptides, polymer, polysaccharide or the polynucleotide and according to circumstances through posttranslational modification.
In addition, comprise following have formula (XXVI) structure and have at least one through the protection carbonyl amino acid:
Wherein:
A is optional, and when existing, it is the low-carbon (LC) alkylidene, be substituted low-carbon (LC) alkylidene, low-carbon (LC) cycloalkylidene, be substituted the low-carbon (LC) cycloalkylidene, the low-carbon (LC) alkenylene, be substituted low-carbon (LC) alkenylene, alkynylene, the inferior assorted alkyl of low-carbon (LC), be substituted inferior assorted alkyl, the inferior Heterocyclylalkyl of low-carbon (LC), be substituted low-carbon (LC) inferior Heterocyclylalkyl, arlydene, be substituted arlydene, inferior heteroaryl, be substituted inferior heteroaryl, alkarylene, be substituted alkarylene, inferior aralkyl or be substituted inferior aralkyl;
B is optional, and when existing, its at one end with contain the connection base that two amine moieties are connected, described connection base is selected from the group that is made up of following each group: the low-carbon (LC) alkylidene, be substituted low-carbon (LC) alkylidene, low-carbon (LC) alkenylene, be substituted the low-carbon (LC) alkenylene, the inferior assorted alkyl of low-carbon (LC), be substituted the low-carbon (LC) Asia mix alkyl ,-O-(alkylidene or be substituted alkylidene)-,-S-(alkylidene or be substituted alkylidene)-,-C (O) R " ,-S (O) k(alkylidene or be substituted alkylidene)-(wherein k is 1,2 or 3) ,-C (O)-(alkylidene or be substituted alkylidene)-,-C (S)-(alkylidene or be substituted alkylidene)-,-NR " (alkylidene or be substituted alkylidene)-,-CON (R ")-(alkylidene or be substituted alkylidene)-,-CSN (R ")-(alkylidene or be substituted alkylidene)-and-N (R ") CO-(alkylidene or be substituted alkylidene)-, each R wherein is " independently for H, alkyl or be substituted alkyl;
Q is
Figure A200680049995D00851
Or
Figure A200680049995D00852
M 2For
Figure A200680049995D00853
Figure A200680049995D00854
Or
Figure A200680049995D00855
Wherein (a) expression and the bond of B group, and (b) bond of expression and each carbonyl;
T 3For bond, C (R) (R), O or S;
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 3And R 4Be independently selected from H, halogen, alkyl, be substituted alkyl, cycloalkyl or be substituted cycloalkyl, perhaps R 3And R 4Or two R 3Group or two R 4Group forms cycloalkyl or Heterocyclylalkyl according to circumstances, and
T 4For the carbonyl-protection base, include but not limited to
Figure A200680049995D00856
Figure A200680049995D00857
Or
Figure A200680049995D00858
Each X wherein 1Be independently selected from the group that forms by following group: O, S, NH, NR ', N-Ac and N-OMe; And X 2For O-R, O-Ac, SR, S-Ac, N (R ') (R '), N (R ') (Ac), N (R ') (OMe) or N 3
Amino acid with formula (XXVI) structure comprises the amino acid of have formula (XXVII), formula (XXVIII) and formula (XXIX) structure:
Figure A200680049995D00861
Wherein:
Each R aBe independently selected from the group that forms by following each group: H, halogen, alkyl, be substituted alkyl ,-N (R ') 2,-C (O) R ' ,-C (O) N (R ') 2,-OR ' and-S (O) kR ', wherein k is 1,2 or 3, and each R ' is H, alkyl independently or is substituted alkyl.
In addition, comprise the following amino acid that has through protecting carbonyl and having formula (XXX) structure:
Figure A200680049995D00862
Wherein:
B is optional, and when existing, it is the connection base that is selected from the group that is made up of following each group: the low-carbon (LC) alkylidene, be substituted low-carbon (LC) alkylidene, low-carbon (LC) alkenylene, be substituted the low-carbon (LC) alkenylene, the inferior assorted alkyl of low-carbon (LC), be substituted the inferior assorted alkyl of low-carbon (LC) ,-O-,-O-(alkylidene or be substituted alkylidene)-,-S-,-S-(alkylidene or be substituted alkylidene)-,-C (O) R " ,-S (O) k-(wherein k is 1,2 or 3) ,-S (O) k(alkylidene or be substituted alkylidene)-,-C (O)-,-NS (O) 2-,-OS (O) 2-,-C (O)-(alkylidene or be substituted alkylidene)-,-C (S)-,-C (S)-(alkylidene or be substituted alkylidene)-,-N (R ')-,-NR '-(alkylidene or be substituted alkylidene)-,-C (O) N (R ')-,-CON (R ')-(alkylidene or be substituted alkylidene)-,-CSN (R ')-,-CSN (R ')-(alkylidene or be substituted alkylidene)-,-N (R ') CO-,-N (R ') CO-(alkylidene or be substituted alkylidene)-,-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 ')-,-C (R ')=N-,-C (R ')=N-N (R ')-,-C (R ') 2-N=N-and-C (R ') 2-N (R ')-N (R ')-, wherein each R ' is H, alkyl independently or is substituted alkyl;
M 1For bond ,-C (R 3) (R 4)-,-O-,-S-,-C (R 3) (R 4)-C (R 3) (R 4)-,-C (R 3) (R 4)-O-,-C (R 3) (R 4)-S-,-O-C (R 3) (R 4)-,-S-C (R 3) (R 4) ,-C (R 3)=C (R 3)-or-C (R 4)=C (R 4)-;
T 3For bond, C (R) (R), O or S;
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;
R 2Be OH, ester protecting group, resin, amino acid, polypeptide or polynucleotide;
R 3And R 4Be independently selected from H, halogen, alkyl, be substituted alkyl, cycloalkyl or be substituted cycloalkyl, perhaps R 3And R 4Or two R 3Group or two R 4Group forms cycloalkyl or Heterocyclylalkyl according to circumstances;
T 4For the carbonyl-protection base, include but not limited to
Figure A200680049995D00871
Figure A200680049995D00872
Or
Figure A200680049995D00873
Each X wherein 1Be independently selected from the group that forms by following group: O, S, NH, NR ', N-Ac and N-OMe; And X 2For O-R, O-Ac, SR, S-Ac, N (R ') (R '), N (R ') (Ac), N (R ') (OMe) or N 3
Each R aBe independently selected from the group that forms by following each group: H, halogen, alkyl, be substituted alkyl ,-N (R ') 2,-C (O) R ' ,-C (O) N (R ') 2,-OR ' and-S (O) kR ', wherein k is 1,2 or 3, and each R ' is H, alkyl independently or is substituted alkyl; And n is 0 to 8.
Comprise following having according to formula (XXX) through protecting the amino acid of carbonyl:
With
Described alpha-non-natural amino acid can be salt form, maybe can incorporate in non-natural amino acid polypeptides, polymer, polysaccharide or the polynucleotide and according to circumstances through posttranslational modification.
One of ordinary skill in the art known synthetic contain carbonyl or the amino acid whose method of dicarbapentaborane.In addition, contain amino acid whose various synthetic being described in No. the 60/638th, 418, the U.S. Provisional Patent Application case of carbonyl or dicarbapentaborane, its mode of quoting in full is incorporated herein.Acetyl group-(+/-)-phenyl alanine and an acetyl group-(+/-)-phenyl alanine synthetic is described in people such as Zhang, Z., and among the Biochemistry 42:6735-6746 (2003), its mode of also quoting is in full incorporated into.
Other limiting examples that contains the dicarbapentaborane alpha-non-natural amino acid is showed among Fig. 3.Contain and amino acid whose non-limiting exemplary synthesizing of dicarbapentaborane be described in herein and be provided among Fig. 5 and Fig. 6.
In certain embodiments, the polypeptide that comprises alpha-non-natural amino acid is carried out chemical modification to produce reactive carbonyl or dicarbapentaborane functional group.For instance, the aldehyde functional group that can be used for linking reaction can be produced by the functional group with adjacent amino and hydroxyl.When bioactive molecule is polypeptide, for example can use terminal serine of N or threonine (it exists usually or can expose via chemistry or enzymatic digestion) to produce aldehyde functional group under the mild oxidation cracking condition, to use periodate.For example referring to people such as Gaertner, Bioconjug.Chem.3:262-268 (1992); Geoghegan, K.﹠amp; Stroh, J., Bioconjug.Chem.3:138-146 (1992); People such as Gaertner, J.Biol.Chem.269:7224-7230 (1994).Yet the known method in affiliated field is confined to the amino acid of peptide or protein N end.，
In addition, for instance, the alpha-non-natural amino acid with adjacent hydroxyl and amino can be incorporated in the polypeptide with the form of " through covering " aldehyde functional group.For instance, the 5-oxylysine has the hydroxyl adjacent with ε amine.The reaction condition that is used to produce aldehyde is usually directed to add the sodium metaperiodate of molar excess to avoid the oxidation of other site in the polypeptide under temperate condition.The pH value of oxidation reaction is generally about 7.0.The sodium metaperiodate that typical reaction relates to about 1.5 molar excess adds in the polypeptide buffer solution, in the dark cultivates subsequently about 10 minutes.For example referring to United States Patent (USP) the 6th, 423, No. 685.
C. the structure of alpha-non-natural amino acid and synthetic: ketone alkynes, class ketone alkynes, through covering ketone alkynes and through protection ketone ethynylene group
Containing the amino acid with the reactive reactive group of class dicarbapentaborane allows to connect molecule via nucleophilic addition.Described electrophilic reaction group comprises ketone alkynyl, class ketone alkynyl (its have be similar to the reactive of ketone alkynyl and structurally be similar to the ketone alkynyl), through covering ketone alkynyl (it can easily change into the ketone alkynyl) or through protection ketone alkynyl (it has the reactivity that is similar to the ketone alkynyl after going to protect).Described amino acid comprises the amino acid of (XXXI) structure that has formula:
Figure A200680049995D00881
Wherein:
A is optional, and when existing, it is the low-carbon (LC) alkylidene, be substituted low-carbon (LC) alkylidene, low-carbon (LC) cycloalkylidene, be substituted the low-carbon (LC) cycloalkylidene, the low-carbon (LC) alkenylene, be substituted low-carbon (LC) alkenylene, alkynylene, the inferior assorted alkyl of low-carbon (LC), be substituted inferior assorted alkyl, the inferior Heterocyclylalkyl of low-carbon (LC), be substituted low-carbon (LC) inferior Heterocyclylalkyl, arlydene, be substituted arlydene, inferior heteroaryl, be substituted inferior heteroaryl, alkarylene, be substituted alkarylene, inferior aralkyl or be substituted inferior aralkyl;
B is optional, and when existing, its at one end with contain the connection base that two amine moieties are connected, described connection base is selected from the group that is made up of following each group: the low-carbon (LC) alkylidene, be substituted low-carbon (LC) alkylidene, low-carbon (LC) alkenylene, be substituted the low-carbon (LC) alkenylene, the inferior assorted alkyl of low-carbon (LC), be substituted the low-carbon (LC) Asia mix alkyl ,-O-(alkylidene or be substituted alkylidene)-,-S-(alkylidene or be substituted alkylidene)-,-C (O) R " ,-S (O) k(alkylidene or be substituted alkylidene)-(wherein k is 1,2 or 3) ,-C (O)-(alkylidene or be substituted alkylidene)-,-C (S)-(alkylidene or be substituted alkylidene)-,-NR " (alkylidene or be substituted alkylidene)-,-CON (R ")-(alkylidene or be substituted alkylidene)-,-CSN (R ")-(alkylidene or be substituted alkylidene)-and-N (R ") CO-(alkylidene or be substituted alkylidene)-, each R wherein is " independently for H, alkyl or be substituted alkyl;
G is
Figure A200680049995D00891
Or
Figure A200680049995D00892
T 4For the carbonyl-protection base, include but not limited to
Figure A200680049995D00893
Figure A200680049995D00894
Or
Figure A200680049995D00895
Each X wherein 1Be independently selected from the group that forms by following each group :-O-,-S-,-N (H)-,-N (R)-,-N (Ac)-and-N (OMe)-; X 2For-OR ,-OAc ,-SR ,-N (R) 2,-N (R) (Ac) ,-N (R) (OMe) or N 3, and wherein each R ' is H, alkyl independently or is substituted alkyl;
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 3And R 4Be H, halogen, low-carbon alkyl or be substituted low-carbon alkyl, perhaps R independently of one another 3And R 4Or two R 3Group forms cycloalkyl or Heterocyclylalkyl according to circumstances.
Amino acid with formula (XXXI) structure comprises the amino acid of have formula (XXXII) and formula (XXXIV) structure:
Each R wherein aBe independently selected from the group that forms by following each group: H, halogen, alkyl, be substituted alkyl ,-N (R ') 2,-C (O) R ' ,-C (O) N (R ') 2,-OR ' and-S (O) kR ', wherein k is 1,2 or 3, and each R ' is H, alkyl independently or is substituted alkyl.
Other limiting examples that contains ketone alkynes alpha-non-natural amino acid is showed among Fig. 4.
D. the structure of alpha-non-natural amino acid and synthetic: ketoamine, class ketoamine, through covering ketoamine and through protection ketoamine group
Containing the amino acid with the reactive reactive group of class dicarbapentaborane allows to connect molecule via nucleophilic addition.Described reactive group comprises ketoamine base, class ketoamine base (its have be similar to the reactive of ketoamine base and structurally be similar to the ketoamine base), through covering ketoamine base (it can easily change into the ketoamine base) or through protection ketoamine base (it has the reactivity that is similar to the ketoamine base after going to protect).Described amino acid comprises the amino acid of (XXXIV) structure that has formula:
Figure A200680049995D00901
Wherein:
A is optional, and when existing, it is the low-carbon (LC) alkylidene, be substituted low-carbon (LC) alkylidene, low-carbon (LC) cycloalkylidene, be substituted the low-carbon (LC) cycloalkylidene, the low-carbon (LC) alkenylene, be substituted low-carbon (LC) alkenylene, alkynylene, the inferior assorted alkyl of low-carbon (LC), be substituted inferior assorted alkyl, the inferior Heterocyclylalkyl of low-carbon (LC), be substituted low-carbon (LC) inferior Heterocyclylalkyl, arlydene, be substituted arlydene, inferior heteroaryl, be substituted inferior heteroaryl, alkarylene, be substituted alkarylene, inferior aralkyl or be substituted inferior aralkyl;
B is optional, and when existing, its at one end with contain the connection base that two amine moieties are connected, described connection base is selected from the group that is made up of following each group: the low-carbon (LC) alkylidene, be substituted low-carbon (LC) alkylidene, low-carbon (LC) alkenylene, be substituted the low-carbon (LC) alkenylene, the inferior assorted alkyl of low-carbon (LC), be substituted the low-carbon (LC) Asia mix alkyl ,-O-(alkylidene or be substituted alkylidene)-,-S-(alkylidene or be substituted alkylidene)-,-C (O) R " ,-S (O) k(alkylidene or be substituted alkylidene)-(wherein k is 1,2 or 3) ,-C (O)-(alkylidene or be substituted alkylidene)-,-C (S)-(alkylidene or be substituted alkylidene)-,-NR " (alkylidene or be substituted alkylidene)-,-CON (R ")-(alkylidene or be substituted alkylidene)-,-CSN (R ")-(alkylidene or be substituted alkylidene)-and-N (R ") CO-(alkylidene or be substituted alkylidene)-, each R wherein is " independently for H, alkyl or be substituted alkyl;
G is
Figure A200680049995D00902
Or
Figure A200680049995D00903
T 1Be the C that is substituted according to circumstances 1-C 4Alkylidene, the C that is substituted according to circumstances 1-C 4Alkenylene or the assorted alkyl that is substituted according to circumstances;
T 4For the carbonyl-protection base, include but not limited to
Figure A200680049995D00904
Or
Figure A200680049995D00906
Each X wherein 1Be independently selected from the group that forms by following each group :-O-,-S-,-N (H)-,-N (R ')-,-N (Ac)-and-N (OMe)-; X 2For-OR ,-OAc ,-SR ' ,-N (R ') 2,-N (R ') (Ac) ,-N (R ') (OMe) or N 3, and wherein each R ' is H, alkyl independently or is substituted alkyl;
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 3And R 4Be H, halogen, low-carbon alkyl or be substituted low-carbon alkyl, perhaps R independently of one another 3And R 4Or two R 3Group forms cycloalkyl or Heterocyclylalkyl according to circumstances.
Amino acid with formula (XXXIV) structure comprises the amino acid of have formula (XXXV) and formula (XXXVI) structure:
Figure A200680049995D00911
Each R wherein aBe independently selected from the group that forms by following each group: H, halogen, alkyl, 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 each R ' is H, alkyl independently or is substituted alkyl.
E. the structure of alpha-non-natural amino acid is with synthetic: contain heterocyclic amino acid
Some embodiment as herein described comprises heterocyclic radical for having, through covering heterocyclic radical (it can easily change into heterocyclic radical) or through the alpha-non-natural amino acid of the side chain of protection heterocyclic radical (its can easily go protection become heterocyclic radical).Described amino acid comprises the amino acid of (XXXVII) structure that has formula:
Figure A200680049995D00912
Wherein:
A is optional, and when existing, it is the low-carbon (LC) alkylidene, be substituted low-carbon (LC) alkylidene, low-carbon (LC) cycloalkylidene, be substituted the low-carbon (LC) cycloalkylidene, the low-carbon (LC) alkenylene, be substituted low-carbon (LC) alkenylene, alkynylene, the inferior assorted alkyl of low-carbon (LC), be substituted inferior assorted alkyl, the inferior Heterocyclylalkyl of low-carbon (LC), be substituted low-carbon (LC) inferior Heterocyclylalkyl, arlydene, be substituted arlydene, inferior heteroaryl, be substituted inferior heteroaryl, alkarylene, be substituted alkarylene, inferior aralkyl or be substituted inferior aralkyl;
B is optional, and when existing, its at one end with contain the connection base that two amine moieties are connected, described connection base is selected from the group that is made up of following each group: the low-carbon (LC) alkylidene, be substituted low-carbon (LC) alkylidene, low-carbon (LC) alkenylene, be substituted the low-carbon (LC) alkenylene, the inferior assorted alkyl of low-carbon (LC), be substituted the low-carbon (LC) Asia mix alkyl ,-O-(alkylidene or be substituted alkylidene)-,-S-(alkylidene or be substituted alkylidene)-,-C (O) R " ,-S (O) k(alkylidene or be substituted alkylidene)-(wherein k is 1,2 or 3) ,-C (O)-(alkylidene or be substituted alkylidene)-,-C (S)-(alkylidene or be substituted alkylidene)-,-NR " (alkylidene or be substituted alkylidene)-,-CON (R ")-(alkylidene or be substituted alkylidene)-,-CSN (R ")-(alkylidene or be substituted alkylidene)-and-N (R ") CO-(alkylidene or be substituted alkylidene)-, and each R wherein is " independently for H, alkyl or be substituted alkyl;
Q is the heterocycle that is substituted according to circumstances or the heteroaryl that is substituted according to circumstances, and wherein each optional substituting group is independently selected from the low-carbon (LC) alkylidene, is substituted the low-carbon (LC) alkylidene, the low-carbon (LC) cycloalkylidene, is substituted low-carbon (LC) cycloalkylidene, low-carbon (LC) alkenylene, is substituted the low-carbon (LC) alkenylene, alkynylene, the inferior assorted alkyl of low-carbon (LC), is substituted inferior assorted alkyl, the inferior Heterocyclylalkyl of low-carbon (LC), is substituted low-carbon (LC) inferior Heterocyclylalkyl, arlydene, is substituted arlydene, inferior heteroaryl, is substituted inferior heteroaryl, alkarylene, is substituted alkarylene, inferior aralkyl or is substituted inferior aralkyl;
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 3And R 4Be H, halogen, low-carbon alkyl or be substituted low-carbon alkyl, perhaps R independently of one another 3And 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, be substituted alkoxyl, alkyl alkoxy, be substituted alkyl alkoxy, polyalkylene oxide, be substituted polyalkylene oxide, aryl, be substituted aryl, heteroaryl, be substituted heteroaryl, alkaryl, be substituted alkaryl, aralkyl, be substituted aralkyl ,-(alkylidene or be substituted alkylidene)-ON (R ") 2,-(alkylidene or be substituted alkylidene)-C (O) SR " ,-(alkylidene or be substituted alkylidene)-S-S-(aryl or be substituted aryl) ,-C (O) R " ,-C (O) 2R " or-C (O) N (R ") 2, each R wherein " independently for hydrogen, alkyl, be substituted alkyl, thiazolinyl, be substituted thiazolinyl, alkoxyl, be substituted alkoxyl, aryl, be substituted aryl, heteroaryl, alkaryl, be substituted alkaryl, aralkyl, be substituted aralkyl;
Or R 5Be L-X, wherein X is selected from the group that is made up of required functional group; And L is optional, and when existing, it is the connection base that is selected from the group that is made up of following each group: alkylidene, be substituted alkylidene, alkenylene, be substituted alkenylene ,-O-,-O-(alkylidene or be substituted alkylidene)-,-S-,-S-(alkylidene or be substituted alkylidene)-,-S (O) k-(wherein k is 1,2 or 3) ,-S (O) k(alkylidene or be substituted alkylidene)-,-C (O)-,-C (O)-(alkylidene or be substituted alkylidene)-,-C (S)-,-C (S)-(alkylidene or be substituted alkylidene)-,-N (R ')-,-NR '-(alkylidene or be substituted alkylidene)-,-C (O) N (R ')-,-CON (R ')-(alkylidene or be substituted alkylidene)-,-CSN (R ')-,-CSN (R ')-(alkylidene or be substituted alkylidene)-,-N (R ') CO-(alkylidene or be substituted alkylidene)-,-N (R ') C (O) O-,-(alkylidene or be substituted alkylidene)-O-N=CR '-,-(alkylidene or be substituted alkylidene)-C (O) NR '-(alkylidene or be substituted alkylidene)-,-(alkylidene or be substituted alkylidene)-S (O) k-(alkylidene or be substituted alkylidene)-S-,-(alkylidene or be substituted alkylidene)-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 each R ' is H, alkyl independently or is substituted alkyl.
Described formation with alpha-non-natural amino acid of formula (XXXVII) structure includes but not limited to that (i) contains the diamines alpha-non-natural amino acid and contain the reaction of dicarbapentaborane reagent or contain diamines alpha-non-natural amino acid and the reaction that contains ketone alkynes reagent; (ii) contain the dicarbapentaborane alpha-non-natural amino acid and contain the reaction of diamines reagent or contain dicarbapentaborane alpha-non-natural amino acid and the reaction that contains ketoamine reagent; (iii) contain ketone alkynes alpha-non-natural amino acid and the reaction that contains diamines reagent; Or (iv) contain the ketoamine alpha-non-natural amino acid and contain the reaction of dicarbapentaborane reagent.
Modify alpha-non-natural amino acid as herein described with described reaction and have any or whole following advantage.The first, diamines in about 5 to about 8 pH value scope (and in other embodiments in about 4 to about 10 pH value scope; In other embodiments in about 3 to about 8 pH value scope; In other embodiments in about 4 to about 9 pH value scope; And in other embodiments in about 4 to about 9 pH value scope; In other embodiments under about 4 pH value; And in other embodiments under about 8 pH value) and contain the condensation of dicarbonyl compound experience to produce heterocycle (comprising nitrogen heterocyclic ring) key.Under these conditions, naturally occurring amino acid whose side chain is not had a reactivity.The second, described selective chemical makes the locus specificity derivatization of recombinant protein become possibility: derivatization protein now can be used as specifies the preparation of homology product.The 3rd, realize that the required temperate condition of diamines as herein described and the reaction that contains the dicarbapentaborane polypeptide as herein described can reversibly destroy the tertiary structure of polypeptide (being to destroy the situation of described tertiary structure except that the reaction purpose certainly) usually.The 4th, reaction at room temperature takes place rapidly, and this allows to use will be at the polypeptide or the reagent of unsettled many types under the higher temperature.The 5th, reaction is easy to take place under aqueous conditions, and this also allows to use the polypeptide and the reagent of incompatible with non-aqueous solution (on any degree).The 6th, even when the ratio of polypeptide or amino acid and reagent is stoichiometry, near-stoichiometric or class stoichiometry, reaction also is easy to take place, thereby does not need to add the product that excessive reagent or polypeptide obtain consumption.The 7th, the design of diamines and dicarbapentaborane part in the visual reactant and regioselectivity and/or regiospecificity ground generation gained heterocycle.At last, diamines with contain the condensation of dicarbapentaborane molecule and be created in heterocycle stable under the biotic factor (comprising nitrogen heterocyclic ring) key.
(i) contain the diamines alpha-non-natural amino acid and contain the reaction of dicarbapentaborane reagent or contain diamines alpha-non-natural amino acid and the reaction that contains ketone alkynes reagent
The alpha-non-natural amino acid that contains two amidos allows to react to form concatenator (including but not limited to the concatenator with PEG or other water-soluble polymer) with various electrophilic groups.The nucleophilicity of two amidos can be under temperate condition effectively and be optionally reacted to form corresponding imine linkage it in the aqueous solution with the various molecules that contain carbonyl or dicarbapentaborane functional group or have similar chemically reactive other functional group.In addition, unique reactive permission of carbonyl or dicarbapentaborane carried out selective modification under the situation that has other amino acid side chain.For example referring to Cornish, people such as V.W., J.Am.Chem.Soc.118:8150-8151 (1996); Geoghegan, K.F.﹠amp; Stroh, J.G., Bioconjug.Chem.3:138-146 (1992); Mahal, people such as L.K., Science 276:1125-1128 (1997).
The described alpha-non-natural amino acid that comprises heterocyclic side chain and have formula (XXXVII) structure comprises the amino acid of have formula (XXXVIII) and formula (XXXIX) structure:
Wherein:
Z 1Be bond, CR 7R 7, O, S, NR ', CR 7R 7-CR 7R 7, CR 7R 7-O, O-CR 7R 7, CR 7R 7-S, S-CR 7R 7, CR 7R 7-NR ', NR '-CR 7R 7
Z 2Be selected from the group that forms by following each group: bond, the C that is substituted according to circumstances 1-C 4Alkylidene, the C that is substituted according to circumstances 1-C 4Alkenylene, the assorted alkyl that is substituted according to circumstances ,-O-,-S-,-C (O)-,-C (S)-and-N (R ')-;
R ' is H, alkyl or is substituted alkyl;
Each R 5Independently for H, alkyl, be substituted alkyl, thiazolinyl, be substituted thiazolinyl, alkynyl, be substituted alkynyl, alkoxyl, be substituted alkoxyl, alkyl alkoxy, be substituted alkyl alkoxy, polyalkylene oxide, be substituted polyalkylene oxide, aryl, be substituted aryl, heteroaryl, be substituted heteroaryl, alkaryl, be substituted alkaryl, aralkyl, be substituted aralkyl ,-(alkylidene or be substituted alkylidene)-ON (R ") 2,-(alkylidene or be substituted alkylidene)-C (O) SR " ,-(alkylidene or be substituted alkylidene)-S-S-(aryl or be substituted aryl) ,-C (O) R " ,-C (O) 2R " or-C (O) N (R ") 2, each R wherein " independently for hydrogen, alkyl, be substituted alkyl, thiazolinyl, be substituted thiazolinyl, alkoxyl, be substituted alkoxyl, aryl, be substituted aryl, heteroaryl, alkaryl, be substituted alkaryl, aralkyl, be substituted aralkyl;
Or R 5Be L-X, wherein X is selected from the group that is made up of required functional group; And L is optional, and when existing, it is the connection base that is selected from the group that is made up of following each group: alkylidene, be substituted alkylidene, alkenylene, be substituted alkenylene ,-O-,-O-(alkylidene or be substituted alkylidene)-,-S-,-S-(alkylidene or be substituted alkylidene)-,-S (O) k-(wherein k is 1,2 or 3) ,-S (O) k(alkylidene or be substituted alkylidene)-,-C (O)-,-C (O)-(alkylidene or be substituted alkylidene)-,-C (S)-,-C (S)-(alkylidene or be substituted alkylidene)-,-N (R ')-,-NR '-(alkylidene or be substituted alkylidene)-,-C (O) N (R ')-,-CON (R ')-(alkylidene or be substituted alkylidene)-,-CSN (R ')-,-CSN (R ')-(alkylidene or be substituted alkylidene)-,-N (R ') CO-(alkylidene or be substituted alkylidene)-,-N (R ') C (O) O-,-(alkylidene or be substituted alkylidene)-O-N=CR '-,-(alkylidene or be substituted alkylidene)-C (O) NR '-(alkylidene or be substituted alkylidene)-,-(alkylidene or be substituted alkylidene)-S (O) k-(alkylidene or be substituted alkylidene)-S-,-(alkylidene or be substituted alkylidene)-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 each R ' is H, alkyl independently or is substituted alkyl;
R 6With each R 7Be independently selected from the group that forms by following each group: H, alkyl, be substituted alkyl, thiazolinyl, be substituted thiazolinyl, alkynyl, be substituted alkynyl, alkoxyl, be substituted alkoxyl, alkyl alkoxy, be substituted alkyl alkoxy, polyalkylene oxide, be substituted polyalkylene oxide, aryl, be substituted aryl, heteroaryl, be substituted heteroaryl, alkaryl, be substituted alkaryl, aralkyl, be substituted aralkyl ,-(alkylidene or be substituted alkylidene)-ON (R ") 2,-(alkylidene or be substituted alkylidene)-C (O) SR " ,-(alkylidene or be substituted alkylidene)-S-S-(aryl or be substituted aryl) ,-C (O) R " ,-C (O) 2R " or-C (O) N (R ") 2, each R wherein " independently for hydrogen, alkyl, be substituted alkyl, thiazolinyl, be substituted thiazolinyl, alkoxyl, be substituted alkoxyl, aryl, be substituted aryl, heteroaryl, alkaryl, be substituted alkaryl, aralkyl or be substituted aralkyl;
Or any two adjacent R 7Base forms 5 yuan to 8 yuan heterocycles, cycloalkyl or aromatic rings that are substituted according to circumstances together; Wherein said optional substituting group is selected from halogen, OH, C 1-6Alkyl, C 1-6Alkoxyl, halogen-C 1-6Alkyl, halogen-C 1-6Alkoxyl, aryl, halogen aryl and heteroaryl;
Condition is Z 1Add Z 2Provide to heterocycle structure and to be no more than 3 annular atomses.
In addition, comprise following amino acid with formula (XL), formula (XLI) and formula (XLII) structure:
Wherein:
Each R aBe independently selected from the group that forms by following each group: H, halogen, alkyl, be substituted alkyl ,-N (R ') 2,-C (O) R ' ,-C (O) N (R ') 2,-OR ' and-S (O) kR ', wherein k is 1,2 or 3, and each R ' is H, alkyl independently or is substituted alkyl.
In addition, comprise following amino acid with formula (XL), formula (XLI) or formula (XLII) structure:
With
Figure A200680049995D00963
(ii) contain the dicarbapentaborane alpha-non-natural amino acid and contain diamines reagent or contain the reaction of ketoamine reagent
Alpha-non-natural amino acid with electrophilic reaction group allows especially to connect via nucleophilic addition the various reactions of molecule.Described electrophilic reaction group comprises dicarbapentaborane (comprising diketo, ketaldonyl, ketone acid base, ketone ester base and ketone thioester substrate), class dicarbapentaborane (it has similarly reactive and structurally be similar to carbonyl with dicarbapentaborane), through covering dicarbapentaborane (it can easily change into dicarbapentaborane) or through protection dicarbapentaborane (its going protection then have similarly reactive) with dicarbapentaborane.The alpha-non-natural amino acid that contains dicarbapentaborane allows to react to form concatenator (including but not limited to the concatenator with PEG or other water-soluble polymer) with various nucleophilic groups.The electrophilicity of dicarbapentaborane makes it can be effectively and optionally contain amine, diamines, ketoamine or other has the molecular reaction of similar chemically reactive functional group with various.
Therefore, some embodiment as herein described comprise heterocyclic radical for having, through covering the alpha-non-natural amino acid of heterocyclic radical (it can easily change into heterocyclic radical) or side chain through protecting two amidos (its going protection then have reactive) to carry out other chemical reaction.Wherein said heterocyclic radical is by containing the dicarbapentaborane alpha-non-natural amino acid and variously containing amine, diamines, ketoamine or other molecular reaction with similar chemically reactive functional group and form.
Described amino acid with formula (XXXVII) structure comprises the amino acid of have formula (XLIII), formula (XLIV), formula (XLV), formula (XLVI), formula (XLVII) and formula (XLVIII) structure:
Figure A200680049995D00971
Z 1Be bond, CR 5R 5, CR 5R 5-CR 5R 5, CR 5R 5-O, O-CR 5R 5, S-CR 5R 5, NR 5-CR 5R 5, CR 5R 5-S, CR 5R 5-NR 5
Z 2Be selected from the group that forms by following each group: the C that is substituted according to circumstances 1-C 3Alkylidene, the C that is substituted according to circumstances 1-C 3Alkenylene, the assorted alkyl and the N that are substituted according to circumstances;
M 2For
Figure A200680049995D00972
Figure A200680049995D00973
Or
Figure A200680049995D00974
Wherein (a) expression and the bond of B group, and (b) bond of indivedual positions in expression and the heterocyclic radical;
M 3For
Figure A200680049995D00981
Or
Figure A200680049995D00982
Wherein (a) expression and the bond of B group, and (b) bond of indivedual positions in expression and the heterocyclic radical;
M 4For
Figure A200680049995D00983
Or
Figure A200680049995D00984
Wherein (a) expression and the bond of B group, and (b) bond of indivedual positions in expression and the heterocyclic radical;
T 3For bond, C (R) (R), O or S; And R is H, halogen, alkyl, is substituted alkyl, cycloalkyl or is substituted cycloalkyl;
R 6Be selected from the group that forms by following each group: H, alkyl, be substituted alkyl, thiazolinyl, be substituted thiazolinyl, alkynyl, be substituted alkynyl, alkoxyl, be substituted alkoxyl, alkyl alkoxy, be substituted alkyl alkoxy, polyalkylene oxide, be substituted polyalkylene oxide, aryl, be substituted aryl, heteroaryl, be substituted heteroaryl, alkaryl, be substituted alkaryl, aralkyl, be substituted aralkyl ,-(alkylidene or be substituted alkylidene)-ON (R ") 2,-(alkylidene or be substituted alkylidene)-C (O) SR " ,-(alkylidene or be substituted alkylidene)-S-S-(aryl or be substituted aryl) ,-C (O) R " ,-C (O) 2R " or-C (O) N (R ") 2, each R wherein " independently for hydrogen, alkyl, be substituted alkyl, thiazolinyl, be substituted thiazolinyl, alkoxyl, be substituted alkoxyl, aryl, be substituted aryl, heteroaryl, alkaryl, be substituted alkaryl, aralkyl and be substituted aralkyl;
Condition is Z 1Add Z 2Heterocycle structure provided be no more than 3 annular atomses, or Z 2Add Z 3Heterocycle structure provided be no more than 3 annular atomses;
R 5For H, alkyl, be substituted alkyl, thiazolinyl, be substituted thiazolinyl, alkynyl, be substituted alkynyl, alkoxyl, be substituted alkoxyl, alkyl alkoxy, be substituted alkyl alkoxy, polyalkylene oxide, be substituted polyalkylene oxide, aryl, be substituted aryl, heteroaryl, be substituted heteroaryl, alkaryl, be substituted alkaryl, aralkyl, be substituted aralkyl ,-(alkylidene or be substituted alkylidene)-ON (R ") 2,-(alkylidene or be substituted alkylidene)-C (O) SR " ,-(alkylidene or be substituted alkylidene)-S-S-(aryl or be substituted aryl) ,-C (O) R " ,-C (O) 2R " or-C (O) N (R ") 2, each R wherein " independently for hydrogen, alkyl, be substituted alkyl, thiazolinyl, be substituted thiazolinyl, alkoxyl, be substituted alkoxyl, 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 required functional group; And L is optional, and when existing, it is the connection base that is selected from the group that is made up of following each group: alkylidene, be substituted alkylidene, alkenylene, be substituted alkenylene ,-O-,-O-(alkylidene or be substituted alkylidene)-,-S-,-S-(alkylidene or be substituted alkylidene)-,-S (O) k-(wherein k is 1,2 or 3) ,-S (O) k(alkylidene or be substituted alkylidene)-,-C (O)-,-C (O)-(alkylidene or be substituted alkylidene)-,-C (S)-,-C (S)-(alkylidene or be substituted alkylidene)-,-N (R ')-,-NR '-(alkylidene or be substituted alkylidene)-,-C (O) N (R ')-,-CON (R ')-(alkylidene or be substituted alkylidene)-,-CSN (R ')-,-CSN (R ')-(alkylidene or be substituted alkylidene)-,-N (R ') CO-(alkylidene or be substituted alkylidene)-,-N (R ') C (O) O-,-(alkylidene or be substituted alkylidene)-O-N=CR '-,-(alkylidene or be substituted alkylidene)-C (O) NR '-(alkylidene or be substituted alkylidene)-,-(alkylidene or be substituted alkylidene)-S (O) k-(alkylidene or be substituted alkylidene)-S-,-(alkylidene or be substituted alkylidene)-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 each R ' is H, alkyl independently or is substituted alkyl.
In addition, the amino acid with formula (XLIII), formula (XLIV), formula (XLV), formula (XLVI), formula (XLVII) or formula (XLVIII) structure comprises following amino acid with formula (XLIX), formula (L), formula (LI), formula (LII), formula (LIII) and formula (LIV) structure:
R wherein aBe independently selected from the group that forms by following each group: H, halogen, alkyl, be substituted alkyl ,-N (R ') 2,-C (O) N (R ') 2,-OR ' and-S (O) kR ', wherein k is 1,2 or 3.
In addition, comprise following amino acid according to formula (XXXVII):
Figure A200680049995D01001
Wherein:
Each R 6Be independently selected from the group that forms by following each group: H, alkyl, be substituted alkyl, thiazolinyl, be substituted thiazolinyl, alkynyl, be substituted alkynyl, alkoxyl, be substituted alkoxyl, alkyl alkoxy, be substituted alkyl alkoxy, polyalkylene oxide, be substituted polyalkylene oxide, aryl, be substituted aryl, heteroaryl, be substituted heteroaryl, alkaryl, be substituted alkaryl, aralkyl, be substituted aralkyl ,-(alkylidene or be substituted alkylidene)-ON (R ") 2,-(alkylidene or be substituted alkylidene)-C (O) SR " ,-(alkylidene or be substituted alkylidene)-S-S-(aryl or be substituted aryl) ,-C (O) R " ,-C (O) 2R " or-C (O) N (R ") 2, each R wherein " independently for hydrogen, alkyl, be substituted alkyl, thiazolinyl, be substituted thiazolinyl, alkoxyl, be substituted alkoxyl, aryl, be substituted aryl, heteroaryl, alkaryl, be substituted alkaryl, aralkyl and be substituted aralkyl.
Also comprise by containing the formed alpha-non-natural amino acid of dicarbapentaborane amino acid and ketoamine reaction with heterocycle side group.Described amino acid comprises the amino acid of have formula (LV) and formula (LVI) structure:
Figure A200680049995D01002
Wherein:
A is optional, and when existing, it is the low-carbon (LC) alkylidene, be substituted low-carbon (LC) alkylidene, low-carbon (LC) cycloalkylidene, be substituted the low-carbon (LC) cycloalkylidene, the low-carbon (LC) alkenylene, be substituted low-carbon (LC) alkenylene, alkynylene, the inferior assorted alkyl of low-carbon (LC), be substituted inferior assorted alkyl, the inferior Heterocyclylalkyl of low-carbon (LC), be substituted low-carbon (LC) inferior Heterocyclylalkyl, arlydene, be substituted arlydene, inferior heteroaryl, be substituted inferior heteroaryl, alkarylene, be substituted alkarylene, inferior aralkyl or be substituted inferior aralkyl;
B is optional, and when existing, its at one end with contain the connection base that two amine moieties are connected, described connection base is selected from the group that is made up of following each group: the low-carbon (LC) alkylidene, be substituted low-carbon (LC) alkylidene, low-carbon (LC) alkenylene, be substituted the low-carbon (LC) alkenylene, the inferior assorted alkyl of low-carbon (LC), be substituted the low-carbon (LC) Asia mix alkyl ,-O-(alkylidene or be substituted alkylidene)-,-S-(alkylidene or be substituted alkylidene)-,-C (O) R " ,-S (O) k(alkylidene or be substituted alkylidene)-(wherein k is 1,2 or 3) ,-C (O)-(alkylidene or be substituted alkylidene)-,-C (S)-(alkylidene or be substituted alkylidene)-,-NR " (alkylidene or be substituted alkylidene)-,-CON (R ")-(alkylidene or be substituted alkylidene)-,-CSN (R ")-(alkylidene or be substituted alkylidene)-and-N (R ") CO-(alkylidene or be substituted alkylidene)-, and each R wherein is " independently for H, alkyl or be 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 3And R 4Be H, halogen, low-carbon alkyl or be substituted low-carbon alkyl, perhaps R independently of one another 3And 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, be substituted alkoxyl, alkyl alkoxy, be substituted alkyl alkoxy, polyalkylene oxide, be substituted polyalkylene oxide, aryl, be substituted aryl, heteroaryl, be substituted heteroaryl, alkaryl, be substituted alkaryl, aralkyl, be substituted aralkyl ,-(alkylidene or be substituted alkylidene)-ON (R ") 2,-(alkylidene or be substituted alkylidene)-C (O) SR " ,-(alkylidene or be substituted alkylidene)-S-S-(aryl or be substituted aryl) ,-C (O) R " ,-C (O) 2R " or-C (O) N (R ") 2, each R wherein " independently for hydrogen, alkyl, be substituted alkyl, thiazolinyl, be substituted thiazolinyl, alkoxyl, be substituted alkoxyl, aryl, be substituted aryl, heteroaryl, alkaryl, be substituted alkaryl, aralkyl, be substituted aralkyl;
Or R 5Be L-X, wherein X is selected from the group that is made up of required functional group; And L is optional, and when existing, it is the connection base that is selected from the group that is made up of following each group: alkylidene, be substituted alkylidene, alkenylene, be substituted alkenylene ,-O-,-O-(alkylidene or be substituted alkylidene)-,-S-,-S-(alkylidene or be substituted alkylidene)-,-S (O) k-(wherein k is 1,2 or 3) ,-S (O) k(alkylidene or be substituted alkylidene)-,-C (O)-,-C (O)-(alkylidene or be substituted alkylidene)-,-C (S)-,-C (S)-(alkylidene or be substituted alkylidene)-,-N (R ')-,-NR '-(alkylidene or be substituted alkylidene)-,-C (O) N (R ')-,-CON (R ')-(alkylidene or be substituted alkylidene)-,-CSN (R ')-,-CSN (R ')-(alkylidene or be substituted alkylidene)-,-N (R ') CO-(alkylidene or be substituted alkylidene)-,-N (R ') C (O) O-,-(alkylidene or be substituted alkylidene)-O-N=CR '-,-(alkylidene or be substituted alkylidene)-C (O) NR '-(alkylidene or be substituted alkylidene)-,-(alkylidene or be substituted alkylidene)-S (O) k-(alkylidene or be substituted alkylidene)-S-,-(alkylidene or be substituted alkylidene)-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 each R ' is H, alkyl independently or is substituted alkyl;
R 6Be selected from the group that forms by following each group: H, alkyl, be substituted alkyl, thiazolinyl, be substituted thiazolinyl, alkynyl, be substituted alkynyl, alkoxyl, be substituted alkoxyl, alkyl alkoxy, be substituted alkyl alkoxy, polyalkylene oxide, be substituted polyalkylene oxide, aryl, be substituted aryl, heteroaryl, be substituted heteroaryl, alkaryl, be substituted alkaryl, aralkyl, be substituted aralkyl ,-(alkylidene or be substituted alkylidene)-ON (R ") 2,-(alkylidene or be substituted alkylidene)-C (O) SR " ,-(alkylidene or be substituted alkylidene)-S-S-(aryl or be substituted aryl) ,-C (O) R " ,-C (O) 2R " or-C (O) N (R ") 2, each R wherein " independently for hydrogen, alkyl, be substituted alkyl, thiazolinyl, be substituted thiazolinyl, alkoxyl, be substituted alkoxyl, aryl, be substituted aryl, heteroaryl, alkaryl, be substituted alkaryl, aralkyl and be substituted aralkyl.
In addition, comprise following amino acid according to formula (LV) or formula (LVI):
Figure A200680049995D01021
Provide among Figure 11 via containing the dicarbapentaborane alpha-non-natural amino acid and synthesize with the non-limiting exemplary of heterocycle alpha-non-natural amino acid that contain that contains the diamines reagent reacting.
(iii) contain ketone alkynes alpha-non-natural amino acid and the reaction that contains diamines reagent
Containing the alpha-non-natural amino acid with the reactive reactive group of class dicarbapentaborane allows to connect molecule via nucleophilic addition.Described electrophilic reaction group comprises ketone alkynyl, class ketone alkynyl (its have be similar to the reactive of ketone alkynyl and structurally be similar to carbonyl), through covering ketone alkynyl (it can easily change into the ketone alkynyl) or through protection ketone alkynyl (it has the reactivity that is similar to the ketone alkynyl after going to protect).The alpha-non-natural amino acid that contains the ketone alkynyl allows to react to form concatenator with (but being not limited to) PEG or other water-soluble polymer with various groups (such as (but being not limited to) two amidos).
Therefore, some embodiment as herein described comprise heterocyclic radical for having, through covering the alpha-non-natural amino acid of heterocyclic radical (it can easily change into heterocyclic radical) or side chain through protecting two amidos (its going protection then have reactive) to carry out other chemical reaction.Wherein said heterocyclic radical is by containing ketone alkynes alpha-non-natural amino acid and variously containing amine, diamines or other molecular reaction with similar chemically reactive functional group and form.
Described amino acid with formula (XXXVII) structure comprise have formula (LVII) to the amino acid of formula (LX) structure:
Figure A200680049995D01031
Wherein:
Z 1Be bond, CR 5R 5, CR 5R 5-CR 5R 5, CR 5R 5-O, O-CR 5R 5, S-CR 5R 5, NR 5-CR 5R 5, CR 5R 5-S, CR 5R 5-NR 5
Z 3Be selected from the group that forms by following each group: bond, the C that is substituted according to circumstances 1-C 4Alkylidene, the C that is substituted according to circumstances 1-C 4Alkenylene, the assorted alkyl that is substituted according to circumstances ,-O-,-S-,-C (O)-,-C (S)-and-N (R ')-;
R 6Be selected from the group that forms by following each group: H, alkyl, be substituted alkyl, thiazolinyl, be substituted thiazolinyl, alkynyl, be substituted alkynyl, alkoxyl, be substituted alkoxyl, alkyl alkoxy, be substituted alkyl alkoxy, polyalkylene oxide, be substituted polyalkylene oxide, aryl, be substituted aryl, heteroaryl, be substituted heteroaryl, alkaryl, be substituted alkaryl, aralkyl, be substituted aralkyl ,-(alkylidene or be substituted alkylidene)-ON (R ") 2,-(alkylidene or be substituted alkylidene)-C (O) SR " ,-(alkylidene or be substituted alkylidene)-S-S-(aryl or be substituted aryl) ,-C (O) R " ,-C (O) 2R " or-C (O) N (R ") 2, each R wherein " independently for hydrogen, alkyl, be substituted alkyl, thiazolinyl, be substituted thiazolinyl, alkoxyl, be substituted alkoxyl, aryl, be substituted aryl, heteroaryl, alkaryl, be substituted alkaryl, aralkyl and be substituted aralkyl;
R 5For H, alkyl, be substituted alkyl, thiazolinyl, be substituted thiazolinyl, alkynyl, be substituted alkynyl, alkoxyl, be substituted alkoxyl, alkyl alkoxy, be substituted alkyl alkoxy, polyalkylene oxide, be substituted polyalkylene oxide, aryl, be substituted aryl, heteroaryl, be substituted heteroaryl, alkaryl, be substituted alkaryl, aralkyl, be substituted aralkyl ,-(alkylidene or be substituted alkylidene)-ON (R ") 2,-(alkylidene or be substituted alkylidene)-C (O) SR " ,-(alkylidene or be substituted alkylidene)-S-S-(aryl or be substituted aryl) ,-C (O) R " ,-C (O) 2R " or-C (O) N (R ") 2, each R wherein " independently for hydrogen, alkyl, be substituted alkyl, thiazolinyl, be substituted thiazolinyl, alkoxyl, be substituted alkoxyl, aryl, be substituted aryl, heteroaryl, alkaryl, be substituted alkaryl, aralkyl, be substituted aralkyl;
Or R 5Be L-X, wherein X is selected from the group that is made up of required functional group; And L is optional, and when existing, it is the connection base that is selected from the group that is made up of following each group: alkylidene, be substituted alkylidene, alkenylene, be substituted alkenylene ,-O-,-O-(alkylidene or be substituted alkylidene)-,-S-,-S-(alkylidene or be substituted alkylidene)-,-S (O) k-(wherein k is 1,2 or 3) ,-S (O) k(alkylidene or be substituted alkylidene)-,-C (O)-,-C (O)-(alkylidene or be substituted alkylidene)-,-C (S)-,-C (S)-(alkylidene or be substituted alkylidene)-,-N (R ')-,-NR '-(alkylidene or be substituted alkylidene)-,-C (O) N (R ')-,-CON (R ')-(alkylidene or be substituted alkylidene)-,-CSN (R ')-,-CSN (R ')-(alkylidene or be substituted alkylidene)-,-N (R ') CO-(alkylidene or be substituted alkylidene)-,-N (R ') C (O) O-,-(alkylidene or be substituted alkylidene)-O-N=CR '-,-(alkylidene or be substituted alkylidene)-C (O) NR '-(alkylidene or be substituted alkylidene)-,-(alkylidene or be substituted alkylidene)-S (O) k-(alkylidene or be substituted alkylidene)-S-,-(alkylidene or be substituted alkylidene)-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 each R ' is H, alkyl independently or is substituted alkyl.
Provide among Figure 13 via containing ketone alkynes alpha-non-natural amino acid and synthesize with the non-limiting exemplary of heterocycle alpha-non-natural amino acid that contain that contains the diamines reagent reacting.
(iv) contain ketoamine alpha-non-natural amino acid and the reaction that contains dicarbapentaborane reagent
Containing the alpha-non-natural amino acid with the reactive reactive group of class dicarbapentaborane allows to connect molecule via nucleophilic addition.Described reactive group comprises ketoamine base, class ketoamine base (its have be similar to the reactive of ketoamine base and structurally be similar to the ketoamine base), through covering ketoamine base (it can easily change into the ketoamine base) or through protection ketoamine base (it has the reactivity that is similar to the ketoamine base after going to protect).The alpha-non-natural amino acid that contains the ketoamine base allows to react to form concatenator with (but being not limited to) PEG or other water-soluble polymer with various groups (such as (but being not limited to) dicarbapentaborane).
Therefore, some embodiment as herein described comprise heterocyclic radical for having, through covering heterocyclic radical (it can easily change into heterocyclic radical) or through the alpha-non-natural amino acid of the side chain of protection heterocyclic radical (its going protection then have reactive) to carry out other chemical reaction.Wherein said heterocyclic radical is by containing the ketoamine alpha-non-natural amino acid and variously containing dicarbapentaborane or other molecular reaction with similar chemically reactive functional group forms.
Described amino acid with formula (XXXVII) structure comprises the amino acid of have formula (LXII) and formula (LXIII) structure:
Figure A200680049995D01041
Wherein:
R 6Be selected from the group that forms by following each group: H, alkyl, be substituted alkyl, thiazolinyl, be substituted thiazolinyl, alkynyl, be substituted alkynyl, alkoxyl, be substituted alkoxyl, alkyl alkoxy, be substituted alkyl alkoxy, polyalkylene oxide, be substituted polyalkylene oxide, aryl, be substituted aryl, heteroaryl, be substituted heteroaryl, alkaryl, be substituted alkaryl, aralkyl, be substituted aralkyl ,-(alkylidene or be substituted alkylidene)-ON (R ") 2,-(alkylidene or be substituted alkylidene)-C (O) SR " ,-(alkylidene or be substituted alkylidene)-S-S-(aryl or be substituted aryl) ,-C (O) R " ,-C (O) 2R " or-C (O) N (R ") 2, each R wherein " independently for hydrogen, alkyl, be substituted alkyl, thiazolinyl, be substituted thiazolinyl, alkoxyl, be substituted alkoxyl, aryl, be substituted aryl, heteroaryl, alkaryl, be substituted alkaryl, aralkyl and be substituted aralkyl;
R 5For H, alkyl, be substituted alkyl, thiazolinyl, be substituted thiazolinyl, alkynyl, be substituted alkynyl, alkoxyl, be substituted alkoxyl, alkyl alkoxy, be substituted alkyl alkoxy, polyalkylene oxide, be substituted polyalkylene oxide, aryl, be substituted aryl, heteroaryl, be substituted heteroaryl, alkaryl, be substituted alkaryl, aralkyl, be substituted aralkyl ,-(alkylidene or be substituted alkylidene)-ON (R ") 2,-(alkylidene or be substituted alkylidene)-C (O) SR " ,-(alkylidene or be substituted alkylidene)-S-S-(aryl or be substituted aryl) ,-C (O) R " ,-C (O) 2R " or-C (O) N (R ") 2, each R wherein " independently for hydrogen, alkyl, be substituted alkyl, thiazolinyl, be substituted thiazolinyl, alkoxyl, be substituted alkoxyl, aryl, be substituted aryl, heteroaryl, alkaryl, be substituted alkaryl, aralkyl, be substituted aralkyl;
Or R 5Be L-X, wherein X is selected from the group that is made up of required functional group; And L is optional, and when existing, it is the connection base that is selected from the group that is made up of following each group: alkylidene, be substituted alkylidene, alkenylene, be substituted alkenylene ,-O-,-O-(alkylidene or be substituted alkylidene)-,-S-,-S-(alkylidene or be substituted alkylidene)-,-S (O) k-(wherein k is 1,2 or 3) ,-S (O) k(alkylidene or be substituted alkylidene)-,-C (O)-,-C (O)-(alkylidene or be substituted alkylidene)-,-C (S)-,-C (S)-(alkylidene or be substituted alkylidene)-,-N (R ')-,-NR '-(alkylidene or be substituted alkylidene)-,-C (O) N (R ')-,-CON (R ')-(alkylidene or be substituted alkylidene)-,-CSN (R ')-,-CSN (R ')-(alkylidene or be substituted alkylidene)-,-N (R ') CO-(alkylidene or be substituted alkylidene)-,-N (R ') C (O) O-,-(alkylidene or be substituted alkylidene)-O-N=CR '-,-(alkylidene or be substituted alkylidene)-C (O) NR '-(alkylidene or be substituted alkylidene)-,-(alkylidene or be substituted alkylidene)-S (O) k-(alkylidene or be substituted alkylidene)-S-,-(alkylidene or be substituted alkylidene)-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 each R ' is H, alkyl independently or is substituted alkyl.
F. the structure of alpha-non-natural amino acid is with synthetic: contain alkene-two keto amino acid
Alpha-non-natural amino acid with electrophilic reaction group allows especially to connect via nucleophilic addition the various reactions of molecule.Described electrophilic reaction group comprises dicarbapentaborane (comprising diketo, ketaldonyl, ketone acid base, ketone ester base and ketone thioester substrate), class dicarbapentaborane (it has similarly reactive and structurally be similar to carbonyl with dicarbapentaborane), through covering dicarbapentaborane (it can easily change into dicarbapentaborane) or through protection dicarbapentaborane (its going protection then have similarly reactive) with dicarbapentaborane.The alpha-non-natural amino acid that contains dicarbapentaborane allows to react to form concatenator (including but not limited to the concatenator with PEG or other water-soluble polymer) with various nucleophilic groups.The electrophilicity of dicarbapentaborane can be reacted to form " based on the key of aldehyde alcohol " or " based on the key that mixes aldehyde alcohol " it in aldehyde alcohol reaction or the reaction of aldehyde alcohol type.
Therefore, some embodiment as herein described is the alpha-non-natural amino acid with the side chain that comprises the group that is produced by related dicarbapentaborane in aldehyde alcohol reaction, the reaction of mixing aldehyde alcohol or the reaction of aldehyde alcohol type.Described amino acid comprises the amino acid of (LXIV) structure that has formula:
Figure A200680049995D01061
Wherein:
A is optional, and when existing, it is the low-carbon (LC) alkylidene, be substituted low-carbon (LC) alkylidene, low-carbon (LC) cycloalkylidene, be substituted the low-carbon (LC) cycloalkylidene, the low-carbon (LC) alkenylene, be substituted low-carbon (LC) alkenylene, alkynylene, the inferior assorted alkyl of low-carbon (LC), be substituted inferior assorted alkyl, the inferior Heterocyclylalkyl of low-carbon (LC), be substituted low-carbon (LC) inferior Heterocyclylalkyl, arlydene, be substituted arlydene, inferior heteroaryl, be substituted inferior heteroaryl, alkarylene, be substituted alkarylene, inferior aralkyl or be substituted inferior aralkyl;
B is optional, and when existing, its at one end with contain the connection base that two amine moieties are connected, described connection base is selected from the group that is made up of following each group: the low-carbon (LC) alkylidene, be substituted low-carbon (LC) alkylidene, low-carbon (LC) alkenylene, be substituted the low-carbon (LC) alkenylene, the inferior assorted alkyl of low-carbon (LC), be substituted the low-carbon (LC) Asia mix alkyl ,-O-(alkylidene or be substituted alkylidene)-,-S-(alkylidene or be substituted alkylidene)-,-C (O) R " ,-S (O) k(alkylidene or be substituted alkylidene)-(wherein k is 1,2 or 3) ,-C (O)-(alkylidene or be substituted alkylidene)-,-C (S)-(alkylidene or be substituted alkylidene)-,-NR " (alkylidene or be substituted alkylidene)-,-CON (R ")-(alkylidene or be substituted alkylidene)-,-CSN (R ")-(alkylidene or be substituted alkylidene)-and-N (R ") CO-(alkylidene or be substituted alkylidene)-, and each R wherein is " independently for H, alkyl or be 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 3And R 4Be H, halogen, low-carbon alkyl or be substituted low-carbon alkyl, perhaps R independently of one another 3And 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, be substituted alkoxyl, alkyl alkoxy, be substituted alkyl alkoxy, polyalkylene oxide, be substituted polyalkylene oxide, aryl, be substituted aryl, heteroaryl, be substituted heteroaryl, alkaryl, be substituted alkaryl, aralkyl, be substituted aralkyl ,-(alkylidene or be substituted alkylidene)-ON (R ") 2,-(alkylidene or be substituted alkylidene)-C (O) SR " ,-(alkylidene or be substituted alkylidene)-S-S-(aryl or be substituted aryl) ,-C (O) R " ,-C (O) 2R " or-C (O) N (R ") 2, each R wherein " independently for hydrogen, alkyl, be substituted alkyl, thiazolinyl, be substituted thiazolinyl, alkoxyl, be substituted alkoxyl, aryl, be substituted aryl, heteroaryl, alkaryl, be substituted alkaryl, aralkyl, be substituted aralkyl;
Or R 5Be L-X, wherein X is selected from the group that is made up of required functional group; And L is optional, and when existing, it is the connection base that is selected from the group that is made up of following each group: alkylidene, be substituted alkylidene, alkenylene, be substituted alkenylene ,-O-,-O-(alkylidene or be substituted alkylidene)-,-S-,-S-(alkylidene or be substituted alkylidene)-,-S (O) k-(wherein k is 1,2 or 3) ,-S (O) k(alkylidene or be substituted alkylidene)-,-C (O)-,-C (O)-(alkylidene or be substituted alkylidene)-,-C (S)-,-C (S)-(alkylidene or be substituted alkylidene)-,-N (R ')-,-NR '-(alkylidene or be substituted alkylidene)-,-C (O) N (R ')-,-CON (R ')-(alkylidene or be substituted alkylidene)-,-CSN (R ')-,-CSN (R ')-(alkylidene or be substituted alkylidene)-,-N (R ') CO-(alkylidene or be substituted alkylidene)-,-N (R ') C (O) O-,-(alkylidene or be substituted alkylidene)-O-N=CR '-,-(alkylidene or be substituted alkylidene)-C (O) NR '-(alkylidene or be substituted alkylidene)-,-(alkylidene or be substituted alkylidene)-S (O) k-(alkylidene or be substituted alkylidene)-S-,-(alkylidene or be substituted alkylidene)-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 each R ' is H, alkyl independently or is substituted alkyl;
T 3For bond, C (R) (R), O or S, and R is H, halogen, alkyl, is substituted alkyl, cycloalkyl or is substituted cycloalkyl.
In addition, comprise the following amino acid that arrives formula (LVII) according to formula (LV):
G. the cellular uptake of alpha-non-natural amino acid
Eukaryotic is when design to the picked-up of alpha-non-natural amino acid and a problem considering usually when selecting (including but not limited to) to be used for incorporating into the alpha-non-natural amino acid of protein.For instance, the high charge density of a-amino acid shows that these compounds can not permeation cell.Natural amino acid is to absorb in the eukaryotic via a series of movement systems based on protein.Can carry out rapid screening is absorbed by cell to evaluate which alpha-non-natural amino acid (if existence).For example referring to, U.S. Patent Publication case No. 2004/198637 (its mode of quoting in full is incorporated herein) and the Liu of " Protein Arrays " for example by name, D.R.﹠amp; Schultz, the toxicological detection among P.G. (1999) the Progress toward the evolution of anorganism with an expanded genetic code.PNAS United States 96:4780-4785.Although be easy to utilize various calibratings to analyze picked-up, design is applicable to that the alternative method of the alpha-non-natural amino acid in cellular uptake path provides the biosynthesis path to produce amino acid in vivo.
Usually, be to produce being enough to carry out effective protein biosynthesis (including but not limited to the n cell amount) but not reaching the concentration that influences other amino acid whose concentration or exhaust the degree of cell resource via the alpha-non-natural amino acid that cellular uptake produced as described herein.The typical concentration that is produced arrives about 0.05mM for about 10mM in this way.
H. the biosynthesis of alpha-non-natural amino acid
Many biosynthesis path has been present in the cell for producing amino acid and other compound.The biological synthesis method of specific alpha-non-natural amino acid may not be present in the natural world (including but not limited to cell), and method and composition as herein described provides described method.For instance, in host cell by adding novel enzymes or changing the biosynthesis path that existing host cell path can produce alpha-non-natural amino acid.Other novel enzymes comprises the enzyme of naturally occurring enzyme or artificial exploitation.For instance, the biosynthesis of p-Aminophenylalanine (providing as the example among the WO 2002/085923 of " In vivo incorporation of unnaturalamino acids " by name) depends on the combination of adding from other organic known enzyme.Can described gene be introduced in the eukaryotic by plasmid transformant with the gene that comprises these enzymes.When expressing in cell, these genes provide the enzymatic path of synthetic required compound.The example of the enzyme type of Jia Ruing is provided in herein according to circumstances.Other enzyme sequence for example sees among the Genbank.Can in the same manner the enzyme of manually developing be added in the cell.In this way, utilize cell machine and cell resource to produce alpha-non-natural amino acid.
Several different methods can be used for producing the novel enzymes that is used for the biosynthesis path or develops existing path.For instance, can (include but not limited to) that recurrence reorganization (recursive recombination) (can obtain) that Inc. developed is used to develop novel enzymes and path on WWW www.maxygen.com as Maxygen.For example referring to Stemmer (1994), Rapid evolution of a protein in vitro by DNA shuffling, Nature 370 (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.Similarly, the DesignPath that according to circumstances Genencor is developed TMIt is engineered that (can obtain on WWW genencor.com) is used for metabolic pathway, includes but not limited to that engineered path is to produce alpha-non-natural amino acid in cell.This technology uses being combined in of new gene (including but not limited to the gene by functioning gene group and molecular evolution and designing institute discriminating) to rebuild existing path in host's organism.Diversa company (can obtain on WWW diversa.com) also provides the technology of rapid screening-gene library and gene path (including but not limited to set up new route) to produce alpha-non-natural amino acid for biosynthesis.
Usually, the alpha-non-natural amino acid that utilizes the biosynthesis path through engineered as described herein to be produced is to produce being enough to carry out effective protein biosynthesis (including but not limited to the n cell amount) but not reaching the concentration that influences other amino acid whose concentration or exhaust the degree of cell resource.In vivo the typical concentration that produces in this way arrives about 0.05mM for about 10mM.With the plasmid transformant that comprises the gene that is used to produce the required enzyme of particular path and after producing alpha-non-natural amino acid, use according to circumstances and in vivo select to grow into the one-step optimization alpha-non-natural amino acid and produce with and cell synthetic at ribosomal protein.
I. other synthetic method
Can use the method described in the affiliated field or use the techniques described herein or its to make up to synthesize alpha-non-natural amino acid as herein described.As auxiliary, provide the various initial electrophilic reagent and the nucleopilic reagent of the required functional group of generation capable of being combined in the following table.The information that is provided plans to illustrate and unrestricted synthetic technology as herein described.
Table 1: the example of covalent bond and its precursor
The covalent bond product Electrophilic reagent Nucleopilic reagent
Carboxylic acid amides Active ester Amine/aniline
Carboxylic acid amides Acid azide Amine/aniline
Carboxylic acid amides Carboxylic acid halides Amine/aniline
Ester Carboxylic acid halides 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
Benzenethiol Aryl halide Mercaptan
Arylamine Aryl halide Amine
Thioether Acridine Mercaptan
Borate Borate Glycol
Carboxylic acid amides Carboxylic acid Amine/aniline
Ester Carboxylic acid Alcohol
Hydrazine Hydrazides Carboxylic acid
N-acylureas or acid anhydride Carbodiimides Carboxylic acid
Ester Diazonium paraffin Carboxylic acid
Thioether Epoxides Mercaptan
Thioether Haloacetamide Mercaptan
Amino triazine The halo triazine Amine/aniline
Triazinyl ether The halo triazine Alcohol/phenol
Amidine Imide ester Amine/aniline
Urea Isocyanates Amine/aniline
Carbamate Isocyanates Alcohol/phenol
Thiocarbamide Different thiocyanic ester Amine/aniline
Thioether Maleimide Mercaptan
Phosphite ester Phosphoramidate Alcohol
Silyl ether Silylation halogen Alcohol
Alkylamine Sulphonic acid ester Amine/aniline
Thioether Sulphonic acid ester Mercaptan
Ester Sulphonic acid ester Carboxylic acid
Ether Sulphonic acid ester Alcohol
Sulfonamide Sulfonic acid halide Amine/aniline
Sulphonic acid ester Sulfonic acid halide Phenol/alcohol
In general, the carbon electrophilic reagent is easy to be comprised that the complementary nucleopilic reagent of carbon nucleophile attacks, and wherein aggressive nucleopilic reagent is transported to the carbon electrophilic reagent with duplet, so that form new key between nucleopilic reagent and carbon electrophilic reagent.
The limiting examples of carbon nucleophile includes but not limited to alkyl, thiazolinyl, aryl and alkynyl Grignard reagent (Grignard); Organolithium; Organic zinc; Alkyl, thiazolinyl, aryl and alkynyl tin reagent (organic stannane); Alkyl, thiazolinyl, aryl and alkynyl borane reagent (organo-borane and organic borate), these carbon nucleophiles have at water or polar organic solvent medium power learns stable advantage.Other limiting examples of carbon nucleophile comprises phosphorus ylide (phosphorus ylid), enol and enolate reagent, and these carbon nucleophiles have the advantage that is easy to relatively by the well-known precursor generation of synthetic organic chemistry those skilled in the art.When being used in combination with the carbon electrophilic reagent, carbon nucleophile produces new carbon-carbon bond between carbon nucleophile and carbon electrophilic reagent.
Be suitable for limiting examples with the non-carbon nucleophile of carbon electrophilic reagent coupling and include but not limited to primary amine and secondary amine, mercaptan, mercaptides and thioether, alcohol, alkoxide, azide, semicarbazides etc.When being used in combination with the carbon electrophilic reagent, these non-carbon nucleophiles produce heteroatomic bond (C-X-C) usually, and wherein X is a hetero atom, includes but not limited to oxygen, sulphur or nitrogen.
VI. the polypeptide that has alpha-non-natural amino acid
For simplicity, prevailingly and/or utilize particular instance to describe the form of the compound described in this section, characteristic and further feature.Yet, basic description or particular instance that form described in this section, characteristic and further feature should not only limit in this section to be provided, but the form described in this section, characteristic and further feature are equally applicable to all compounds in formula I-LXVII scope, are included in any minor or specific compound in this specification, claims and this paper formula I-LXVII scope described in graphic.
Composition as herein described and method are incorporated in the polypeptide at least one alpha-non-natural amino acid into.Alpha-non-natural amino acid can be present in any position on the polypeptide, comprises any terminal position or any interior location of polypeptide.Alpha-non-natural amino acid preferably can not destroy the activity and/or the tertiary structure of polypeptide with respect to naturally occurring homologous amino acid polypeptide, unless the destruction of described activity and/or tertiary structure is one of purpose of alpha-non-natural amino acid being incorporated into polypeptide.In addition, not exclusively causing under the situation that activity and/or tertiary structure are destroyed, alpha-non-natural amino acid is incorporated in the polypeptide and can (for example, be controlled the therapeutic efficiency of polypeptide with respect to the activity that naturally occurring homologous amino acid polypeptide changes polypeptide to a certain extent; Improve the security features of polypeptide; Regulate pharmacokinetics, pharmacology and/or the pharmacodynamics (for example, increase water-soluble, biological usability, increase serum half-life, increase the treatment half life period, regulate immunogenicity, regulate biologically active or prolong circulation timei) of polypeptide; For polypeptide provides other functional group; Label, mark or detectable signal are incorporated in the polypeptide; The stalling characteristic of convenient polypeptide; Any combination with above-mentioned change) and/or tertiary structure.The change of described activity and/or tertiary structure is generally and realizes one of described target of incorporating into, also can have minimal effects to the activity and/or the tertiary structure of polypeptide but alpha-non-natural amino acid incorporated in the polypeptide with respect to naturally occurring homologous amino acid polypeptide.Correspondingly, think non-natural amino acid polypeptides; The composition that comprises non-natural amino acid polypeptides; The method for preparing described polypeptide and peptide composition; The method of described polypeptide of purifying, separation and sign and peptide composition; And the method for using described polypeptide and peptide composition is all in the scope of this disclosure.In addition, also non-natural amino acid polypeptides as herein described and another polypeptide (for example comprising non-natural amino acid polypeptides or naturally occurring amino acid polypeptide) can be engaged.
Non-natural amino acid polypeptides as herein described can produce by biosynthesis or abiotic synthetic method.The biological synthesis method meaning is meant any method of utilizing translation system (cell or acellular), comprises using at least a in the following assembly: polynucleotide, codon, tRNA and ribosome.The abiotic synthetic method meaning refers to not utilize any method of translation system, and the method can further be divided into the method for utilizing solid-state peptide synthetic method, solid-phase peptide synthetic method; Utilize the method for at least a enzyme; Do not utilize the method for at least a enzyme.In addition, in this divided method any can be overlapping and many methods can utilize the combination of these divided methods.
Method as herein described, composition, strategy and technology are not limited to particular type, kind or the family of polypeptide or protein.In fact, almost any polypeptide all can comprise at least one alpha-non-natural amino acid as herein described.Only for instance, polypeptide can with the therapeutic protein homology that is selected from the group that forms by required polypeptide.In relevant or other embodiment, non-natural amino acid polypeptides also can with any polypeptide member homology of somatotropin supergene family.
Can be in addition other is further modified or can use non-natural amino acid polypeptides under without the situation of further modifying non-natural amino acid polypeptides as described in local as this disclosure.Alpha-non-natural amino acid is incorporated in the polypeptide and can be carried out for multiple purpose, include but not limited to customize the change of protein structure and/or function; The accessibility of varying sized, acidity, nucleophilicity, hydrogen bond, hydrophobicity, protease target site; Targeting moiety (including but not limited to be used for the polypeptide array) etc.That the polypeptide that comprises alpha-non-natural amino acid can have an enhancing or even brand-new catalysis or biophysical properties.Only for instance, can change following characteristic in the protein by alpha-non-natural amino acid is forgiven: toxicity, bio distribution, architectural characteristic, spectral characteristic, chemistry and/or photochemical properties, catalytic capability, half life period (including but not limited to serum half-life), with the ability of other molecular reaction (including but not limited to covalently or non-covalently) etc.Composition with the polypeptide that comprises at least one alpha-non-natural amino acid can be used for (including but not limited to) novel therapeutic agents, diagnosticum, catalyzing enzyme, industrial enzyme, conjugated protein (including but not limited to antibody) and includes but not limited to protein structure and the research of functional study.For example referring to Dougherty, (2000) Unnatural Amino Acids as Probes of Protein Structure and Function, Current Opinion in Chemical Biology.4:645-652.
In addition, the side chain of alpha-non-natural amino acid component can be polypeptide multiple other functional group is provided in the polypeptide; (but not as restriction) only for instance, the side chain of alpha-non-natural amino acid part can comprise any in the following required functional group in the polypeptide.
On the one hand, composition comprises at least a polypeptide with at least one (including but not limited 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 more) alpha-non-natural amino acid.Described alpha-non-natural amino acid can be identical or different.In addition, can be in polypeptide 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20 or more different loci place comprise 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20 or more differences or identical alpha-non-natural amino acid.On the other hand, composition comprises the polypeptide that at least one that exist in the polypeptide (but being less than all) specific amino acids replaces through alpha-non-natural amino acid.For given polypeptide with an above alpha-non-natural amino acid, alpha-non-natural amino acid can be identical or different (only for instance, polypeptide can comprise the alpha-non-natural amino acid that two or more are dissimilar, perhaps can comprise two identical alpha-non-natural amino acids).For the appointment polypeptide with two above alpha-non-natural amino acids, alpha-non-natural amino acid can be identical or different, or the combination of a plurality of alpha-non-natural amino acids of identical category alpha-non-natural amino acid different with at least one.
Although can be (only for instance via the solid-phase peptide synthetic method, on hard resin), the solution phase peptide synthetic method and/or under the auxiliary situation of no enzyme through the embodiment of the synthetic non-natural amino acid polypeptides as herein described of chemical method, but other embodiment of non-natural amino acid polypeptides as herein described allows via cell membrane, cell extract or lysate system or via in vivo system's (only for instance, using protokaryon or eukaryotic cell machine) is next synthetic.In other or extra embodiment, one of key character of non-natural amino acid polypeptides as herein described is that it can utilize ribosome to be synthesized.In other or extra embodiment of non-natural amino acid polypeptides as herein described, combination that can be by including but not limited to hard resin, under the auxiliary situation of no enzyme, assist and/or make up to synthesize non-natural amino acid polypeptides via the method for system in vivo via ribosome.
Via ribosome and/or in vivo the synthetic non-natural amino acid polypeptides of system have with at hard resin or there be not the distinct advantage of non-natural amino acid polypeptides and the feature of being synthesized under the auxiliary situation of enzyme.These advantages or feature comprise different Impurity Distribution: utilize ribosomal system and/or in vivo system will have the impurity that comes from used biosystem, comprise host cell proteins matter, membrane portions and lipid; And can comprise that from the Impurity Distribution of utilizing hard resin and/or not having an auxiliary system of enzyme organic solvent, protecting group, resin material, coupling reagent and other are used for the chemicals of synthesis program.The isotopic pattern that can reflect in addition, the raw material that cell utilizes via the isotopic pattern that uses ribosome and/or the non-natural amino acid polypeptides that in vivo system synthesized; On the other hand, on hard resin and/or the isotopic pattern that does not have a non-natural amino acid polypeptides that synthesize under the auxiliary situation of enzyme can reflect the amino acid whose isotopic pattern that is utilized in synthesizing.In addition, via using ribosome and/or alpha-non-natural amino acid that in vivo system synthesized can not to contain amino acid whose D isomer in fact, and/or can easily inner cysteine amino acids be incorporated in the polypeptide structure, and/or can seldom provide internal amino acid disappearance polypeptide.On the other hand, via hard resin and/or do not use the non-natural amino acid polypeptides that is synthesized under the situation of enzyme can have higher amino acid D content of isomer and/or lower inside cysteine amino acids content and/or higher internal amino acid disappearance polypeptide percentage.In addition, one of ordinary skill in the art can distinguish and use ribosome and/or non-natural amino acid polypeptides that in vivo system synthesize and by hard resin and/or do not use the non-natural amino acid polypeptides that is synthesized under the situation of enzyme.
VII. the composition and the method that comprise nucleic acid and oligonucleotides
A. be used for general recombinant nucleic acid method herein
In numerous embodiment of methods described herein and composition, recombination method separates with using, clone and change the nucleic acid of the polypeptide (comprising for example GH polypeptide) that coding paid close attention to usually.Described embodiment is used for the process that (including but not limited to) protein expression or generation come from variant polypeptides, derivative, expression cassette or other sequence.In certain embodiments, the sequence with coded polypeptide is operably connected to allogeneic promoter.
This paper also describes the cell that can produce non-natural amino acid polypeptides, wherein at least one alpha-non-natural amino acid on the polypeptide comprise have dicarbapentaborane, diamines, heterocycle (comprising nitrogen heterocyclic ring) key or based on the side chain of the key of aldehyde alcohol.Described cell uses method as herein described or its variant to produce described non-natural amino acid polypeptides, and produces at least one alpha-non-natural amino acid with biological synthesis method.Can use the techniques described herein, method, composition and strategy or its variant to produce the cell of at least one alpha-non-natural amino acid of biosynthesis.
Can be based on the amino acid sequence of parent polypeptide, and change 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) and come composite coding to comprise the nucleotide sequence of the polypeptide of alpha-non-natural amino acid.Can pass through direct mutagenesis modified nucleotide sequence expediently according to conventional methods.Perhaps, nucleotide sequence can be prepared by chemosynthesis, and described chemosynthesis includes but not limited to by using oligonucleotide synthesizer (wherein oligonucleotides is to design according to required amino acid sequence of polypeptide) and preferably being chosen in those favourable codons in the host cell of generation recombinant polypeptide.For instance, can or engage some small oligonucleotides that the part of the required polypeptide of coding is synthesized and assembled in chain reaction by PCR, joint.For example referring to people such as Barany, Proc.Natl.Acad, Sci.88:189-193 (1991); U.S.6,521,427, it is incorporated herein with way of reference.
Alpha-non-natural amino acid method and composition as herein described utilizes genetic recombination to learn the routine techniques in field.The basic article that discloses the general using method of alpha-non-natural amino acid method and composition as herein described comprises people such as Sambrook, Molecular Cloning, A Laboratory Manual (the 3rd edition, 2001); Kriegler, Gene Transfer andExpression:A Laboratory Manual (1990); With Current Protocols in Molecular Biology (people such as Ausubel compiles 1994).
The common article of describing molecular biotechnology 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) 1-3 volume .ColdSpring Harbor Laboratory, Cold Spring Harbor, New York, people such as 1989 (" Sambrook ") and CurrentProtocols in Molecular Biology.F.M.Ausubel compile Current Protocols, Greene PublishingAssociates, Inc. and John Wiley ﹠amp; Sons, the co-partnership company of Inc., (1999 supplementary issue) (" Ausubel ").Use, promotor and many other relevant problems, the described relevant problem that mutagenesis, carrier described in these articles relate to the generation that (including but not limited to) comprises the selection codon, quadrature tRNA, quadrature synzyme and its right polynucleotide that are used to produce the protein that comprises alpha-non-natural amino acid.
The multiclass method of mutagenesis is used for alpha-non-natural amino acid method and composition as herein described to be used for various purposes, include but not limited to produce novel synzyme or tRNA, make tRNA molecule sudden change, make the coding synzyme mutant polynucleotide, produce the tRNA library, produce the synzyme library, produce select codon, insert coding the selection codon of the alpha-non-natural amino acid in the protein of paying close attention to or the polypeptide.Described method of mutagenesis includes but not limited to that direct mutagenesis, random point mutagenesis, homologous recombination, DNA reorganization or other recurrence method of mutagenesis, chimeric construct, use contain the mutagenesis of the template of uracil, oligonucleotides directed mutagenesis, the mutagenesis of thiophosphate modifying DNA, use mutagenesis etc. or its any combination of breach duplex DNA.Other proper method comprises a mispairing reparation, uses mutagenesis, restricted selection and the restricted purifying of repair-deficiency type host strain, deletion mutagenesis, synthesize mutagenesis, bifilar fracture reparation etc. by total gene.(including but not limited to) relates to chimeric mutagenesis of constructing body and is also included within the alpha-non-natural amino acid method and composition as herein described.In one embodiment, can instruct mutagenesis by the natural natural Given information (including but not limited to sequence comparison, physical characteristic, crystal structure etc.) of molecule that exists of molecule or change or sudden change that exists.
Being seen herein article and these and other relative program of case description.Following discloses case and list of references that out of Memory sees in the literary composition to be quoted: people such as Ling, Approaches to DNA mutagenesis:an overview, AnalBiochem.254 (2): 157-178 (1997); People such as Dale, Oligonucleotide-directed random mutagenesising the phosphorothioate method, Methods Mol.Biol.57:369-374 (1996); Smith, In vitromutagenesis, Ann.Rev-Genet.19:423-462 (1985); Botstein ﹠amp; 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, Nucleic Acids ﹠amp; Molecular Biology (D.M.J. compiles for Eckstein, F. and Lilley, SpringerVerlag, Berlin)) (1987); Kunkel, Rapid and efficient site-specific mutagenesis withoutphenotypic selection, Proc.Natl.Acad.Sci.USA 82:488-492 (1985); People such as Kunkel, Rapid andefficient site-specific mutagenesis without phenotypic selection, Methods in Enzymol.154,367-382 (1987); People such as Bass, Mutant Trp repressors with new DNA-binding specificities, Science 242:240-245 (1988); Methods in Enzymol.100:468-500 (1983); Methods inEnzymol.154:329-350 (1987); Zoller ﹠amp; Smith, Oligonucleotide-directed mutagenesis usingM13-derived vectors:an efficient and general procedure for the production of pointmutations in any DNA fragment, Nucleic Acids Res.10:6487-6500 (1982); Zoller ﹠amp; Smith, Oligonucleotide-directed mutagenesis of DNA fragments cloned into M13 vectors, Methodsin Enzymol.100:468-500 (1983); Zoller ﹠amp; Smith, Oligonucleotide-directed mutagenesis:asimple method using two oligonucleotide primers and a single-stranded DNA template, Methods in Enzymol.154:329-350 (1987); People such as Taylor, The use ofphosphorothioate-modified DNA in restriction enzyme reactions to prepare nicked 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 ﹠amp; 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 presenceof ethidium 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 ﹠amp; 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 M13 vectors, Nucl.Acids Res.13:4431-4443 (1985); Carter, Improved oligonucleotide-directed mutagenesis using M13 vectors, Methods in Enzymol.154:382-403 (1987); Eghtedarzadeh ﹠amp; Henlkoff, 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.Loud.A 317:415-423 (1986); People such as Nambiar, Total synthesis and cloning of a gene coding for theribonuclease S protein, Science 223:.1299-1301 (1984); Sakmar and Khorana, Total synthesisand expression of a gene for the alpha-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 mutagenesis bymicroscale ' 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); Sieber waits the people, Nature Biotechnology, and 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 numerous described methods is found in Methods in Enzymology the 154th volume, and it also describes the useful control of the trouble shooter problem that causes for various method of mutagenesis.
Method and composition as herein described also comprise use via quadrature tRNA/RS to the eukaryotic host cell of incorporating alpha-non-natural amino acid in vivo into, non-eukaryotic host cell and organism.Host cell is through the polynucleotide corresponding with polypeptide as herein described or comprise that the body (including but not limited to and polypeptide corresponding vectors as herein described that it can be for example cloning vector or expression vector) of constructing with the corresponding polynucleotide of polypeptide as herein described carries out genetic engineering modified (including but not limited to conversion, transduction or transfection).For instance, quadrature tRNA, quadrature tRNA synzyme and the coding region of desiring the protein of derivatization are operably connected with the gene expression control element that works in required host cell.Carrier can be for example form of plasmid, Coase plasmid (cosmid), phage, bacterium, virus, naked polynucleotide or binding polynucleotide.Can carrier be introduced in cell and/or the microorganism by standard method; described method comprises electroporation (people such as Fromm; Proc.Natl.Acad.Sci.USA 82; 5824 (1985)), by viral vector infection, in beads or particle matrix or from the teeth outwards with the (people such as Klein of the small-particle high speed ballistic penetration with nucleic acid; Nature 327.70,70-73 (1987)) etc.
Can in the conventional nutrient medium that is applicable to through adjusting such as the activity of screening step, activation promotor or selection transformant, cultivate through engineered host cell.These cells can be cultivated in the transgenosis organism according to circumstances.Other the useful list of references that is used for (including but not limited to) cell separation and cultivation (for example being used for separate nucleic acid subsequently) comprises Freshney (1994) Culture of Animal Cells, a Manual of Basic Technique, the 3rd edition, Wiley-Liss, New York and the list of references of wherein being quoted; People such as Payne (1992) Plant Cell and Tissue Culture inLiquid Systems John Wiley ﹠amp; 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 use target nucleic acid is introduced some kinds of well-known methods in the cell, wherein any all can be used in the method and composition as herein described.These methods comprise: recipient cell infects with the fusion of bacterium protoplast, electroporation, particle bombardment that contain DNA with through viral vectors (further discussing in this article) etc.Bacterial cell can be used for increasing and contains the number that the DNA corresponding with polypeptide as herein described constructs the plasmid of body.Make bacterial growth to exponential phase and can be by the plasmid in the several different methods separation of bacterial known in the affiliated field (for example referring to Sambrook).In addition, can buy a plurality of kits with plasmid purification from bacterium (for example referring to all from the EasyPrep of Pharmacia Biotech TM, FlexiPrep TMStrataClean from Stratagene TMWith QIAprep from Qiagen TM).Subsequently further operation through separate and the plasmid of purifying to produce other plasmid, it is used for transfectional cell or incorporates relevant carriers into the infection organism.Typical carriers contains transcribes with translation termination, transcribes and translation initiation sequence and the promotor that can be used for regulating and control the particular target expression of nucleic acids.Carrier comprises the universal expression box according to circumstances, and it contains at least one independent terminator sequence, the permission expression cassette is in eukaryotic or prokaryotic or the sequence (including but not limited to shuttle vector) of duplicating among both and be used for prokaryotic system and the selected marker of eukaryotic system.Carrier is applicable at prokaryotic, eukaryotic or preferably duplicates and integrate among both.Referring to Gillam ﹠amp; 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 together above).For example, ATCC (for example people's (volume) such as The ATCC Catalogue of bacteria andbacteriophage (1992) Gherna that published by ATCC) provides the bacterium that can be used for cloning and the catalogue of phage.Be used to check order, other base program and the basic theory of clone and molecular biological others consider also to see the 2nd edition Scientific American Books of people (1992) Recombinant DNA such as Watson, NY.In addition, basically any nucleic acid is (with any labeling nucleic acid almost, standard or non-standard) all can any customization or standard from multiple commercial source order, these commercial source are such as Midland Certified Reagent Company (Midland, TX, mcrc.com), The Great American Gene Company (Ramona, CA, can on genco.com, obtain by the WWW), ExpressGen Inc. (Chicago, IL, can on expressgen.com, obtain by the WWW), (Alameda is CA) with many other sources for OperonTechnologies Inc..
B. select codon
The genetic code subframe of the selection codon expansion protein biosynthesis machine of being contained in the method and composition as herein described.For instance, select codon to include but not limited to the codon, rare codon etc. of three unique base codons, nonsense codon (such as terminator, it includes but not limited to amber codon (UAG) or opal codon (UGA)), non-natural codon, four or more base.The number range that can introduce the selection codon in required gene or the polynucleotide is very wide, its include but not limited to the coding polypeptide that at least a portion is paid close attention to single polynucleotide in exist one or more, two or more, more than three or three, 4,5,6,7,8,9, more than 10 or 10.
In one embodiment, described method relates to the selection codon that uses as the terminator that is used for incorporating into one or more alpha-non-natural amino acids in vivo.For instance, produce the O-tRNA of identification terminator (including but not limited to UAG), and make its aminoacylization by O-RS with required alpha-non-natural amino acid.This O-tRNA also can't help naturally occurring host's aminoacyl tRNA synthetase and discerns.Conventional direct mutagenesis be used in institute pay close attention in the polypeptide the site of paying close attention to introducing terminator (including but not limited to UAG).For example referring to Sayers, people such as J.R., (1988), 5 ', 3 ' Exonuclease in phosphorothioate-based oligonucleotide-directed mutagenesis.Nucleic Acids Res, 16 (3): 791-802.When the nucleic acid of O-RS, O-tRNA and polypeptide that coding is paid close attention in vivo makes up, respond the UAG codon and incorporate alpha-non-natural amino acid into to obtain containing the polypeptide of alpha-non-natural amino acid in specified location.
Also available rare codon coding alpha-non-natural amino acid.For instance, during arginine concentration in reducing the reaction of protein synthesis in vitro, to insert Ala through the synthetic tRNA of alanine acyl groupization effective for using to have confirmed rare arginine codon AGG.For example referring to people such as Ma, Biochemistry.32:7939 (1993).In the case, synthesizing tRNA competes with the natural tRNAArg that exists that exists as the minor materials in the Escherichia coli.Some organisms do not use all triplet codons.Utilize the sub-AGA of not designated pin in the micrococcus luteus (Micrococcus luteus) to transcribe/translate in vitro and inserted amino acid in the extract.For example referring to Kowal and Oliver, Nucl.Acid.Res., 25:4685 (1997).Can produce component of the present invention to use these rare codons in vivo.
Can under the situation of significantly not disturbing eukaryotic host cell, in vivo carry out incorporating into of alpha-non-natural amino acid.For instance, owing to depend on competition between O-tRNA (including but not limited to that amber suppresses tRNA) and the eucaryon releasing factor (including but not limited to eRF) (its combine and starts the ribosome release peptide of growing) for the inhibition efficient of UAG codon, so can regulate inhibition efficient by the expression of (including but not limited to) increase O-tRNA and/or inhibition tRNA with terminator.
Select codon also to comprise the prolongation codon, it includes but not limited to the codon of four or more base, such as, the codon of four, five, six or six above bases.The example of four base codons includes but not limited to AGGA, CUAG, UAGA, CCCU etc.The example of five base codons includes but not limited to AGGAC, CCCCU, CCCUC, CUAGA, CUACU, UAGGC etc.The feature of method and composition as herein described comprises the prolongation codon that use suppresses based on frameshit.The codon of four or more base can (include but not limited to) that one or more alpha-non-natural amino acids insert in the same protein.For instance, exist sudden change O-tRNA (including but not limited to) (for example to have anticodon loop, have the anticodon loop of 8-10 nucleotide at least) specific frameshift suppressor tRNA) situation under, four or more base codon is read as single amino acid.In other embodiments, at least one four base codon of anticodon loop decodable code (including but not limited to), at least one five base codon or at least one hexabasic basic codon or more polybase base codon.Owing to there are 256 kinds of four possible base codons, so can use the codon of four or more base multiple alpha-non-natural amino acid of in same cell, encoding.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:Selection of Efficient Suppressorsof Four-base Codons and Identification of " Shifty " Four-base Codons with a LibraryApproach in Escherichia coli.J.Mol.Biol.307:755-769.
For instance, used in vitro biological synthesis method alpha-non-natural amino acid to be incorporated in the protein with four base codons.For example referring to people such as Ma, (1993) Biochemistry, 32:7939-7945; With people such as Hohsaka, (1999) J.Am.Chem.Soc, 121:34-40.CGGG and AGGU are used for incorporating the NBD derivative of 2-naphthyl alanine and lysine into streptavidin simultaneously with the frameshift suppressor tRNA of two kinds of chemical acylation in vitro.For example referring to people such as Hohsaka, (1999) J.Am.Chem.Soc, 121:12194-12195.In vivo in the research, people such as Moore check that the tRNALeu derivative with NCUA anticodon suppresses the ability of UAGN codon (N can be U, A, G or C), and find that tetrad UAGA can be decoded with 13% to 26% efficient by the tRNALeu with UCUA anticodon, does not wherein almost have decoding in 0 or-1 framework.Referring to people such as Moore, (2000) J.Mol.Biol., 298:195-205.In one embodiment, the prolongation codon based on rare codon or nonsense codon can be used for method and composition as herein described, it can be reduced in other missense that does not need site and read over frameshit and suppress.
For giving fixed system, select codon also can comprise a kind of in the natural three base codons, wherein in origin system do not use (or seldom using) natural base codon.For instance, these system and/or three base codons that comprise the tRNA that lacks the natural three base codons of identification are the system of rare codon.
Select codon to comprise the non-natural base-pair according to circumstances.These non-natural base-pairs further expand existing hereditary code.Extra base-pair is increased to 125 with the number of triplet codon from 64.The characteristic of the 3rd base-pair comprise stable and optionally base pairing, by polymerase with high fidelity effectively enzymatic incorporate among the DNA and effectively lasting primer extension after synthetic newborn non-natural base-pair.The description that can be used for the non-natural base-pair of method and composition comprises for example people such as Hirao, (2002) An unnatural base pair for incorporating amino acid analoguesinto 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 case is listed in hereinafter.
For in vivo using, non-natural nucleoside can see through film and through phosphorylation to form corresponding triphosphate.In addition, the hereditary information of increase is stable and not destroyed by cellular enzymes.People's such as Benner previous effort utilization is different from the hydrogen bond pattern of standard Watson-Crick centering, and its most noticeable example is that iso-C:iso-G is right.For example referring to people such as Switzer, (1989) J.Am.Chem.Soc, 111:8322-8322; With 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 colleague confirm that the alternative hydrogen bond that interacts of the hydrophobic filling between the base drives the formation of base-pair.Referring to Kool, (2000) Curr.Opin.Chem.Biol, 4:602-608; With Guckian and Kool, (1998) Angew.Chem.Int.Ed.Engl., 36 (24): 2825-2828.In the process of being devoted to develop the non-natural base-pair that satisfies all above-mentioned requirements, Schultz, Romesberg and colleague are systematically synthetic and studied a series of non-natural hydrophobicity bases.It is stable to find that PICS:PICS self contrasts natural base-pair, and can incorporate among the DNA effectively by the Klenow fragment (KF) of e. coli dna polymerase I.For example referring to people such as McMinn, (1999) J.Am.Chem.Soc, 121:11585-11586; And people such as Ogawa, (2000) J.Am.Chem.Soc, 122:3274-3278.Can be by KF with right for biological function enough efficient and the synthetic 3MN:3MN self of selectivity.For example referring to people such as Ogawa, (2000) J.Am.Chem.Soc, 122:8803-8804.Yet two kinds of bases are all served as the chain terminator that further duplicates.Mutation DNA polymerase is developed recently, and it is right that it can be used for duplicating PICS self.In addition, reproducible 7AI self is right.For example referring to people such as Tae, (2001) J.Am.Chem.Soc, 123:7439-7440.Also researched and developed novel metal base-pair Dipic:Py, its form in conjunction with Cu (II) back stablize right.Referring to people such as Meggers, (2000) J.Am.Chem.Soc, 122:10714-10715.Because prolong codon and non-natural codon inherently with natural codon quadrature, so alpha-non-natural amino acid method as herein described can utilize this characteristic to produce quadrature tRNA from it.
Translation bypath system (translational bypassing 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 do not translated into protein.Described sequence contain serve as induce ribosome jump over sequence clue and continue to insert the structure of the translation in downstream.
In certain embodiments, in method as herein described and/or composition the protein of paying close attention to or polypeptide (or its part) be by nucleic acid coding.Usually, nucleic acid comprises at least one and 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, selects codon more than ten or ten.
Can use method mutagenesis coding that one of ordinary skill in the art describe down at " mutagenesis and other Protocols in Molecular Biology " as everyone knows and in this article the gene of the protein of paying close attention to or polypeptide to comprise that for example one or more are used to incorporate into the selection codon of alpha-non-natural amino acid.For instance, make the nucleic acid mutagenesis of the protein of paying close attention to select codon, thereby incorporating into of one or more alpha-non-natural amino acids is provided to comprise one or more.Method and composition as herein described comprises any this kind variant (including but not limited to mutant) form of any protein that for example comprises at least one alpha-non-natural amino acid.Similarly, method and composition as herein described also comprises corresponding nucleic, that is, have one or more codings or allow in vivo to incorporate into any nucleic acid of the selection codon of one or more alpha-non-natural amino acids.
Can easily make the nucleic acid molecules sudden change of the polypeptide (only for instance, comprising the GH polypeptide) that coding pays close attention to introduce cysteine with any desired location place at polypeptide.Cysteine is widely used in to be paid close attention to reactive molecule, water-soluble polymer, protein or multiple other molecule introducing on the protein.Under be suitable for as everyone knows in the field incorporating cysteine in the desired location of polypeptide method, such as at United States Patent (USP) the 6th, 608, those methods and the standard induced-mutation technique described in No. 183 (mode of quoting in full is incorporated herein).Can will use described cysteine to introduce and utilize technology and alpha-non-natural amino acid as herein described to introduce and utilize technical combinations to use.
VIII. in vivo produce the polypeptide that comprises alpha-non-natural amino acid
For simplicity, prevailingly and/or utilize particular instance to describe the in vivo generation of the polypeptide that comprises alpha-non-natural amino acid described in this section.Yet, basic description or particular instance that in vivo the producing of the polypeptide that comprises alpha-non-natural amino acid described in this section should not only limit in this section to be provided, but the polypeptide that comprises alpha-non-natural amino acid described in this section in vivo produce all compounds be equally applicable in formula I-LXVII scope, be included in any minor or specific compound in this specification, claims and this paper formula I-LXVII scope described in graphic.
Can use modified tRNA and tRNA synzyme to produce polypeptide as herein described in vivo to add or to replace in the naturally occurring system not amino acids coding.
The method of using in the naturally occurring system amino acids coding not to produce tRNA and tRNA synzyme for example is described in the United States Patent (USP) the 7th of " In vivo incorporation of unnatural amino acids " by name, 045, the United States Patent (USP) the 7th of No. 337 and by name " Methods and compositions for the production of orthogonal tRNA-aminoacyl tRNAsynthetase pairs ", 083, in No. 970, the mode that described patent is quoted in full is incorporated herein.These methods relate to and produce that to be independent of for translation system be the work machine translator of (and therefore be sometimes referred to as " quadrature ") of endogenic synzyme and tRNA.In one embodiment, translation system comprises the polynucleotide of coded polypeptide; Polynucleotide can be the mRNA that is transcribed by corresponding DNA, and perhaps mRNA can be from rna virus vector; Polynucleotide comprises the also corresponding selection codon in angle of striking that designs in advance with alpha-non-natural amino acid in addition.Translation system comprise in addition at and also comprise the tRNA of alpha-non-natural amino acid in due course, wherein tRNA is to above-mentioned selection codon tool specificity or the above-mentioned selection codon of specific recognition; In other embodiments, alpha-non-natural amino acid is through aminoacylization.Alpha-non-natural amino acid comprises the alpha-non-natural amino acid with structure of arbitrary formula among the formula I-LXVII as herein described.In other or extra embodiment, translation system comprises the specific amino acyl synthetase of tRNA tool, and in other embodiments, translation system comprises quadrature tRNA and quadrature aminoacyl tRNA synthetase.In other or extra embodiment, translation system comprise following at least one: comprise above-mentioned polynucleotide (only for instance, dna form) plasmid, comprises above-mentioned polynucleotide (only for instance, dna form) genomic DNA or wherein be integrated with the genomic DNA of above-mentioned polynucleotide (in other embodiments, being integrated into stable integration).In other or extra embodiment of translation system, select codon to be selected from: amber codon, ochre codon, opal codon, unique codon, rare codon, non-natural codon, five base codons and four base codons by the molecular group of following password.In other or extra embodiment of translation system, tRNA is for suppressing tRNA.In other or extra embodiment, non-natural amino acid polypeptides is synthetic by ribosome.
In other or extra embodiment, translation system comprises quadrature tRNA (O-tRNA) and quadrature aminoacyl tRNA synthetase (O-RS).Usually, O-RS preferentially makes the O-tRNA aminoacylization that has at least one alpha-non-natural amino acid in the translation system, and O-tRNA discerns at least one not by the selection codon that other tRNA discerned in the described system.Therefore, translation system responds coded selection codon with in the polypeptide that is produced in the alpha-non-natural amino acid insertion system, thereby amino acid " replacement " is gone in a certain position of coded polypeptide.
Described multiple quadrature tRNA and the aminoacyl tRNA synthetase that is used for specific synthesizing amino acid is inserted polypeptide in the affiliated field, and it is applicable to usually in the method as herein described to produce non-natural amino acid polypeptides as herein described.For instance, ketone group 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 United States Patent (USP) the 7th of " In vivo incorporation ofunnatural amino acids " by name, 045, the United States Patent (USP) the 7th of No. 337 and by name " Methods and compositions forthe production of orthogonal tRNA-arninoacyl tRNA synthetase pairs ", the amino acid sequence that is disclosed in 083, No. 970 (mode of quoting is in full separately incorporated this paper into).The corresponding O-tRNA molecule that uses with O-RS also is described in the United States Patent (USP) the 7th of " In vivo incorporation of unnatural amino acids " by name, 045, the United States Patent (USP) the 7th of No. 337 and by name " Methods and compositions for the production oforthogonal tRNA-aminoacyl tRNA synthetase pairs ", 083, in No. 970, the mode that described patent is quoted is in full incorporated this paper into.In addition, people J.Am.Chem.Soc.2003 such as Mehl; People such as 125:935-939 and Santoro Nature Biotechnology in October, 2002; 20:1044-1048 (mode of quoting in full is incorporated herein) discusses screening technique and aminoacyl tRNA synthetase and the tRNA molecule that is used for p-Aminophenylalanine is incorporated into polypeptide.
Be applicable to that the exemplary O-tRNA sequence in the described method includes but not limited to as being called the United States Patent (USP) the 7th of " Methodsand compositions for the production of orthogonal tRNA-arninoacyl tRNA synthetase pairs " herein, 083, the nucleotide sequence SEQ ID NO:1-3 that is disclosed in No. 970, described patent is incorporated herein by reference.To the United States Patent (USP) 7th of right other case description of the specific O-tRNA/ aminoacyl tRNA synthetase of specific alpha-non-natural amino acid tool in " In vivo incorporation of unnatural amino acids " by name; 045; in No. 337, its mode of quoting in full is incorporated herein.Incorporate ketone group containing amino acid into and contain the amino acid whose O-RS of azido and O-tRNA is described in Chin in saccharomyces cerevisiae, people such as J.W. are among the Science 301:964-967 (2003).
The use of O-tRNA/ aminoacyl tRNA synthetase relates to specific cryptosystem of selecting the coding alpha-non-natural amino acid.Although can use any codon, generally need to select seldom or never to be used for to express the codon of the cell of O-tRNA/ aminoacyl tRNA synthetase.Only for instance, exemplary codon comprises nonsense codon, such as terminator (amber, ochre and opal); The codon of four or more base; Seldom use or without other natural three base codons that use.
Can use method of mutagenesis (including but not limited to site-specific mutagenesis, cassette mutagenesis, restricted selectivity mutagenesis etc.) known in the affiliated field that specific selectivity codon is introduced in the appropriate location of polynucleotide coding sequence.
Be used to produce the protein synthesis machine component that can be used for incorporating into alpha-non-natural amino acid (such as O-RS, O-tRNA and quadrature O-tRNA/O-RS to) method 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 that is used in vivo incorporating into alpha-non-natural amino acid is described in No. the 7th, 045,337, the United States Patent (USP) of " In vivoincorporation of unnatural amino acids " by name, and its mode of quoting in full is incorporated herein.Be used for selecting organism in vivo the right method of quadrature tRNA-tRNA synzyme used of translation system also be described in the United States Patent (USP) the 7th of " In vivo incorporation of unnatural amino acids " by name, 045, the United States Patent (USP) the 7th of No. 337 and by name " Methods and compositions for the production of orthogonaltRNA-aminoacyl tRNA synthetase pairs ", 083, in No. 970, and the mode that described patent is quoted in full is incorporated herein.In addition, the open case WO of the PCT of by name " Site Specific Incorporation of Keto Amino Acidsinto Proteins " No. 04/035743 (its mode of quoting in full is incorporated herein) describe be used to incorporate into the amino acid whose quadrature RS of ketone group and tRNA right.It is right that the open case WO of the PCT of by name " Expanding the Eukaryotic GeneticCode " No. 04/094593 (its mode of quoting in full is incorporated herein) describes the quadrature RS and the tRNA that are used for non-naturally encoded amino acid is incorporated into eukaryotic host cell.
The method that produces at least a reorganization quadrature aminoacyl tRNA synthetase (O-RS) comprises: (a) produce (sudden change according to circumstances) the RS library that derives from least a aminoacyl tRNA synthetase (RS) by first organism, described first organism includes but not limited to protokaryon organism (only for instance, Methanococcus jannaschii, thermophilic autotrophy methagen, Halophiles, Escherichia coli, super hyperthermophilic archaeon strain, strong thermophilic coccus, hole are got over the fireball bacterium, thermophilic spring is given birth to archeobacteria, extreme thermophilic bacteria etc.) or eucaryon organism; (b) in RS (RS according to circumstances suddenlys change) library, select (and/or screening) to exist under the situation of alpha-non-natural amino acid and natural amino acid and make the member of quadrature tRNA (O-tRNA) aminoacylization, thereby the set of active (sudden change according to circumstances) RS is provided; And/or (c) in described set, select (according to circumstances by negative selection) under the situation that does not have alpha-non-natural amino acid, preferentially to make the active RS (including but not limited to the RS that suddenlys change) of O-tRNA aminoacylization, thereby provide described at least a reorganization O-RS; Wherein said at least a reorganization O-RS preferentially makes the O-tRNA aminoacylization with alpha-non-natural amino acid.
In one embodiment, RS is non-activity RS.Can produce non-activity RS by making active RS sudden change.Only for instance, can by make at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6 or become different aminoacids (including but not limited to alanine) to produce non-activity RS at least about 10 or more a plurality of amino acid mutation.
The known various technology in field produce sudden change RS library under can using, and described technology includes but not limited to the appropriate design based on the three-dimensional RS structure of protein, or in the mutagenesis of RS nucleotide at random or in the appropriate design technology.Only for instance, can produce sudden change RS by locus specificity sudden change, random mutation, the multifarious recombination mutation of generation, chimeric other the known method of body, appropriate design and described herein or affiliated field of constructing.
In one embodiment, in RS (RS according to circumstances suddenlys change) library, select active member (and/or screening) (include but not limited to exist under the situation of alpha-non-natural amino acid and natural amino acid and make quadrature tRNA (O-tRNA) aminoacyl person) to include but not limited to: the positive to be selected or introduce in a plurality of cells in selection markers (including but not limited to antibiotics resistance gene etc.) and (sudden change according to circumstances) RS library wherein positive select and/or selection markers comprises at least one selection codon and (includes but not limited to amber codon, ochre codon, the opal codon, unique codon, rare codon, the non-natural codon, five base codons and four base codons); Described a plurality of cell is grown existing under the situation of selective agent; Differentiate the cell of (or showing specific reaction) of survival under the situation that has selective agent and/or selective agent by described at least one the selection codon that suppresses in positive selection or the selection markers, thereby the subclass through positive selection cell of the set that contains activity (sudden change according to circumstances) RS is provided.Can change selective agent and/or selective agent concentration according to circumstances.
On the one hand, positive selectable marker is that chloramphenicol (chloramphenicol) acetyltransferase (CAT) gene and selection codon are the amber terminator in the CAT gene.Other selected marker includes but not limited in neomycin (neomycin) resistant gene, blasticidin-S (blasticidin) resistant gene, hygromycin (hygromycin) resistant gene or the affiliated field any other available resistant gene well-known and that describe.According to circumstances, positive selectable marker is that beta-lactamase gene and selection codon are the amber terminator in the beta-lactamase gene.On the other hand, the positive-selecting mark comprises fluorescence or luminous selection markers or based on the selection markers (including but not limited to cell surface marker) of compatibility.
In one embodiment, negatively in set select or screen active RS (sudden change according to circumstances) (including but not limited under the situation that does not have alpha-non-natural amino acid, preferentially make the active RS of O-tRNA aminoacylization) to include but not limited to: with feminine gender selection or selection markers with select from the positive or the set of activity (the suddenling change according to circumstances) RS of screening is introduced in second organic a plurality of cells, wherein negative selection or selection markers comprise at least one and select codon (include but not limited to antibiotics resistance gene, it includes but not limited to chloramphenicol acetyltransferase (CAT) gene); And discriminating survival or the reaction of demonstration specificity screening in being supplemented with first medium of alpha-non-natural amino acid and selective agent or selective agent, but in second medium that does not replenish alpha-non-natural amino acid and selective agent or selective agent, can not survive or show the cell of specific reaction, thereby survivaling cell or the screening cell with described at least a reorganization O-RS is provided.Only for instance, the CAT authentication schemes serves as positive the selection and/or negative screening according to circumstances in measuring suitable O-RS recombinant.For instance, duplicating clone's set in containing on the CAT growth plate of (it comprises at least one and selects codon) under the situation that has or do not exist one or more alpha-non-natural amino acids according to circumstances.Therefore only think and to contain reorganization O-RS containing the bacterium colony of growing on the flat board of alpha-non-natural amino acid.On the one hand, change the concentration of selecting (and/or screening) agent.In certain aspects, first and second organism difference.Therefore, first and/or second organism comprises according to circumstances: prokaryotes, eucaryote, mammal, Escherichia coli, fungi, yeast, archeobacteria, eubacteria, plant, insect, protist etc.In other embodiments, selection markers comprises fluorescence or luminous selection markers or based on the selection markers of compatibility.
In another embodiment, screening or selection in set (including but not limited to negative the selection) active (sudden change according to circumstances) RS includes but not limited to: the positive certainly set of selecting step (b) isolating active sudden change RS; The set of feminine gender selection or selection markers and activity (sudden change according to circumstances) RS is introduced in second organic a plurality of cells, wherein negative selection or selection markers comprise at least one and select codon (include but not limited to comprise the toxicity marker gene that at least one selects codon, it includes but not limited to ribalgilase barnase gene); And discriminating survival or the reaction of demonstration specificity screening in first medium that does not replenish alpha-non-natural amino acid, but in being supplemented with second medium of alpha-non-natural amino acid, can not surviving or show the cell of specificity screening reaction, thereby survivaling cell or screening cell with at least a reorganization O-RS are provided, and wherein said at least a reorganization O-RS is to alpha-non-natural amino acid tool specificity.On the one hand, described at least one selection codon comprises two or more selection codons approximately.These embodiment can comprise that according to circumstances described at least one selection codon comprises two or more and selects codon, and the situation of first and second organism difference (including but not limited to that each organism is (including but not limited to) prokaryotes, eucaryote, mammal, Escherichia coli, fungi, yeast, archeobacteria, eubacteria, plant, insect, protist etc. according to circumstances).In addition, some aspects comprise that negative selection marker comprises the ribalgilase barnase gene situation of (it comprises at least one and selects codon).Others comprise that selection markers comprises fluorescence or luminous selection markers according to circumstances or based on the situation of the selection markers of compatibility.Among the embodiment in this article, screening and/or the variation of selecting to comprise screening according to circumstances and/or selecting strict degree.
In another embodiment, the method that is used to produce at least a reorganization quadrature aminoacyl tRNA synthetase (O-RS) can further comprise: (d) separate described at least a reorganization O-RS; (e) produce the second group of O-RS (sudden change according to circumstances) that derives from described at least a reorganization O-RS; And (f) repeating step (b) and (c) up to the sudden change O-RS that obtains to comprise the ability that preferentially makes the O-tRNA aminoacylization.According to circumstances, step (d)-(f) is repeated (including but not limited to) at least about twice.On the one hand, can produce the second group of sudden change O-RS that derives from least a reorganization O-RS by mutagenesis (including but not limited to random mutagenesis, site-specific mutagenesis, reorganization or its combination).
The strict degree of selection/screening step in the said method (include but not limited to positive selections/screening step (b), negative selections/screening step (c) or the positive and feminine gender selection/screening step (b) and (c)) comprises that according to circumstances change selects/screen strict degree.In another embodiment, positive selections/screening step (b), negative selections/screening step (c) or the positive and feminine gender selection/screening step (b) and (c) comprise the operation report gene, wherein reporter gene be by fluorescent activation cell sorting method (FACS) detection or wherein reporter gene be by luminous detection.According to circumstances, reporter gene be showed on the cell surface, phage display is first-class and select according to compatibility that relates to alpha-non-natural amino acid or analog or catalytic activity.In one embodiment, the sudden change synzyme be showed on the cell surface, phage display is first-class.
The method that is used for generation reorganization quadrature tRNA (O-tRNA) includes but not limited to: (a) produce the sudden change tRNA library that derives from least a tRNA (including but not limited to suppress tRNA) by first organism; (b) in described library, select (including but not limited to negative the selection) or screening under not existing from the situation of the first organic RS by (sudden change according to circumstances) tRNA from second organic aminoacyl tRNA synthetase (RS) aminoacylization, thereby tRNA is provided the set of (sudden change according to circumstances); And (c) in described tRNA (sudden change) according to circumstances set, select or screening by through introducing the member of quadrature RS (O-RS) aminoacylization, thereby at least a reorganization O-tRNA is provided; Wherein said at least a reorganization O-tRNA identification selection codon and non-origin are from the effective identification of the second organic RS and by the preferential aminoacylization of O-RS.In certain embodiments, described at least a tRNA is for inhibition tRNA and/or comprise the uniqueness three base codons with 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, ochre codon or opal terminator.In one embodiment, the improvement of reorganization O-tRNA with orthogonality.Should be appreciated that in certain embodiments O-tRNA need not to modify and introduces first organism from second organism according to circumstances.In various embodiments, first and second organism is identical or different and be selected from (including but not limited to) prokaryotes (including but not limited to Methanococcus jannaschii, thermophilic autotrophy methagen, Escherichia coli, Halophiles etc.), eucaryote, mammal, fungi, yeast, archeobacteria, eubacteria, plant, insect, protist etc. according to circumstances.In addition, reorganization tRNA is by alpha-non-natural amino acid aminoacylization according to circumstances, and wherein alpha-non-natural amino acid is that in vivo natural biological is synthetic or by the genetically manipulated biosynthesis.According to circumstances alpha-non-natural amino acid is added at least the first or second organic growth medium, wherein said alpha-non-natural amino acid can reach suitable IC to be incorporated in the non-natural amino acid polypeptides allowing.
On the one hand, in described library, select (including but not limited to negative the selection) or screening to comprise: toxicity marker gene and (sudden change according to circumstances) tRNA library are introduced from second organic a plurality of cells by (sudden change according to circumstances) tRNA (step (b)) of aminoacyl tRNA synthetase aminoacylization, its toxic marker gene comprises at least one and selects codon (or cause the gene or the essential gene of organism of toxigenicity agent or inhibitor (static agent), wherein said marker gene comprises at least one and selects codon); With the selection survivaling cell, wherein survivaling cell contains the set of (sudden change according to circumstances) tRNA that comprises at least one quadrature tRNA or non-functional tRNA.For instance, can select survivaling cell 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.In another embodiment of methods described herein, the toxicity marker gene is a ribalgilase barnase gene, and wherein ribalgilase barnase gene comprises at least one amber codon.Ribalgilase barnase gene can comprise two or more amber codons according to circumstances.
In another embodiment, selection or screening can be comprised by the member through introducing quadrature RS (O-RS) aminoacylization in the set of (sudden change according to circumstances) tRNA: in introducing the set of positive selection or selection markers gene and O-RS and (suddenling change according to circumstances) tRNA from second organic a plurality of cells, wherein positive mark's gene comprises drug resistance gene (it includes but not limited to the beta-lactamase gene, it comprises at least one and selects codon, such as at least one amber terminator) or essential gene of organism or the gene that makes the toxic agents detoxifcation; With differentiate survival or the screening cell under the situation that has selective agent or selective agent (including but not limited to antibiotic), grow; thereby the set of the cell with described at least a reorganization tRNA is provided, and wherein said at least a reorganization tRNA is by the O-RS aminoacylization and responds described at least one selection codon and with in the translation product of aminoacid insertion by positive mark's gene code.In another embodiment, change the concentration of selective agent and/or selective agent.
Be provided for producing the right method of specificity O-tRNA/O-RS.Described method includes but not limited to: the library that (a) is produced the sudden change tRNA that derives from least a tRNA by first organism; (b) in described library negative select or screening under not existing from the situation of the first organic RS by (sudden change according to circumstances) tRNA from second organic aminoacyl tRNA synthetase (RS) aminoacylization, thereby the set of (sudden change according to circumstances) tRNA is provided; (c) in the set of (sudden change) according to circumstances tRNA, select or screening by through introducing the member of quadrature RS (O-RS) aminoacylization, thereby at least a reorganization O-tRNA is provided.Described at least a reorganization O-tRNA identification selection codon and non-origin are from the effective identification of the second organic RS and by the preferential aminoacylization of O-RS.Described method comprises that also (d) produced the library of (sudden change according to circumstances) RS that derives from least a aminoacyl tRNA synthetase (RS) by the 3rd organism; (e) in sudden change RS library, select or screening exists under the situation of alpha-non-natural amino acid and natural amino acid and preferentially makes the member of described at least a reorganization O-tRNA aminoacylization, thereby the set of active (sudden change according to circumstances) RS is provided; (f) activity (sudden change according to circumstances) RS that preferentially makes described at least a reorganization O-tRNA aminoacylization under the situation that does not have alpha-non-natural amino acid is selected or screened to feminine gender in described set; thereby provide described 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 tool specific reorganization O-RS and described at least a reorganization O-tRNA to comprising.Comprise in scope as herein described and the method that the specificity O-tRNA/O-RS that is produced by method as herein described is right.For instance, specificity O-tRNA/O-RS is right to comprising (including but not limited to) mutRNATyr-mutTyrRS, such as mutRNATyr-SS12TyrRS to, mutRNALeu-mutLeuRS to, mutRNAThr-mutThrRS to, mutRNAGlu-mutGluRS equity.In addition, described method comprises the situation of first identical with the 3rd organism (it includes but not limited to Methanococcus jannaschii).
In method as herein described, also comprise the right method of quadrature tRNA-tRNA synzyme of selecting to be used for the second organic in vivo translation system.Described method includes but not limited to: marker gene, tRNA and the aminoacyl tRNA synthetase (RS) that separates from first organism or obtain are introduced from second organic first group of cell; Marker gene and tRNA are introduced from the second organic replicating cell group; Be chosen in that survivaling cell in nonviable first group in the replicating cell group or screening show the specificity screening reaction but cell that this reaction can not be provided in the replicating cell group, wherein first group is to grow under the situation that has selective agent or selective agent with the replicating cell group, and wherein to comprise the quadrature tRNA-tRNA synzyme that is used for the second organic in vivo translation system right for survival or screening cell.In one embodiment, relatively and select or screening comprises in vivo complementary calibrating.Can change the concentration of selective agent or selective agent.
Organism as herein described comprises multiple organism and multiple combination.In one embodiment, organism is the protokaryon organism according to circumstances, and it includes but not limited to that Methanococcus jannaschii, thermophilic autotrophy methagen, Halophiles, Escherichia coli, super hyperthermophilic archaeon strain, strong thermophilic coccus, hole are got over the fireball bacterium, thermophilic spring is given birth to archeobacteria, extreme Thermophilic Bacteria etc.Perhaps, organism is the eucaryon organism, it includes but not limited to plant (including but not limited to complicated plant, such as monocotyledon or dicotyledon), algae, protist, fungi (including but not limited to yeast etc.), animal (including but not limited to mammal, insect, arthropods etc.) etc.
A. the expression in non-eucaryote and the eucaryote
The technology that is disclosed in this section can be applicable to express non-natural amino acid polypeptides as herein described in non-eucaryote and eucaryote.
Be to obtain the high level expression of cloned polynucleotide, the polynucleotide subclone of the required polypeptide of will encode usually instructs the strong promoter of transcribing to containing, transcribes/expression vector of the ribosome bind site of the sub and nucleic acid of coded protein (if with regard to) the confession translation initiation of translation termination in.Suitable bacterium promotor for example is described in philtrums such as people such as Sambrook and Ausubel.
The bacterial expression system that is used for express polypeptide can obtain (people such as Palva, Gene 22:229-235 (1983) (including but not limited to) Escherichia coli, bacillus (Bacillussp.), pseudomonas fluorescens, pseudomonas aeruginosa, pseudomonas putida and salmonella (Salmonella); People such as Mosbach, Nature 302:543-545 (1983)).The kit that is used for these expression systems is on sale on the market.The eukaryotic expression system of mammalian cell, yeast and insect cell is on sale on the market.In the situation that quadrature tRNA and aminoacyl tRNA synthetase (this paper other places are described) is used for express polypeptide, the host cell that is used to express is to use the ability of quadrature component to be selected according to it.Exemplary host cell comprises Gram-positive bacteria (including but not limited to bacillus pumilus (B.brevis) or hay bacillus (B.subtilis) or streptomycete (Streptomyces)) and Gram-negative bacteria (Escherichia coli or pseudomonas fluorescens, pseudomonas aeruginosa, pseudomonas putida), and yeast and other eukaryotic.Can use as described herein and comprise the right cell of O-tRNA/O-RS.
Eukaryotic host cell as described herein or non-eukaryotic host cell provide the synthetic ability that the polypeptide that comprises alpha-non-natural amino acid of consumption is arranged more greatly.On the one hand, composition include but not limited to according to circumstances at least about 10 micrograms, at least about 50 micrograms, at least about 75 micrograms, at least about 100 micrograms, at least about 200 micrograms, at least about 250 micrograms, at least about 500 micrograms, at least about 1 milligram, at least about 10 milligrams, at least about 100 milligrams, at least about 1 gram or the polypeptide that comprises alpha-non-natural amino acid more, the amount (details about recombinant protein preparation and purifying is provided in herein) that can be realized by polypeptide preparation method in vivo maybe.On the other hand, polypeptide is according to circumstances with in (including but not limited to) cell lysates, buffer solution, every liter of (including but not limited to) in medicine buffer solution or other liquid suspension (including but not limited to that volume all is that about 1nl is to about 100L or more anywhere) is at least about 10 microgram polypeptide, every liter at least about 50 microgram polypeptide, every liter at least about 75 microgram polypeptide, every liter at least about 100 microgram polypeptide, every liter at least about 200 microgram polypeptide, every liter at least about 250 microgram polypeptide, every liter at least about 500 microgram polypeptide, every liter is present in the composition at least about 10 milligrams of polypeptide or higher concentration at least about 1 milligram of polypeptide or every liter.Producing a large amount of (include but not limited to greater than usually can obtainable amount with other method (including but not limited in vitro translate)) protein in comprising the eukaryotic of at least one alpha-non-natural amino acid is the feature of method as herein described, technology and composition.
Eukaryotic host cell as described herein or non-eukaryotic host cell provide biosynthesis that the ability of the polypeptide that comprises alpha-non-natural amino acid of consumption is arranged more greatly.For instance, can be in cell extract, cell lysates, medium, (including but not limited to) in the buffer solutions etc. is at least about 10 micrograms per litre, at least about 50 micrograms per litre, at least about 75 micrograms per litre, at least about 100 micrograms per litre, at least about 200 micrograms per litre, at least about 250 micrograms per litre or at least about 500 micrograms per litre, at least about 1 mg/litre, at least about 2 mg/litre, at least about 3 mg/litre, at least about 4 mg/litre, at least about 5 mg/litre, at least about 6 mg/litre, at least about 7 mg/litre, at least about 8 mg/litre, at least about 9 mg/litre, at least about 10 mg/litre, at least about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 200, about 300, about 400, about 500, about 600, about 700, about 800, about 900 mg/litre, about 1 grams per liter, about 5 grams per liters, about 10 grams per liters or bigger protein concentration produce the polypeptide that comprises alpha-non-natural amino acid.
1. expression system, cultivation and separate
The technology that is disclosed in this section can be applicable to expression system, the cultivation of non-natural amino acid polypeptides as herein described and separates.Non-natural amino acid polypeptides can be expressed in the suitable expression system (including but not limited to yeast, insect cell, mammalian cell and bacterium) of any number.The description of exemplary expression system is provided in herein.
Yeast as used herein, term " yeast " comprises any in each primary yeast of gene that can express the coding non-natural amino acid polypeptides.These yeast include but not limited to ascosporogenous yeast (ascosporogenous yeast) (Endomycetale (Endomycetales)), basidiospore yeast (basidiosporogenous yeast) and belong to the yeast of imperfect fungus (Fungiimperfecti) (gemma guiding principle (Blastomycetes)) class.Ascosporogenous yeast is divided into two sections: Spermophthoraceae (Spermophthoraceae) and Saccharomycetaceae (Saccharomycetaceae).The latter is made up of four subfamilies, be Schizosaccharomycoideae (for example, Schizosaccharomyces (Schizosaccharomyces)), Nadsonioideae (Nadsonioideae), Lipomycoideae and Saccharomycoideae (for example, pichia (Pichia), Kluyveromyces (Kluyveromyces) and saccharomyces (Saccharomyces)).The basidiospore yeast comprises that Leucosporidium (Leucosporidium), Rhodosporidium (Rhodosporidium), lock are thrown saccharomyces (Sporidiobolus), powder saccharomyces (Filobasidium) deceived by line and incense ashes are intended lock load Pseudomonas (Filobasidiella).The yeast that belongs to imperfect fungus (gemma guiding principle) class is divided into two sections: Sporobolomycetaceae (Sporobolomycelaceae) (for example, Sporobolomyces (Sporoholomyces) and Bullera (Bullera)) and Cryptococcaceae (Cryptococcaceae) (for example, candida (Candida)).
In certain embodiments, with pichia, Kluyveromyces, Blastocystis (Saccharomyces), Schizosaccharomyces (Schizosaccharomyces), Hansenula (Hansenula), species in Torulopsis (Torulopsis) and the candida are used for method as herein described, in technology and the composition, described species include but not limited to pichia pastoris phaff (P.pastoris), paddy Le Shi yeast (P.guillerimondii), saccharomyces cerevisiae, Ka Ersibai yeast (S.carlsbergensis), saccharomyces diastaticus (S.diastaticus), Douglas yeast (S.douglasii), kluyveromyces (S.kluyveri), this yeast of promise (S.norbensis), ellipsoideus yeast (S.oviformis), Kluyveromyces lactis (K.lactis), Kluyveromyces fragilis (K.fragilis), Candida albicans (C.albicans), maltose Candida (C.maltosa) and saccharomyces hansenii (H.polymorpha).
The suitable yeast that selection is used to express non-natural amino acid polypeptides is in one of ordinary skill in the art's technical scope.When selecting the yeast host that is used to express, suitable host can include but not limited to show the yeast host with for example good secretion capacity, the active and overall steadiness of low proteolytic.Yeast can obtain from multiple source usually, include but not limited to that University of California's biophysics and medical physics are yeast genes preservation center (California Berkeley) (Yeast GeneticStock Center, Department of Biophysics and Medical Physics, University of California (Berkeley, and U.S. typical case (the Americanpe Culture Collection (" ATCC ") (Manassas, VA)) of DSMZ (" ATCC ") (Manassas, northern Virginia) CA)).
Term " yeast host " or " yeast host cell " comprise the yeast that can be used as or be used as the acceptor of recombinant vector or other transfer DNA.Described term comprises the offspring of the original yeast host cell that receives recombinant vector or other transfer DNA.Should be appreciated that because accidental sudden change or deliberately sudden change, the offspring of single mother cell on the form or with the genomic DNA of original parent complementation or total DNA on may may not be identical.With the parent of desiring to characterize with correlation properties (such as the nucleotide sequence that has the coding non-natural amino acid polypeptides) enough the offspring of similar mother cell be included among the offspring who defines indication thus.
The expression that comprises extrachromosomal replication or integration vector and conversion carrier have been researched and developed to be used for being transformed into many yeast hosts.For instance, researched and developed and be used for following each organic expression vector: saccharomyces cerevisiae (people such as Sikorski,
Figure A200680049995D0131175057QIETU
(1998) 122:19; People such as Ito, J.
Figure A200680049995D0131175132QIETU
(1983) 153:163; People such as Hinnen,
Figure A200680049995D0131175215QIETU
Figure A200680049995D0131175231QIETU
USA (1978) 75:1929); Candida albicans (people such as Kurtz,
Figure A200680049995D0131175336QIETU
(1986) 6:142); Maltose Candida (people such as Kunze, J. (1985) 25:141); Saccharomyces hansenii (people such as Gleeson, J.
Figure A200680049995D0131175442QIETU
(1986) 132:3459; People such as Roggenkamp,
Figure A200680049995D0131175505QIETU
(1986) 202:302); Kluyveromyces fragilis (people such as Das, J.
Figure A200680049995D0131175521QIETU
(1984) 158:1165); Kluyveromyces lactis (people such as De Louvencourt, J. (1983) 154:737; People such as Van den Berg,
Figure A200680049995D0131175557QIETU
(1990) 8:135); Paddy Le Shi yeast (people such as Kunze, J.
Figure A200680049995D0131175610QIETU
(1985) 25:141); Pichia pastoris phaff (United States Patent (USP) the 5th, 324, No. 639; The 4th, 929, No. 555; With the 4th, 837, No. 148; People such as Cregg,
Figure A200680049995D0131175631QIETU
(1985) 5:3376); Schizosaccharomyces pombe (Schizosaccharomycespombe) (people such as Beach,
Figure A200680049995D0131175649QIETU
(1982) 300:706) conciliate fat Ye Shi yeast (Y.lipolytica); Aspergillus nidulans (A.nidulans) (people such as Ballance,
Figure A200680049995D0131175718QIETU
(1983) 112:284-89; People such as Tilburn,
Figure A200680049995D0131175738QIETU
(1983) 26:205-221; With people such as Yelton,
Figure A200680049995D0131175828QIETU
USA (1984) 81:1470-74); Aspergillus niger (A.niger) (Kelly and Hynes, EMBO J. (1981) 4:475-479); Trichoderma reesei (T.reesia) (EP 0 244 234); With the filamentous fungi such as neurospora (Neurospora), Penicillium notatum (Penicillium), curved neck mould (Tolypocladium) (WO 91/00357), wherein the mode quoted in full of each document is incorporated herein.
The control sequence that is used for yeast vector includes but not limited to from the promoter region such as following gene: alcohol dehydrogenase (ADH) (EP 0 284 044); Enolase; Glucokinase; The G-6-P isomerase; Glyceraldehyde-3-phosphate-dehydrase (GAP or GAPDH); Hexokinase; Phosphofructokinase; The 3-phoshoglyceric acid mutase; And pyruvate kinase (PyK) (EP 0 329 203).The yeast PHO5 gene of coding acid phosphatase also can provide usefulness promoter sequence (people such as Miyanohara,
Figure A200680049995D0131175906QIETU
USA (1983) 80:1).Other the suitable promoter sequence that is used for yeast host can comprise glycerol 3-phosphate acid kinase (people such as Hitzeman, J.
Figure A200680049995D0131175933QIETU
(1980) 255:(4): 12073-12080) and other glycolytic enzyme (such as pyruvate decarboxylase, phosphotriose isomerase and glucose phosphate isomerase (people such as Holland,
Figure A200680049995D0132155018QIETU
(1978) 17 (23): 4900-4907; People such as Hess, J.ADV.
Figure A200680049995D0132155041QIETU
(1969) promotor 7:149-167)).Have brought out Yeast promoter by other advantage of transcribing of growth conditions control and can comprise the promoter region of the enzyme of alcohol dehydrogenase 2, different cell pigment C, acid phosphatase, metallothionein, glyceraldehyde-3-phosphate dehydrogenase, the digestive enzyme relevant and responsible maltose and galactose utilization with nitrogen metabolism.The suitable carrier and the promotor that are used for Yeast expression are further described among the EP 0,073 657.
The yeast enhancer 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 district of another Yeast promoter, produces synthetic hybrid promoter.The example of described hybrid promoter comprises the ADH regulating and controlling sequence that is connected with GAP transcription activating district.Referring to United States Patent (USP) the 4th, 880, No. 734 and the 4th, 876, No. 197, its mode of quoting in full is incorporated herein.Other example of hybrid promoter comprises the promotor that the transcription activating district by the regulating and controlling sequence of ADH2, GAL4, GAL10 or PHO5 gene and glycolytic enzyme gene (such as GAP or PyK) forms.Referring to EP 0 164 556.In addition, Yeast promoter can comprise having and combines with the yeast rna polymerase and the naturally occurring promotor in the non-yeast source of initial ability of transcribing.
Other control element that can constitute the part of Yeast expression carrier for example comprises terminator (people such as Holland, the J. from GAPDH or enolase gene
Figure A200680049995D0132155110QIETU
(1981) 256:1385).In addition, the origin of replication from 2 μ plasmids source is applicable to yeast.The suitable selection gene that is used for yeast is an existing trpl gene in the yeast plasmid.Referring to people such as Tschumper,
Figure A200680049995D0132155159QIETU
(1980) 10:157; People such as Kingsman,
Figure A200680049995D0132155159QIETU
(1979) 7:141.The trpl gene pairs lacks the yeast of the ability of growing in tryptophan mutant strain provides selected marker.Similarly, Leu2 defective yeast bacterial strain (ATCC 20,622 or 38,626) is to be replenished by the known plasmid that has the Leu2 gene.
The method that foreign DNA is introduced in the yeast host includes but not limited to transform spheroplast or transforms the complete yeast host cell of handling through alkaline kation.For instance, can be according to people such as Hsiao,
Figure A200680049995D0132155223QIETU
People such as USA (1979) 76:3829 and Van Solingen, J.
Figure A200680049995D0132155243QIETU
(1977) method described in the 130:946 is carried out the conversion of yeast.Yet, also can as
Figure A200680049995D0132155252QIETU
Deng the people,
Figure A200680049995D0132155320QIETU
: A
Figure A200680049995D0132155335QIETU
(2001) common described use is introduced other method in the cell with DNA in, such as merging by nuclear injection, electroporation or protoplast.Can use the known standard technique of those skilled in the art to come the culture yeasts host cell subsequently.
Other method of expressing heterologous albumen is described in the U.S. Patent Publication case No. 20020055169 in yeast host cell; United States Patent (USP) the 6th, 361, No. 969; 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. reexamination 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 0 480 480; WO90/10277; EP 0 460 071; EP 0 340 986; EP 0 329 203; EP 0 324 274; In EP 0 164 556.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, Methods in Enzymology (1990) 185:3-7, the mode of quoting in full separately is incorporated herein.
During the amplification stage of using standard feed batch fermentation method, the yeast host bacterial strain can be grown in fermentation tank.Fermentation process can be used for explaining that the carbon of specific yeast host utilizes the path or expresses the difference of control model.Only for instance, the fermentation of saccharomycete yeast host (for example may need single glucose charging, compound nitrogen source, caseic hydrolysate) and multivitamin replenish, and the methylotrophic yeast pichia pastoris phaff may need glycerine, methyl alcohol and the charging of trace mineral, but only needs simple ammonium (nitrogen) salt to carry out optimum growh and expression.For example referring to United States Patent (USP) the 5th, 324, No. 639; People such as Elliott, J.Protein Chem. (1990) 9:95; With people such as Fieschko, Biotech.Bioeng. (1987) 29:1113, the mode of quoting in full separately is incorporated herein.
Yet these fermentation process can have and some irrelevant common feature of used yeast host strain.For instance, during the amplification stage, the nutrient (being generally carbon) of limiting growth can be added in the fermentation tank to allow maximum growth.In addition, fermentation process uses usually through being designed to contain the fermentation medium of capacity carbon, nitrogen, basic salt, phosphorus and other micro-nutrient (vitamin, trace mineral and salt etc.).The case description of fermentation medium that is applicable to Pichia pastoris is in United States Patent (USP) the 5th, 324, and No. 639 and the 5th, 231, in No. 178, the mode of quoting in full separately is incorporated herein.
The insect cell term " insect host " or " insect host cell " that infect baculoviral are meant the insect that can be used as or be used as the acceptor of recombinant vector or other transfer DNA.Described term comprises the offspring of the protentomon host cell of transfection.Should be appreciated that because accidental sudden change or deliberately sudden change, the offspring of single mother cell on the form or with the genomic DNA of original parent complementation or total DNA on may may not be identical.With the parent of desiring to characterize with correlation properties (such as the nucleotide sequence that has the coding non-natural amino acid polypeptides) enough the offspring of similar mother cell be included among the offspring who defines indication thus.
One of ordinary skill in the art are used to express the selection of the suitable insect cell of required polypeptide as everyone knows.Some kinds of insect species are fully described in affiliated field and on sale on the market, it includes but not limited to Aedes aegypti (Aedesaegypti), silkworm (Bombyx mori), Drosophila melanogaster (Drosophila melanogaster), the greedy noctuid (Spodoptera frugiperda) in meadow and cabbage looper (Trichoplusia ni).When the insect host of selecting to be used to express, suitable host can include but not limited to show especially have good secretion capacity, those hosts of the active and overall steadiness of low proteolytic.Insect can obtain from multiple source usually, include but not limited to that University of California's biophysics and medical physics are insect genes preservation center (California Berkeley) (Insect Genetic Stock Center, Department ofBiophysics and Medical Physics, University of California (Berkeley, CA)); With U.S. typical case (the American Type Culture Collection (" ATCC ") (Manassas, VA)) of DSMZ (" ATCC ") (Manassas, northern Virginia).
Usually, the component that infects the insect expression system of baculoviral comprises: transfer vector (being generally bacterial plasmid), and it contains the genomic fragment of baculoviral and is used to insert the convenient restriction site of the heterologous gene of desire expression; Wild-type baculovirus, its have with transfer vector in the sequence (this allow heterologous gene homologous recombination in the baculoviral genome) of baculoviral specific fragment homology; And suitable insect host cell and growth medium.Under become known for carrier construction, transfectional cell in the field, select patch, make material, method and technology that cell grows etc. and the handbook that can use these technology of description in culture.
After inserting heterologous gene in the transfer vector, with carrier and the transfection of wild-type virus genome in insect host cell, wherein carrier and viral genome reorganization.Expression is through packing recombinant virus and discriminating and purification of Recombinant body patch.Be used for the material of baculoviral/insect cell expression system and method can kit form available from for example Invitrogen Corp. (Carlsbad, CA).Illustrative technique is described in
Figure A200680049995D0134155610QIETU
Figure A200680049995D0134155639QIETU
In the 1555th phase (1987), it is incorporated herein by reference.Also referring to
Figure A200680049995D0134155741QIETU
Figure A200680049995D0134155828QIETU
(1995);
Figure A200680049995D0134155842QIETU
Deng the people, 16.9-16.11 (1994);
Figure A200680049995D0134155919QIETU
With (1992); With
Figure A200680049995D0134155951QIETU
Deng the people,
Figure A200680049995D0134160004QIETU
(1992).
Use baculoviral/insect cell expression system to prepare various heterologous proteins and be described in, and described technology can be through revising to prepare non-natural amino acid polypeptides as herein described below with reference in the document.For example referring to, United States Patent (USP) the 6th, 368, No. 825; The 6th, 342, No. 216; The 6th, 338, No. 846; The 6th, 261, No. 805; The 6th, 245, No. 528; The 6th, 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; WO02/06305; WO 01/90390; 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 92/02628; 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 the mode of quoting in full separately is incorporated herein.
The carrier that can be used in baculoviral/insect cell expression system includes but not limited to express and transfer vector from the insect that baculoviral autographa california (Autographa californica) nuclear polyhedrosis virus (AcNPV) obtains, and it is the virus expression carrier that does not rely on auxiliary cell.From then on the virus expression carrier that obtains of system uses the strong virus polyhedrin gene promoter to drive expression of heterologous genes usually.Usually referring to people such as Reilly,
Figure A200680049995D0135160120QIETU
Figure A200680049995D0135160134QIETU
(1992).
Before alien gene being inserted in the shaft-like viral genome, the said components that will comprise promotor, targeting sequencing (in case of necessity), the coded sequence of being paid close attention to and transcription terminator usually is assembled into middle dislocation and constructs in the body (intermediatetransplacement construct) (transfer vector).Middle dislocation is constructed body and is remained on usually and can stablize in the replicon (such as extra-chromosomal element (for example, plasmid)) that remains among the host (such as bacterium).Replicon will have dubbing system, therefore allow it to remain in the suitable host that supplies clone and amplification usefulness.More particularly, plasmid can contain the polyhedrin polyadenylation signal (people such as Miller,
Figure A200680049995D0135160154QIETU
42:177) and be used for selecting and the anti-ampicillin of protokaryon (ampicillin) of breeding (amp) gene and origin of replication (1988) Escherichia coli.
A kind of transfer vector commonly used that alien gene is introduced among the AcNPV is pAc373.Also known many other carriers of one of ordinary skill in the art are designed, it comprises for example pVL985, and it becomes ATT with the polyhedrin initiation codon by ATG, and it introduces 32 base-pair places, ATT downstream with the BamHI cloning site.Referring to Luckow and Summers, Virology 170:31 (1989).Other commercially available carrier for example comprise PBlueBac4.5/V5-His, pBlueBacHis2, pMelBac, pBlueBac4.5 (Invitrogen Corp., Carlsbad, CA).
After inserting heterologous gene, with transfer vector and wild-type baculovirus genome cotransfection in the insect cell host.The exemplary methods of allogeneic dna sequence DNA being introduced in the required site of baculoviral is described in
Figure A200680049995D0135160227QIETU
With
Figure A200680049995D0135160234QIETU
The 1555th phase (1987); People such as Smith,
Figure A200680049995D0135160327QIETU
Figure A200680049995D0135160340QIETU
(1983) 3:2156; Luckow and Summers,
Figure A200680049995D0135160353QIETU
(1989) among the 170:31-39.For instance, can be by in the gene of homology dual crossing reorganization insertion such as polyhedron gene; Also can insert in the required baculoviral gene in engineered restriction enzyme sites.Referring to people such as Miller,
Figure A200680049995D0135160408QIETU
(1989) 11 (4): 91.
Can use
Figure A200680049995D0135160431QIETU
With
Figure A200680049995D0135160450QIETU
(1995); Mann and King, J.
Figure A200680049995D0135160521QIETU
(1989) method described in the 70:3501 realizes transfection by electroporation.Perhaps, liposome can be used for recombinant expression carrier and baculoviral transfection insect cell.For example referring to, people such as Liebman,
Figure A200680049995D0135160541QIETU
(1999) 26 (1): 36; People such as Graves,
Figure A200680049995D0135160558QIETU
(1998) 37:6050; People such as Nomura, J.
Figure A200680049995D0135160621QIETU
(1998) 273 (22): 13570; People such as Schmidt,
Figure A200680049995D0135160635QIETU
(1998) 12:323; People such as Siffert,
Figure A200680049995D0135160647QIETU
(1998) 18:45; People such as TILKINS,
Figure A200680049995D0135160717QIETU
Figure A200680049995D0135160729QIETU
145-154 (1998); People such as Cai,
Figure A200680049995D0135160747QIETU
(1997) 10:263; People such as Dolphin, (1997) 17:491; People such as Kost,
Figure A200680049995D0135160822QIETU
(1997) 190:139; People such as Jakobsson, J. (1996) 271:22203; People such as Rowles, J.
Figure A200680049995D0135160846QIETU
(1996) 271 (37): 22376; People such as Reverey, J.
Figure A200680049995D0136160902QIETU
(1996) 271 (39): 23607-10; People such as Stanley, J.
Figure A200680049995D0136160948QIETU
(1995) 270:4121; People such as Sisk, J.
Figure A200680049995D0136161000QIETU
(1994) 68 (2): 766; With people such as Peng,
Figure A200680049995D0136161015QIETU
(1993) 14 (2): 274.Commercially available liposome for example comprises
Figure A200680049995D01361
With
Figure A200680049995D01362
(Invitrogen, Corp., Carlsbad, CA).In addition, also can use calcium phosphate transfection.Referring to
Figure A200680049995D0136161041QIETU
39
Figure A200680049995D0136161100QIETU
(1995); Kitts, NAR (1990) 18 (19): 5667; And Mann and King, J.
Figure A200680049995D0136161115QIETU
(1989) 70:3501.
Rhabdovirus expression vector contains bacilliform virus promoter usually.Bacilliform virus promoter is can combine with the baculoviral RNA polymerase and start code sequence (for example, structural gene) downstream (3 ') is transcribed into any dna sequence dna of mRNA.Promotor will have usually and 5 of coded sequence ' the hold transcription initiation region of approaching placement.This transcription initiation region generally includes RNA polymerase binding site and transcription initiation site.Bacilliform virus promoter also can have the second area that is called enhancer, and when existing, it is usually at the tip of structural gene.In addition, expression can or be a composition through regulation and control.
The structural gene of transcribing in a large number latter stage in infectious cycle provides useful especially promoter sequence.Example comprise be derived from the coding viral polyhedron albumen gene (
Figure A200680049995D0136161156QIETU
Deng the people, The Regulation of Baculovirus Gene Expression,
Figure A200680049995D0136161219QIETU
(1986); EP 0 127 839 and 0 155 476) and the gene of coding p10 albumen (people such as Vlak, J.
Figure A200680049995D0136161242QIETU
(1988) sequence 69:765).
Be packaged into the rhabdovirus expression vector that forms recently in the infectious recombinant baculovirus and subsequently can be by such as people such as Miller, (1989) 4:91;
Figure A200680049995D0136161322QIETU
Figure A200680049995D0136161335QIETU
The patch that the technology of technology comes purifying to grow described in the 1555th phase (1987).
Developed the recombination rhabdovirus expression vector that is used for infecting some kinds of insect cells.For instance, developed the recombinant baculovirus that is particularly useful for Aedes aegypti (ATCC CCL-125 number), silkworm (ATCC CRL-8910 number), Drosophila melanogaster (No. the 1963rd, ATCC), the greedy noctuid in meadow and cabbage looper.Referring to WO 89/046,699; Wright,
Figure A200680049995D0136161349QIETU
(1986) 321:718; People such as Carbonell, J.
Figure A200680049995D0136161359QIETU
(1985) 56:153; People such as Smith, MOL.CELL.BIOL. (1983) 3:2156.Usually referring to people such as Fraser,
Figure A200680049995D0136161421QIETU
BIOL (1989) 25:225.More particularly, the cell-line that is used for the rhabdovirus expression vector system generally includes but is not limited to Sf9 (noctuid is coveted on the meadow) (ATCC CRL-1711 number), Sf21 (noctuid is coveted on the meadow) (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 at the cell of the direct expression of rhabdovirus expression vector heterologous polypeptide and amalgamation and expression and medium on sale on the market.
Well-known bacterial expression technology in the field under Escherichia coli, pseudomonad and other prokaryotes.Variety carrier can be used in the bacterial host.Carrier can be single copy or low or high multi-copy vector.Carrier can be used for the clone and/or expresses.In view of exist about the commercial applicability of enriching document, many carriers of carrier and even the handbook of carrier and its restriction endonuclease map and feature is described, need not extensive discussions in this article.As everyone knows, carrier is usually directed to allow the mark selected, and these marks can provide cytotoxic agent resistance, former nutrition or immunity.Usually have a plurality of marks, it provides different characteristic.
The bacterium promotor is for being transcribed into any dna sequence dna of mRNA in conjunction with bacteria RNA polymerase and start code sequence (for example structural gene) downstream (3 ').Promotor will have usually and 5 of coded sequence ' the hold transcription initiation region of approaching placement.This transcription initiation region generally includes RNA polymerase binding site and transcription initiation site.The bacterium promotor also can have the second area that is called operon, and it can be overlapping with the adjacent R NA polymerase binding site point that starting rna synthesizes.Operon allows negative regulation (can induce) to transcribe, and this is because gene inhibition albumen can be in conjunction with operon and suppressed transcribing of specific gene thus.The constructive expression can be taken place under the situation that does not have negative regulatory element (such as operon).In addition, can realize just regulating and control by the gene activation protein binding sequence, when existing, described sequence is usually near (5 ') of RNA polymerase binding sequence.The example of gene activation albumen is metabolite activated protein (CAP), its help the transcribing of lac operon in the initial Escherichia coli (E.coli) [people such as Raibaud, (1984) 18:173].Therefore, regulating and expressing can be forward or negative sense, strengthens thus or weakens and transcribe.
The sequence of coding metabolic pathway enzyme provides useful especially promoter sequence.Example comprise derive from the glycometabolism enzyme (such as galactose, lactose (lac) [people such as Chang,
Figure A200680049995D0137161513QIETU
(1977) 198:1056] and maltose) promoter sequence.Other example comprises promoter sequence [people such as Goeddel, the Nuc. that derives from biosynthetic enzyme (such as tryptophan (trp))
Figure A200680049995D0137161529QIETU
(1980) 8:4057; People such as Yelverton,
Figure A200680049995D0137161541QIETU
(1981) 9:731; United States Patent (USP) the 4th, 738, No. 921; IFNPub the 036 No. 776 and the 121 No. 775, the mode of quoting in full separately is incorporated herein].People such as beta galactosidase (bla) promoter systems [Weissmann (1981) " The cloning of interferon and othermistakes. " Interferon 3 (I.Gresser volume)], phage PL[Shimatake, NATURE (1981) 292:128] and T5[United States Patent (USP) the 4th, 689, No. 406] (described document quote in full separately mode be incorporated herein) promoter systems also provides the promoter sequence of usefulness.The method for optimizing of being contained utilizes strong promoter (such as the T7 promotor) to produce to induce high-caliber polypeptide herein.The example of these carriers includes but not limited to from the pET29 series of Novagen and the pPOP carrier described in the WO99/05297 (its mode of quoting in full is incorporated herein).These expression systems produce the polypeptide of high-load in the host, and do not damage host cell survival ability or growth parameter(s).
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, thereby produce synthetic hybrid promoter [United States Patent (USP) the 4th, 551, No. 433].For instance, the heterozygosis trp-lac promotor that the tac promotor is made up of trp promotor and lac operon sequence, its by lac repressor regulation and control [people such as Amann,
Figure A200680049995D0137161636QIETU
(1983) 25:167; People such as de Boer,
Figure A200680049995D0137161651QIETU
(1983) 80:21].In addition, the bacterium promotor can comprise the natural promotor that exists of non-bacterial origin, and it has with the bacteria RNA polymerase and combines and initial ability of transcribing.Also non-bacterial origin natural can be existed promotor and compatible RNA polymerase coupling to produce the high level expression of some genes in prokaryotes.Phage t7 RNA polymerase/promoter systems is example [people such as Studier, the J. of coupling promoter systems
Figure A200680049995D0138162130QIETU
(1986) 189:113; People such as Tabor, Proc Natl.Acad.Sci. (1985) 82:1074].In addition, hybrid promoter also can be made of (No. the 267 851, the open case in Europe) phage promoter and Escherichia coli operon zone.
Except that functional promoter sequence, effectively ribosome bind site also is used in and expresses alien gene in the prokaryotes.In Escherichia coli, ribosome bind site be called Shine-Dalgarno (SD) sequence and the length that comprises initiation codon (ATG) and be positioned at upstream from start codon 3-11 nucleotide place be 3-9 nucleotide sequence [people such as Shine,
Figure A200680049995D0138162207QIETU
(1975) 254:34].Think that the SD sequence promotes mRNA and ribosomal combination the [people such as Steitz by the base pairing between SD sequence and 3 of the Escherichia coli 16S rRNA ' end, " Genetic signals and nucleotidesequences in messenger RNA ", Biological Regulation and Development:Gene Expression (Ed.R.F.Goldberger, 1979)].The eukaryotic gene and the prokaryotic gene [people such as Sambrook, " Expression of cloned genes in Escherichia coli ", Molecular Cloning:ALaboratory Manual, 1989] that have weak ribosome bind site for expression.
Term " bacterial host " or " bacterial host cell " are meant the bacterium that can be used as or be used as the acceptor of recombinant vector or other transfer DNA.Described term comprises the offspring of the primitive bacteria host cell of transfection.Should be appreciated that because accidental sudden change or deliberately sudden change, the offspring of single mother cell on the form or with the genomic DNA of original parent complementation or total DNA on may may not be identical.Be included among the offspring of this definition indication with the offspring of the fully similar mother cell of desiring to characterize of parent with correlation properties (such as the nucleotide sequence that has the required polypeptide of coding).
One of ordinary skill in the art are used to express the selection of the suitable host bacteria of required polypeptide as everyone knows.When selecting for the bacterial host of expressing, suitable host can include but not limited to show have in the following feature at least a and preferably have in the following feature host of at least two kinds: (especially) good inclusion body forms ability, low proteolytic activity, good secretion capacity, good soluble protein and produces ability and overall steadiness.Bacterial host can obtain from multiple source usually, include but not limited to that University of California's biophysics and medical physics are bacterial gene preservation center (California Berkeley) (Bacterial Genetic Stock Center, Department of Biophysics and Medical Physics, Universityof California (Berkeley, and U.S. typical case (the American Type Culture Collection (" ATCC ") (Manassas, VA)) of DSMZ (" ATCC ") (Manassas, northern Virginia) CA)).The bacterium that derives from the bacterium of K bacterial strain (for example W3110) or derive from B bacterial strain (for example BL21) is used in the fermentation of industry/medicine usually.These bacterial strains are well-known and stable and particularly useful because of its growth parameter(s).In addition, these bacterial strains are avirulence, and for safety and environment reason, it is commercial quite important.In an embodiment of described herein and the method that contains, escherichia coli host includes but not limited to bacterial strain BL21, DH10B or derivatives thereof.In another embodiment of described herein and the method that contains, escherichia coli host is that protease lacks bacterial strain, and it includes but not limited to OMP-and LON-.In another embodiment, bacterial host is a pseudomonas, such as pseudomonas fluorescens, pseudomonas aeruginosa and pseudomonas putida.Another example of pseudomonad expression strain is Pseudomonas fluorescence biotype I, bacterial strain MB101 (DowChemical).
Cell or expression of cell lines system cells or expression of cell lines system are meant cell, cell-line and the transgenosis organism (comprising amphibian, reptile, birds and mammal) of the gene that can express the coding non-natural amino acid polypeptides.In addition, the transgenosis organism is expressed and can be comprised the polypeptide (such as milk or egg) that produces the secretion or the form of drainage, can be collected it, and can use affiliated field and standard method as herein described to extract and be further purified expressed non-natural amino acid polypeptides in case of necessity.
The example of useful host cell and/or cell-line includes but not limited to Vero cell, HeLa cell, COS cell, Chinese hamster ovary (CHO) cell-line, W138, BHK, COS-7,293, HepG2, Balb/3T3, RIN, MT2, mouse NS0 and other myeloma cell line, hybridoma and xenogenesis hybridoma (heterohybridoma) cell-line, lymphocyte, fibroblast, Sp2/0 and mdck cell.The cell-line that is applicable to serum free medium is also available, and described cell-line is beneficial to the secreted protein of purifying from cell culture medium because of there not being serum proteins.A kind of this type of example is (but being not limited to) serum-free EBNA-1 cell-line (people such as Pham, (2003) Biotechnol.Bioeng.84:332-42).In addition, can select to regulate that insetion sequence is expressed or modify and the host cell strain of processed gene product in required mode.Described modification to protein (for example glycosylation) and processing (for example cracking) are most important for the function of protein.Different host cells, cell-line, host system or organism at the translation of protein after processing and modification have characteristic and specific mechanism.Can select suitable cell, cell-line, host system or organism to guarantee correctly to modify and process expressed extraneous protein.
The multiple choices system be can use, herpes simplex virus thymidine kinase base, hypoxanthine guanine phosphoribosyltransferase, adenine phosphoribosyl transferase and/or the dihydrofolate reductase gene in tk-, hgprt-, aprt-or dhfr-cell respectively included but not limited to.In addition, can with the antimetabolite resistance with the gpt that give the mycophenolic acid resistance of electing, give the neo of aminoglycoside G418 resistance and the basis of giving the hygro of hygromycin resistance.Well-known and visual various preparation Considerations (including but not limited to the simplicity of host cell type, required posttranslational modification, carrier selection, preparative-scale, preparation cost and purifying) are used other selective system for one of ordinary skill in the art.
Form the recombinant host cell strain (that is, expression has been constructed body introduce in the host cell and separate have the host cell that body is constructed in suitable expression) after, cultivate the recombinant host cell strain being suitable for producing under the condition of polypeptide.The method of cultivating the recombinant host cell strain will depend on that the expression that is utilized constructs the character of body and the identity of host cell.Usually well-known method is cultivated the recombinant host bacterial strain in the affiliated field of use.Recombinant host cell is normally cultivated in the liquid nutrient medium of fill-in and is cultivated containing absorbable carbon source, nitrogenous source and inorganic Yanyuan and contain well-known other protein in vitamin, amino acid, growth factor and the affiliated field according to circumstances.The liquid nutrient medium that is used to cultivate host cell can contain antibiotic or antifungal agent according to circumstances to prevent the growth of unwanted microorganism and/or compound (including but not limited to be used to select contain the antibiotic of the host cell of expression vector).
Recombinant host cell pattern is in batches or continuously cultivated, and wherein the collection of cell collection (at required polypeptide under the situation of cell inner accumulated) or culture supernatants is pattern in batches or continuously.For in prokaryotic host cell, preparing preferred batch culture and cell collection.Realizing under the situation of protein expression via cell or expression of cell lines system, can make cell proliferation with various modes in vitro, non-anchor the dependent cell (non-anchorage dependent cell) that includes but not limited between most of culture period, in suspension, to grow all the time, maybe need to be attached on the solid matrix anchorage dependence cell (that is the cell of single layer type growth) for its breeding.Non-anchorage dependence or suspension culture from the cell-line of continuous generation are the most widely used modes that produces cell and cellular products on a large scale.And, can select cell type and breeding pattern based on various preparation Considerations as indicated above.
In one embodiment, be to express in the recombination system purifying non-natural amino acid polypeptides as herein described afterwards.Can be by the known several different methods purified polypeptide from host cell or medium in affiliated field.Usually, the many polypeptide that produced in the bacterial host cell have weak dissolubility or soluble (being the inclusion body form).In one embodiment, utilize the known method of method disclosed herein and affiliated field, can easily in polypeptide, carry out the aminoacid replacement of selecting for the deliquescent purpose of the polypeptide that increases the reorganization generation.Under the situation of insoluble polypeptide, can collect polypeptide from the host cell lysate by centrifugal or filtration, and can then carry out homogenizing of cell thereafter.Under situation with weak deliquescent polypeptide, can add include but not limited to polymine (PEI) compound to induce the precipitation of part soluble polypeptide.Subsequently can be by centrifugal or filter collecting precipitation polypeptide expediently.Can use the well-known several different methods of one of ordinary skill in the art that recombinant host cell is broken or homogenize to discharge inclusion body from cell interior.Can use well-known technology to carry out breaking of host cell or homogenize, these technology include but not limited to that enzymatic cell rupture, sonication, Du Ensi homogenize (dounce homogenization) or high pressure discharges and breaks.In an embodiment of described herein and the method that contains, use the high pressure release tech that e. coli host cell is broken to discharge the polypeptide inclusion body.When handling the polypeptide inclusion body, advantageously make repeating to minimize so that maximize the inclusion body productive rate and can not causing damage of the time of homogenizing owing to factor such as dissolving, mechanical shearing or proteolysis.
Under can using subsequently in the known numerous suitable solubilizer in field any dissolves insoluble or the precipitation polypeptide.For instance, utilize urea or guanidine hydrochloride to dissolve polypeptide.The volume of dissolving polypeptide is minimized, thereby can use the batch size preparation that is convenient to operation in enormous quantities.At recombinant host can volume be that this factor can be important in the large-scale commercial applications device of batch growth of thousands of liters.In addition, when in the large-scale commercial applications device, making polypeptide,, if possible, should avoid to damage the harsh chemicals (harshchemicals) or the polypeptide product itself of machine and container so particularly for human medical usage.Confirm that in described herein and the method that contains relatively mild denaturant urea can replace harsh denaturant guanidine hydrochloride to be used to dissolve the polypeptide inclusion body.The use of urea significantly reduces the hurtful risk of being utilized in the manufacturing of polypeptide and purge process of stainless steel equipment in effective dissolving polypeptide inclusion body.
Under the situation of soluble polypeptide, peptide can be secreted in periplasmic space or the medium.In addition, soluble peptide can be present in the cytoplasm of host cell.Can before carrying out purification step, concentrate soluble peptide.Can use the concentrated soluble peptide of standard technique (including but not limited to the techniques described herein) from for example cell lysates or medium.In addition, standard technique (including but not limited to the techniques described herein) can be used for that host cell is broken and discharges soluble peptide from the cytoplasm of host cell or periplasmic space.
When preparation is the polypeptide of fusion form, preferably remove fusion sequence.Can realize the removal of fusion sequence by the method that includes but not limited to enzymatic lysis or chemical cracking, wherein preferred enzymatic lysis.Can use the well-known method of one of ordinary skill in the art to realize the enzymatic removal of fusion sequence.The selection that is used to remove the enzyme of fusion sequence will be by the identity decision of fusion, and reaction condition will be specified by the selection of enzyme.Can use the reagent that includes but not limited to cyanogen bromide, TEV protease and other reagent to realize chemical cracking.According to circumstances by well-known method from the fusion sequence of cracking purifying through the cracking polypeptide.These methods will be by the identity and the characteristic decision of fusion sequence and polypeptide.The method that is used for purifying can include but not limited to size exclusion chromatography, hydrophobic interaction chromatograph, ion-exchange chromatography or dialysis or its any combination.
Also according to circumstances purified polypeptide from protein solution, to remove DNA.Can remove DNA by the known any appropriate methodology in affiliated field (including but not limited to precipitation or ion-exchange chromatography).In one embodiment, by removing DNA with nucleic acid precipitating reagent (such as (but being not limited to) protamine sulfate) precipitation.Can use and include but not limited to that well-known standard method centrifugal or that filter makes polypeptide separate with deposit D NA.Polypeptide is used for the treatment of in the human situation in desire, the removal of host's nucleic acid molecules is a key factor, and method as herein described drops to pharmaceutically acceptable content with host cell DNA.
The method that is used for small-scale or large scale fermentation also can be used for protein expression, and described method includes but not limited to fermentation tank, vibration flask, fluidized bed bio reactor, hollow-fiber bioreactor, rolls bottle culture systems and tank diameter bioreactor system.In these methods each can be in batches, feedback expects that mode process carries out in batches or continuously.
Usually the human form of non-natural amino acid polypeptides as herein described is reclaimed in the standard method under can using in the field.For instance, can with medium or cell lysates be centrifugal or filter to remove cell fragment.Can or be diluted to volume required or saturating the filter in the suitable buffer solution with supernatant concentration to regulate preparation for being further purified.Being further purified of non-natural amino acid polypeptides as herein described includes but not limited to separate desamidization and the polypeptide variants of cutting short form from corresponding complete form.
Below in the exemplary program any all can be used for purifying non-natural amino acid polypeptides as herein described: affinity chromatography; Anion or cation-exchange chromatography (use (including but not limited to DEAE SEPHAROSE); Silica gel chromatograph; Reversed-phase HPLC; Gel filtration (using (including but not limited to) SEPHADEX G-75); Hydrophobic interaction chromatograph; Size exclusion chromatography; The metallo-chelate chromatogram; Ultrafiltration/saturating filter; Precipitation with alcohol; Ammonium sulfate precipitation; Chromatofocusing; Displcement chromatography; Electrophoretic procedures (including but not limited to the isoelectric focusing of preparation type); Differential solvability (including but not limited to ammonium sulfate precipitation); SDS-PAGE; Or extraction; Or its any combination.
Known according to one of ordinary skill in the art and use standardization program, can with the polypeptide contained in the method and composition as herein described (include but not limited to comprise alpha-non-natural amino acid polypeptide, at the antibody of the polypeptide that comprises alpha-non-natural amino acid, comprise alpha-non-natural amino acid polypeptide in conjunction with the collocation thing) part or be purified to homogeneous in fact.Therefore, can reclaim and purifying polypeptide as herein described by in well-known numerous methods in the affiliated field any, these methods include but not limited to ammonium sulfate or precipitation with alcohol, acid or alkali extraction, column chromatography, affinity column chromatography, anion or cation-exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatograph, hydroxylapatite chromatography, agglutinin chromatogram, gel electrophoresis and its any combination.In case of necessity, when the mature protein that preparation correctly folds, can use the protein refolding step.High performance liquid chromatography (HPLC), affinity chromatography or other appropriate methodology can be used for the highly purified final purification step of needs.In one embodiment, the antibody that will make at alpha-non-natural amino acid the polypeptide of alpha-non-natural amino acid (or comprise) is as the purifying based on compatibility of purified reagent (including but not limited to) with the polypeptide that is used to comprise one or more alpha-non-natural amino acids.In case of necessity,, according to circumstances polypeptide is used for multiple application, includes but not limited to as the immunogene of examining and determine component, therapeutic agent, prophylactic, diagnosticum, research reagent and/or producing as antibody at partial purification or after reaching homogeneous.
Except other list of references of being mentioned herein, under well-known multiple purifying/protein folding method in the field, include but not limited to described in the following document those: R.Scopes, Protein Purification, Springer-Verlag, N.Y. (1982); Deutscher, Methods in Enzymology the 182nd volume: Guide to Protein Purification.Academic Press, Inc.N.Y. (1990); Sandana, (1997) Bioseparation of Proteins.AcademicPress, Inc.; People such as Bollag (1996) Protein Methods, the 2nd edition Wiley-Liss, NY; Walker, (1996) The Protein Protocols Handbook Humana Press, NJ, Harris and Angal, (1990) ProteinPurification Applications:A Practical Approach IRL Press at Oxford, Oxford, England; Harris and Angal, Protein Purification Methods:A Practical Approach IRL Press at Oxford, Oxford, England; Scopes, the 3rd edition Springer Verlag of (1993) Protein Purification:Principles and Practice, NY; Janson and Ryden, (1998) Protein Purification:Principles.HighResolution Methods and Applications, the 2nd edition Wiley-VCH, NY; And Walker (1998), ProteinProtocols on CD-ROM Humana Press, NJ; And the list of references of wherein being quoted.
An advantage that produces the polypeptide that comprises at least one alpha-non-natural amino acid in eukaryotic host cell or non-eukaryotic host cell is that common described polypeptide will be folding with its native conformation.Yet in some embodiment of methods described herein and composition, after synthetic, expression and/or purifying, polypeptide can have the conformation different with the required conformation of related polypeptide.In aspect methods described herein and composition one, through marking protein according to circumstances through sex change and renaturation subsequently.This optional sex change and renaturation are that known method realizes in the affiliated field of utilization, include but not limited to by chaperone (chaperonin) is added pay close attention in the polypeptide and by polypeptide being dissolved in the chaotropic agent that includes but not limited to guanidine hydrochloride and utilizing protein disulfide isomerase.
In general, sometimes need sex change and reduce through express polypeptide and make polypeptide be folded into preferred conformation more subsequently.For instance, can by guanidine, urea, DTT, DTE and/or chaperone are added to realize in the translation product of being paid close attention to described folding again.The method of one of ordinary skill in the art's reduction as everyone knows, sex change and recombinant protein matter is (referring to people such as above-mentioned list of references and Debinski, (1993) J.Biol.Chem., 268:14065-14070; Kreitman and Pastan (1993) Bioconjug.Chem., 4:581-585; And people such as Buchner, (1992) Anal.Biochem., 205:263-270).For instance, people such as Debinski describes the sex change and the reduction of inclusion body protein matter among guanidine-DTE.Protein can be folding again in containing the arginic potential buffer solution of (including but not limited to) oxidized glutathione and L-.Folding again reagent can flow or otherwise move contact with one or more polypeptide or other expression product, perhaps one or more polypeptide or other expression product can flow or otherwise move with fold reagent again and contact.
Produce at protokaryon under the situation of non-natural amino acid polypeptides, consequent polypeptide possible errors folds and thereby lacks biologically active or have the biologically active of reduction.Can come the biologically active of recoverin matter by " folding again ".In one embodiment, by using one or more chaotropic agents (including but not limited to urea and/or guanidine) for example and can reducing reductant (including but not limited to dithiothreitol (DTT), DTT or 2 mercapto ethanol (2-ME)) dissolving (wherein polypeptide is also soluble), expansion and the reducing polypeptide chain of disulfide bond the polypeptide of false folding is folded again.Under the chaotropic agent of intermediate concentration, add oxidant (including but not limited to oxygen, cystine or cystamine) subsequently, it allows to form disulfide bond again.Under can using in the field known standard method will not fold or the polypeptide of false folding folds again, such as United States Patent (USP) the 4th, 511, No. 502, the 4th, 511, No. 503 and the 4th, those methods described in 512, No. 922, the mode that described patent is quoted separately in full is incorporated herein.Polypeptide also can be folded to form heterodimer or heteromultimeric altogether with other protein.After folding again or folding altogether, be further purified polypeptide according to circumstances.
Can use multiple technologies to realize the purifying of non-natural amino acid polypeptides, include but not limited to the techniques described herein, for example hydrophobic interaction chromatograph, size exclusion chromatography, ion-exchange chromatography, RPLC, affinity chromatography etc. or its any combination.Other purifying also can comprise the step of drying or deposition and purification protein.
Behind the purifying, non-natural amino acid polypeptides exchanged in the different buffer solutions and/or concentrate by in the several different methods known in the affiliated field any, described method includes but not limited to filter and dialyses.The hGH that provides as single protein purification can experience gathering 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% pure or high-purity more.No matter the definite numerical value of the purity of non-natural amino acid polypeptides how, non-natural amino acid polypeptides is for as medical product or be used for the further processing (including but not limited to link with water-soluble polymer such as PEG) all enough pure.
In certain embodiments, can under the situation that does not have other active component or protein (except that excipient, supporting agent and stabilizing agent, seralbumin etc.), the non-natural amino acid polypeptides molecule be used as therapeutic agent, and in some embodiment of non-natural amino acid polypeptides molecule, it can be compound with another polypeptide or polymer.
2. the purifying of non-natural amino acid polypeptides
General purification process: the technology that is disclosed in this section can be applicable to the general purifying of non-natural amino acid polypeptides as herein described.
Can any suitable ordered pair comprise the cytoplasm of periplasmic space, host cell of cell lysates extract, medium, inclusion body, the host cell of required polypeptide or other material or any mixtures of polypeptides that is produced by any separating step is carried out in the multiple separating step any, described separating step includes 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 repetition.
Be used to carry out the equipment of described technology herein and other necessary material on sale on the market.Pump, fraction collector, monitor, logger and whole system can be from for example Applied Biosystems (Foster City, CA), Bio-RadLaboratories, Inc. (Hercules, CA) and Amersham Biosciences, Inc. (Piscataway NJ) obtains.The chromatographic material that includes but not limited to exchange base material, medium and buffer solution also can obtain from these companies.
Can use special equipment (such as pump) more promptly realize in balance and the described herein column chromatography process other step (such as the washing and elution).Commercially available pump includes but not limited to
Figure A200680049995D01441
Pump P-50, peristaltic pump P-1, pump P-901 and pump P-903 (Amersham Biosciences, Piscataway, NJ).
The example of fraction collector comprises RediFrac fraction collector, FRAC-100 and FRAC-200 fraction collector and SUPERFRAC
Figure A200680049995D01442
Fraction collector (Amersham Biosciences, Piscataway, NJ).Blender also can be used for forming pH value and linear concentration gradient.Commercially available blender comprise gradient mixer GM-1 and pipe-line mixer (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 A200680049995D01443
S II, monitor UV-M II, monitor UV-900, monitor UPC-900, monitor pH/C-900 and conductivity monitor (AmershamBiosciences, Piscataway, NJ).In fact, whole system is on sale on the market, comprises (Piscataway, NJ) various from AmershamBiosciences System.
In an embodiment of methods described herein and composition, for example, can then in the TRIS buffer solution that contains reductant (such as DTT), dilute reducing polypeptide under the suitable pH value and make its sex change by at first in urea, making the purified polypeptide sex change of gained.In another embodiment, polypeptide be with between about 2M to the concentration range sex change in the urea element between about 9M, then under about 5.0 pH values in about 8.0 the scope, in the TRIS buffer solution, dilute.Can cultivate the folding again mixture of this embodiment subsequently.In one embodiment, at room temperature will fold mixture again cultivated 4 hours to 24 hours.Subsequently can be with the further isolated or purified of mixtures of polypeptides of reduction and sex change.
As described herein, before carrying out any later separation step, can adjust the pH value of first mixtures of polypeptides.In addition, can use under in the field known technology concentrate first mixtures of polypeptides or its any subsequent mixtures.In addition, the elution buffer solution that can use the well-known technology of one of ordinary skill in the art will comprise first mixtures of polypeptides or its any subsequent mixtures changes the buffer solution that is applicable to next separating step into.
The technology that is disclosed in ion-exchange chromatography this section can be applicable to the chromatography of ions of non-natural amino acid polypeptides as herein described.
In one embodiment and as optional additional step, can carry out ion-exchange chromatography to first mixtures of polypeptides.Usually referring to
Figure A200680049995D0145163014QIETU
(catalog number (Cat.No.) 18-1114-21, Amersham Biosciences (Piscataway, NJ)).Commercially available ion exchange column comprises
Figure A200680049995D01451
With Post (Amersham Biosciences, Piscataway, NJ).These posts utilize strong anion exchanger, such as Q
Figure A200680049995D01453
Fast Flow, Q
Figure A200680049995D01454
High Performance and Q
Figure A200680049995D01455
XL; Strong cation exchanger is such as SP
Figure A200680049995D01456
High Performance, SP
Figure A200680049995D01457
FastFlow and SP
Figure A200680049995D01458
XL; Weak anion exchanger is such as DEAE
Figure A200680049995D01459
Fast Flow; And weak cation exchanger, such as CM
Figure A200680049995D014510
Fast Flow (Amersham Biosciences, Piscataway, NJ).Can carry out anion or cation exchange column chromatography to separate purified in fact polypeptide to polypeptide in any stage of purge process.Can use any suitable cation exchange matrix to carry out the cation-exchange chromatography step.That useful cation exchange matrix includes but not limited to is fibrous, porous, atresia, particulate, bead or cross-linked cationic exchange base material.These cation exchange host materials include but not limited to the compound of cellulose, agarose, glucan, polyacrylate, polyethylene, polystyrene, silica, polyethers or any above-mentioned material.After polypeptide being adsorbed onto on the cation exchange matrix, can contact from described matrix, to replace polypeptide with buffer solution by making matrix with enough high pH value or ion strength, come the purified in fact polypeptide of elution.The suitable buffer solution that is used for the high pH value elution of purified in fact polypeptide includes but not limited to that concentration arrives at least about the citrate in the 100mM scope, phosphate, formates, acetate, HEPES and MES buffer solution at least about 5mM.
Reverse-phase chromatography: the technology that is disclosed in this section can be applicable to the reverse-phase chromatography of non-natural amino acid polypeptides as herein described.
Can carry out RP-HPLC with protein purification according to the known suitable scheme of one of ordinary skill in the art.For example referring to people such as Pearson,
Figure A200680049995D0146163340QIETU
(1982) 124:217-230 (1982); People such as Rivier, J.
Figure A200680049995D0146163351QIETU
(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, can use and have multiple length and (include but not limited at least about C 3Arrive at least about C 30, at least about C 3Arrive at least about C 20Or at least about C 3Arrive at least about C 18) the silica resins derived therefrom of alkyl functional group.Perhaps, can use the polymerism resin.For instance, can use TosoHaas AmberchromeCG1000sd resin, it is a styrenic polymer resins.Also can use cyano group or polymerism resin with multiple alkyl chain length.In addition, available solvent wash RP-HPLC post such as ethanol.The suitable elution buffer solution that contains ion-pairing agent and organic modifiers (such as methyl alcohol, isopropyl alcohol, oxolane, acetonitrile or ethanol) can be used for from RP-HPLC post elution polypeptide.The most frequently used ion-pairing agent includes 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 gradients or isocratic condition to carry out elution, wherein preferably reduce the gradient condition of disengaging time and reduction peak width.Another kind method relates to uses two kinds of gradients with different solvents concentration range.The example that is used for suitable elution buffer solution herein can include but not limited to ammonium acetate and acetonitrile solution.
The hydrophobic interaction chromatograph purification technique: the technology that is disclosed in this section can be applicable to the hydrophobic interaction chromatograph purifying of non-natural amino acid polypeptides as herein described.
Can carry out hydrophobic interaction chromatograph (HIC) to polypeptide.Usually referring to
Figure A200680049995D0146163426QIETU
Figure A200680049995D0146163445QIETU
(it is incorporated herein by reference for catalog number (Cat.No.) 18-1020-90, AmershamBiosciences (Piscataway, NJ)).Suitable HIC matrix can include but not limited to the matrix through the alkyl or aryl replacement, such as the matrix that replaces through butyl, hexyl, octyl group or phenyl, comprise agarose, Sepharose, Ago-Gel, cellulose, silica, glucan, polystyrene, poly-(methacrylate) matrix and mixed mode resin (including but not limited to polyethyene diamine resin or poly-(methacrylate) matrix that replaces through butyl or phenyl).The commercially available source of hydrophobic interaction column chromatography includes but not limited to With
Figure A200680049995D01462
Post (Amersham Biosciences, Piscataway, NJ).Briefly, before loading, 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 HIC post.Can be with ammonium sulfate as the buffer solution that loads the HIC post.After loading polypeptide, can use standard buffer solution and condition to come column scrubber removing unwanted material subsequently, but polypeptide is stayed on the HIC post.Available about 3 standard buffer solutions to about 10 column volumes (such as containing the HEPES buffer solution that EDTA and concentration are lower than the ammonium sulfate of level pad, or acetate/sodium chloride buffer solution) come the elution polypeptide.Also can use the linear salt gradient of the continuous reduction of for example using the potassium phosphate gradient to come the elution peptide molecule.Can for example concentrate eluate subsequently by filtering (such as saturating filter or ultrafiltration).Can utilize filter to remove the salt that is used for the elution polypeptide.
Other purification technique: the technology that is disclosed in this section can be applicable to other purification technique of non-natural amino acid polypeptides as herein described.
Can to first mixtures of polypeptides or its any subsequent mixtures use for example gel filtration (
Figure A200680049995D0147163523QIETU
Figure A200680049995D0147163533QIETU
(catalog number (Cat.No.) 18-1022-18, Amersham Biosciences, Piscataway, NJ), its mode of quoting in full is incorporated herein), hydroxylapatite chromatography (is fit to matrix and includes but not limited to HA-Ultrogel, High Resolution (Calbiochem), CHT pottery hydroxyapatite (BioRad), Bio-GelHTP hydroxyapatite (BioRad)), HPLC, expanded bed adsorption, ultrafiltration, saturating filter, another separating step of freeze-drying etc. is to remove any excess salt and to replace described buffer solution to be used for next separating step or even the finally allotment of drug products with being fit to buffer solution.Can use various technology (including but not limited to the techniques described herein) to monitor the polypeptide productive rate of (comprising purified in fact polypeptide) herein in described each step.These technology also are used in the productive rate of the purified in fact polypeptide of assessment behind the last separating step.For instance, can use have multiple alkyl chain length some anti-phase high pressure liquid chromatography post (such as cyano group RP-HPLC, C 18RP-HPLC) any and among cation exchange HPLC and the gel filtration HPLC monitored the productive rate of polypeptide.
Also can use the affinity purification technology to come the purity of purifying or enhancing non-natural amino acid polypeptides preparation.Affinity purification utilizes antibody, acceptor, agglutinin and/or increases specific other molecule of purifying.Protein formulation passes the matrix that contains specific antibody of being seen epi-position tool on target protein or the target protein or in the target protein or molecule, and subsequently with the target protein elution that kept to reclaim highly purified protein formulation.The expression that also can engineeredly be used to produce non-natural amino acid polypeptides is constructed body to add affinity tag (such as myc epi-position, GST fusion or His label) and to utilize corresponding myc antibody, glutathione resin or Ni resin to carry out affinity purification respectively.The use of described antibody, part and affinity tag is for instance and can not limits the selection of the affinity purification that can be used for non-natural amino acid polypeptides.Can use multiple affinity molecule and matrix (that is, post, bead, slurries etc.) and be that affiliated field is well-known.
Can use such as the standard technique of SDS-PAGE or by using Western blotting and ELISA calibrating to measure polypeptide and measure purity.For instance, can produce polyclonal antibody at reclaiming the protein that separates from negative control culture propagation and cation exchange.Antibody also can be used for surveying the existence of contaminative host cell proteins matter.
In certain embodiments, behind each purification step the productive rate of polypeptide can be polypeptide in the raw material 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 the C4 alkyl chain.Polypeptide and the difference that is based on hydrophobic interaction intensity separating of protein impurities.Carry out elution with the acetonitrile gradient in rare trifluoroacetic acid.Use stainless steel column (filling about 2.8) to be prepared type HPLC to about 3.2 liters of Vydac C4 silica gel.Come acidifying hydroxyapatite Ultrogel eluate and it is loaded on the Vydac C4 post by adding trifluoroacetic acid.For washing and elution, use the acetonitrile gradient in rare trifluoroacetic acid.Collect elution part and with phosphate buffer it is neutralized immediately.Collect in the polypeptide elution part in the IPC limit.
DEAE Sepharose (Pharmacia) material is by forming with surperficial covalently bound diethylamino ethyl (DEAE) group of Ago-Gel bead.Mediate combining of polypeptide and DEAE group by ionic interaction.Acetonitrile and trifluoroacetic acid do not have the ground of delay and pass through post.After washing these materials off, remove trace impurity by acetate buffer column scrubber with low pH value.Subsequently with neutral phosphor phthalate buffer column scrubber and with the buffer solution elution polypeptide of ion strength with increase.With DEAE Sepharose fast flow packed column.The adjustable column volume arrives in the scope of about 10 milligrams of polypeptide at every milliliter of gel about 3 to guarantee the polypeptide useful load.Water and level pad (sodium phosphate/potassium) column scrubber.The HPLC eluate elution part that loading compiles and use the level pad column scrubber.Use lavation buffer solution (sodium acetate buffer) column scrubber subsequently, then wash with level pad.Subsequently, with elution buffer solution (sodium chloride, sodium phosphate/potassium) polypeptide is collected in the single elution part from the post elution and according to the standard elution curve.The eluate of DEAE Sepharose post is adjusted to the appointment conductivity.Be aseptically filled in fe fluon (Teflon) bottle gained drug substance and storage under-70 ℃.
Can use several different methods and procedure qualification to contain the productive rate and the purity of the polypeptide of one or more alpha-non-natural amino acids, include but not limited to SDS-PAGE, Western blotting, mass spectrum, substance assistant laser desorpted/MALDI-MS (MALDI-MS), liquid chromatography/mass spectrometry, isoelectric focusing, analytic type anion exchange, chromatofocusing and circular dichroism with the associating of protein staining method.For instance, described method and the program that is used for profiling protein matter includes but not limited to Bradford calibrating, SDS-PAGE and silver dyeing SDS-PAGE, coomassie dyeing SDS-PAGE.Other method includes but not limited to remove endotoxic step.Endotoxin is for being positioned at the lipopolysaccharides (LPS) on Gram-negative (Gram-negative) the host cell outer side form of (such as, Escherichia coli).The method that is used to reduce endotoxin content includes but not limited to use the purification technique of silica supporter, glass dust or hydroxyapatite; Reverse-phase chromatography, affinity chromatography, size exclusion chromatography, anion-exchange chromatography; Hydrophobic interaction chromatograph; The combination of these methods etc.May need to revise or other method from the polypeptide of being paid close attention to, to remove pollutant (such as moving protein altogether).The method that is used to measure endotoxin content is for known to the one of ordinary skill in the art and include but not limited to LAL (Limulus Amebocyte Lysate, LAL) calibrating.
In certain embodiments, the amino acid (being included in any minor or specific compound in the formula I-LXVII scope) of formula I-LXVII can be incorporated in the polypeptide by biological synthesis method, thereby be produced non-natural amino acid polypeptides.In other embodiments, described amino acid is incorporated into specific site place in the polypeptide.In other embodiments, use translation system that described amino acid is incorporated in the polypeptide.In other embodiments, described translation system comprises: (i) polynucleotide of coded polypeptide, wherein said polynucleotide comprise the also corresponding selection codon in angle of striking with above-mentioned amino acid whose design in advance; (ii) comprise described amino acid whose tRNA, wherein said tRNA is to selecting codon tool specificity.In other embodiment of described translation system, the mRNA of polynucleotide in translation system, producing.In other embodiment of described translation system, translation system comprises plasmid or the phage that comprises polynucleotide.In other embodiment of described translation system, translation system comprises the genomic DNA that comprises polynucleotide.In other embodiment of described translation system, with the polynucleotide stable integration in genomic DNA.In other embodiment of described translation system, translation system comprises being selected from the specific tRNA of selection codon tool by the molecular group of following password: amber codon, ochre codon, opal codon, unique codon, rare codon, non-natural codon, five base codons and four base codons.In other embodiment of described translation system, tRNA is for suppressing tRNA.In other embodiment of described translation system, translation system comprises the tRNA at above-mentioned amino acid aminoacylization.In other embodiment of described translation system, translation system comprises the specific amino acyl synthetase of tRNA tool.In other embodiment of described translation system, translation system comprises quadrature tNRA and quadrature aminoacyl tRNA synthetase.In other embodiment of described translation system, polypeptide is synthetic by ribosome, 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.In other embodiments, cell is Bacillus coli cells, yeast cells, the cell from pseudomonas, mammalian cell, plant cell or insect cell.In other embodiment of described translation system, 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, cell, yeast cells, mammalian cell, plant cell or insect cell from pseudomonas.In other embodiments, at least a portion polypeptide is that synthetic or its combination comes syntheticly by solid phase or solution phase peptide, and in other embodiments, further comprises polypeptide is engaged with another polypeptide.In other embodiments, the amino acid of formula I-LXVII (being included in any minor or specific compound in the formula I-LXVII scope) can be incorporated in the polypeptide by biological synthesis method, wherein said polypeptide for the protein of the therapeutic protein homology that is selected from the group that forms by required polypeptide.
B. in vivo posttranslational modification
By produce the polypeptide of paying close attention to at least one alpha-non-natural amino acid in eukaryotic, described polypeptide is modified after can comprising eukaryotic translation.In certain embodiments, polypeptide comprises at least one alpha-non-natural amino acid and at least one by the posttranslational modification that eukaryotic carries out in vivo, and wherein said posttranslational modification is not to be undertaken by prokaryotic.For instance, posttranslational modification comprises (including but not limited to) acetylization, acidylate, lipid-modified, palmitoylation, palmitate addition, phosphorylation, the modification of glycolipid key, glycosylation etc.On the one hand, posttranslational modification comprises by GlcNAc-asparagine key oligosaccharides (is included but not limited to (GlcNAc-Man) 2-Man-GlcNAc-GlcNAc)) link together with asparagine.Referring to table 1, its N that lists eukaryotic protein connects some examples (the also residue that can exist other not show) of oligosaccharides.On the other hand, posttranslational modification comprises by GalNAc-serine or GalNAc-threonine key or GlcNAc-serine or GlcNAc-threonine key oligosaccharides (including but not limited to Gal-GalNAc, Gal-GlcNAc etc.) and serine or threonine is linked together.
The 1.01st joint table 1: the example of the oligosaccharides that connects by the GlcNAc key
Figure A200680049995D01501
On the other hand, posttranslational modification comprises the proteolytic treatment of precursor (including but not limited to calcitonin precursor, CGRP precursor, Pre Pro PTH, preproinsulin, proinsulin, pre-pro-opiomelanocortin (prepro-opiomelanocortin), POMC etc.); Be assembled into many subunits protein or big molecule assembling thing; Translate another site in the cell (include but not limited to organelle,, or pass through secretory pathway) such as endoplasmic reticulum, Golgi body (golgi apparatus), nuclear, lysosome, peroxisome, mitochondria, chloroplast, vacuole etc.In certain embodiments, protein comprises secretion or positioning sequence, epi-position label, FLAG label, polyhistidyl label, GST fusion etc.
An advantage of alpha-non-natural amino acid is that it provides the extra chemical part that can be used for adding additional molecules.These modifications can in vivo be carried out or in vitro carry out at eucaryon or non-eukaryotic.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 covalent bond that the current major part reaction that is used for selective modification protein relates between nucleophilic and the electrophilic reaction collocation thing forms, and includes but not limited to the reaction of α-Lu Daitong and histidine or cysteine side chain.In these situations, selectivity is by the quantity of nucleophilic residues in the protein and accessibility decision.Described herein or use in the polypeptide that method as herein described produces, can be in vitro or in vivo use other to have more optionally reaction, include but not limited to the reaction of non-natural dicarbapentaborane amino acid and diamines.Illustrative example is found in below with reference in the document.For example referring to people such as Cornish, (1996) J.Am.Chem.Soc.118:8150-8151; People such as Mahal, (1997) Science.276:1125-1128; People such as Wang, (2001) Science 292:498-500; 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.Sci100:56-61; People such as Zhang, (2003) Biochemistry.42:6735-6746; With people such as Chin, (2003) Science.300:964-967.This makes it possible to the almost any protein of plurality of reagents selected marker that comprises fluorogen, crosslinking agent, sugar derivatives and cytotoxicity molecule.Also referring to No. the 6th, 927,042, the United States Patent (USP) of " Glycoproteinsynthesis " by name of on January 16th, 2003 application, it is incorporated herein by reference.Posttranslational modification (including but not limited to by azido amino acid) also can engage (Staudinger ligation) (including but not limited to use triaryl phosphine reagent) by Staudinger and carries out.For example referring to people such as Kiick, (2002) Incorporation of azides intorecombinant proteins for chemoselective modification by the Staudinger ligtation, PNAS99 (1): 19-24.
IX. produce the alternative system of non-natural amino acid polypeptides
Used some kinds of strategies in non-recombinant hosts cell, mutagenesis host cell or cell free system, alpha-non-natural amino acid is introduced in the protein.The alternative system that is disclosed in this section can be applicable to produce non-natural amino acid polypeptides as herein described.For instance, amino acid can make that through the reactive side chain derivatization such as Lys, Cys and Tyr lysine is converted into N 2-acetyl group-lysine.Chemosynthesis also provides the direct method of incorporating alpha-non-natural amino acid into.Utilize the newly-developed that enzymatic engages and native chemical engages of fragments of peptides, might make than larger protein.For example referring to P.E.Dawson and S.B.H.Kent, Annu.Rev.Biochem, 69:923 (2000).The chemistry peptide engages and the native chemical joint 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, its mode of quoting in full is incorporated herein.Used and will be added to the general in vitro biological synthesis method that to support in the biosynthetic in vitro extract of protein with the inhibition tRNA of chemical mode acidylate through required alpha-non-natural amino acid 100 are incorporated in the multiple proteins of virtually any size almost on above alpha-non-natural amino acid locus specificity ground.For example referring to 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-specific incorporation of unnatural aminoacids into proteins, Science 244:182-188 (1989); And J.D.Bain, C.G.Glabe, T.A.Dix, A.R.Chamberlin, E.S.Diala, Biosynthetic site-specific incorporation of a non-natural amino acidinto a polypeptide, J.Am.Chem.Soc.111:8013-8014 (1989).Multiple functional group has been introduced in the protein research for protein stability, protein folding, enzyme mechanism and signal transduction.
Proposed to be called in vivo method that selection pressure incorporates into to utilize the scrambling of wild type synzyme.For example referring to N.Budisa, C.Minks, S.Alefelder, W.Wenger, F.M.Dong, L.Moroder and R.Huber, FASEBJ., 13:41-51 (1999).Make and supply the specific natural amino acid whose associated metabolic auxotrophic strain that the path is closed to cell and grow in the minimal medium of the natural amino acid that contains limited concentration, and transcribing of target gene checked.When the stable growth phase began, natural amino acid was depleted and replace through the alpha-non-natural amino acid analog.To the feasible protein accumulation that contains the non-natural analog of inducing of recombinant protein expression.For instance, used this strategy that adjacent fluorophenylalanine, a fluorophenylalanine and P-fluoropnenylalanine are incorporated in the protein, and it shows two characteristic acromions that are easy to differentiate in UV spectrum, for example referring to C.Minks, R.Huber, L.Moroder and N.Budisa, Anal.Biochem., 284:29-34 (2000); Used the fluoroform methyllanthionine to replace methionine in the phage T4 lysozyme to study the interaction of itself and chitosan oligosaccharide part by 19FNMR, for example referring to H.Duewel, E.Daub, V.Robinson and J.F.Honek, Biochemistry, 36:3404-3416 (1997); And incorporated trifluoro leucine in place leucine into, thereby the heat endurance of leucine zipper protein and chemical stability are increased.For example referring to 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, selenomethionine and telluro methionine are incorporated in the various recombinant proteins to promote the phased soln in the X-ray crystallography.For example referring to 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).Also incorporate methionine analog effectively into, thereby allow protein to be carried out other modification by chemical mode with alkene or alkynes functional group.For example referring to J.C.M.van Hest 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 (2000); And K.L. Kiick and D.A.Tirrell, Tetrahedron, 56:9487-9493 (2000); United States Patent (USP) the 6th, 586, No. 207; No. the 2002/0042097th, U.S. Patent Publication case, its mode of quoting in full is incorporated herein.
The identification of aminoacyl tRNA synthetase to the alpha-non-natural amino acid analog is depended in the success of this method, and described synzyme needs high selectivity to guarantee the fidelity of protein translation usually.A kind of mode of expanding the scope of this method is to relax the substrate specificity of aminoacyl tRNA synthetase, and this realizes under a limited number of situation.Only for instance, in Escherichia coli phenylalanyl tRNA synzyme (PheRS), replace Ala with Gly 294Can increase the size of substrate binding pocket, and cause the acidylate of fenclonine (p-Cl-Phe) tRNAPhe.Referring to, M.Ibba, P.Kast and H.Hennecke, Biochemistry, 33:7107-7112 (1994).The coli strain that has this sudden change PheRS allows to incorporate into fenclonine or bromophenyl alanine is substituted phenyl alanine.For example referring to 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, demonstration allows to incorporate azatyrosine effectively into than tyrosine near the point mutation Phe130Ser of the amino acid binding site of Escherichia coli tyrosyl-tRNA synzyme.Referring to F.Hamano-Takaku, T.Iwama, S.Saito-Yano, K.Takaku, Y.Monden, M.Kitabatake, D.Soll and S.Nishimura, J.Biol.Chem., 275 (51): 40324-40328 (2000).
Alpha-non-natural amino acid is incorporated into the another kind of strategy in the protein has check and correction mechanism for modification synzyme in vivo.These synzyme can not be distinguished structurally with the similar amino acid of homology natural amino acid and therefore with its activation.This mistake is separately obtaining proofreading and correct on the site, and this makes mispairing amino acid removal of acylation from tRNA to keep the fidelity of protein translation.If it is active that synzyme loses check and correction, the analogue of so wrong activation can be avoided editting function and be merged in.Recently used valyl base tRNA synzyme (ValRS) to confirm this method.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 make the tRNAVal mistake aminoacylization with Cys, Thr or aminobutyric acid (Abu); Subsequently by these non-homogeneous amino acid of edit field hydrolysis.After making the escherichia coli chromosome random mutagenesis, be chosen in the mutant Escherichia coli bacterial strain that has sudden change in the editor site of ValRS.This editor's deficiency ValRS makes tRNAVal that Cys is housed mistakenly.Since Abu and Cys spatially similar (Cys-SH group warp-CH in Abu 3Therefore displacement), when this mutant Escherichia coli bacterial strain being existed grow under the situation of Abu, the ValRS that suddenlys change also incorporates Abu in the protein into.Mass spectral analysis is presented at the valine of each valine position about 24% of native protein and replaces through Abu.
Solid phase synthesis and semisynthesis have also allowed the synthetic multiple proteins that contains new amino acid.For instance, referring to following discloses case and the following list of references wherein quoted: 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., Bonn, 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, Ace 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 Biochem, 255-301 (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 for TotalSynthesis of Ribonuclease A with Unnatural Catalytic Residues, Science, 266,243-247 (1994).
Used chemical modification will comprise in the multiple non-natural side chain introducing protein of co-factor, spin labeling and oligonucleotides in vitro.For example referring to, Corey, D.R., Schultz, P.G.Generation of a hybrid sequence-specificsingle-stranded deoxyribonuclease, Science, 238,1401-1403 (1987); Kaiser, E.T., LawrenceD.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 of enyzme active sites, Science, 226 .505-511 (1984); Neet, K.E., Nanci A, Koshland, D.E.Properties ofthiol-subtilisin, J Biol.Chem, 243 (24): 6392-6401 (1968); Polgar, L.B., M.L.A new enzymecontaining 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 the biological synthesis method of the aminoacyl tRNA that modifies with chemical mode to be used for some kinds of biophysics probes are incorporated in vitro synthetic protein.Referring to following discloses case and the list of references wherein quoted: Brunner, J.New Photolabeling and crosslinMng methods, Aium.Rev Biochem, 483-514 (1993); And Krieg, U.C., Walter, P., Hohnson, A.E.Photocrosslinking of the signal sequence ofnascent preprolactin of the 54-kilodalton polypeptide of the signal recognition particle, Proc.Natl.Acad.Sci, 83,8604-8608 (1986).
Before confirmed,, can in vitro alpha-non-natural amino acid locus specificity ground be incorporated in the protein by adding the protein synthesis reaction of gene programming with the inhibition tRNA of chemical mode aminoacylization through containing required amber nonsense mutation.Use these methods, can use concerning specific amino acids, to be auxotrophic bacterial strain, with the several amino acids in 20 kinds of common amino acids of close structure homologue replacement, for example, with fluorophenylalanine substituted benzene alanine.For example referring to 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, Science 268:439-42 (1995); Bain, J.D., Glabe, C.G., Dix, T.A., Chamberlin, A.R., Diala, E.S.Biosynthetic site-specific Incorporation of a non-natural amino acid into apolypeptide, J.Am Chem Soc, 111:8013-8014 (1989); People such as N.Budisa, FASEB be (1999) J.13:41-51; Ellman, J.A., Mendel, D., Anthony-Cahill, S., Noren, C.J., Schultz, P.G. Biosyntheticmethod for introducing unnatural amino acids site-specifically into proteins, Methods in Enz., the 202nd volume, 301-336 (1992); And Mendel, D., Cornish, V.W.﹠amp; Schultz, P.G.Site-DirectedMutagenesis with an Expanded Genetic Code, Annu Rev Biophys.Biomol Struct.24,435-62 (1995).
For instance, preparation is discerned the inhibition tRNA of terminator UAG and is made its aminoacylization with alpha-non-natural amino acid with chemical mode.Use conventional direct mutagenesis in protein gene the site of paying close attention to introduce terminator TAG.For example referring to Sayers, J.R., Schmidt, W.Eckstein, F.5 ', 3 ' Exonuclease inphosphorothioate-based oligonucleotide-directed mutagenesis, Nucleic Acids Res, 16 (3): 791-802 (1988).Transcribe when acyl group inhibition tRNA and mutator gene are combined in vitro/translation system in the time, respond the UAG codon and incorporate alpha-non-natural amino acid into, obtain containing described amino acid whose protein in specified location.Use [ 3H]-experiment of Phe and use the experiment confirm of 'alpha '-hydroxy acids, only incorporate at UAG codon appointed positions place amino acid needed and not any other site in protein incorporate this amino acid into.For example referring to, people such as Noren, with above; 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 intoproteins, Science, 255,197-200 (1992).
Also used the microinjection technology that alpha-non-natural amino acid is incorporated in the protein.For example referring to, 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).Xenopus leavis oocytes (Xenopus oocyte) and the following two kinds of RNA materials that in vitro produce are injected altogether: have at concern amino acid position place the UAG terminator the coding target protein mRNA and through the amber inhibition tRNA of required alpha-non-natural amino acid aminoacylization.The machine translator of egg mother cell inserts the specified position of UAG with alpha-non-natural amino acid subsequently.This method has allowed not to be suitable for usually the in vitro in vivo structure-functional study of the conformity membrane albumen of expression system.Example includes but not limited to fluorescence amino acid is incorporated in tachykinin neurokinin-2 acceptor to come measuring distance by FRET (fluorescence resonance energy transfer), for example referring to G.Turcatti, K.Nemeth, M.D.Edgerton, U.Meseth, F.Talabot, M.Peitsch, J.Knowles, H.Vogel and A.Chollet, J.Biol.Chem., 271 (33): 19991-19998 (1996); Incorporate the residue of biotinylation amino acid into to differentiate that the surface exposes in the ion channel, for example referring to J.P.Gallivan, H.A.Lester and D.A.Dougherty, Chem.Biol., 4 (10): 739-749 (1997); Use is covered the Tyrosine analog with the conformation change in the real-time monitoring ion channel through cage, for example referring to J.C.Miller, and 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 to be used to probe into the ion channel main chain of its door control mechanism with change.For example referring to 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).
In vivo the ability of directly alpha-non-natural amino acid being incorporated in the protein provides multiple advantage, includes but not limited to the high yield, technology simplification of mutain, in cell or may study the possibility and the purposes of these mutains in therapeutic treatment of mutain in Living Organism.The alpha-non-natural amino acid that will have various sizes, acidity, nucleophilicity, hydrophobicity and other character the ability in the protein of being included in can greatly be expanded the rational faculty and the ability of operon protein structure systematically, thereby detects novel protein or organism that protein function and generation have novel characteristics.
Incorporate into specifically in the once trial of p-F-Phe in the site, the yeast amber is suppressed tRNAPheCUA/ phenylalanyl tRNA synzyme to being used for p-F-Phe resistance, Phe auxotroph coli strain.For example referring to R.Furter, Protein Sci., 7:419-426 (1998).
The expression of using acellular (in vitro) translation system to obtain required polynucleotide also is possible.Translation system can be cell or cell free translation system, and can be protokaryon or eukaryotic translation system.The cell translation system includes but not limited to full cell preparation, such as the permeation cell or the cell culture that required nucleotide sequence can be transcribed into mRNA and translation mRNA.Cell free translation system is in number of different types on sale and well-known and system on the market.The example of cell free system includes but not limited to the prokaryotic lysate, such as the Escherichia coli lysate; With the eukaryotic lysate, such as wheat germ extract, insect cell lysate, rabbit granulophilocyte lysate, rabbit oocyte lysate and human cell's lysate.When gained protein through glycosylation, phosphorylation or when otherwise modifying, because many modifications only may take place in eukaryotic system, so eucaryon extract or lysate can be preferably.Some are arranged at (Promega on sale on the market in these extracts and the lysate; Madison, Wis.; Stratagene; La Jolla, Calif.; Amersham; Arlington Heights, Ill.; GIBCO/BRL; Grand Island, N.Y.).Film extract (such as the dog pancreatic extract that contains mucous membrane) is also available, and it can be used for translating secretory protein.In can comprising that mRNA is as template (in vitro translation) or DNA these systems as template (combined live in-vitro transcription and translation), in vitro synthetic is to be instructed by ribosome.Carry out considerable trial and researched and developed the cell-free protein expression system.For example referring to, Kim, D.M. and J.R.Swartz, Biotechnology and Bioengineering, 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 andBioengineering, 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; WO00/55353; WO 90/05785, and its mode of quoting in full is incorporated herein.The another kind of method that can be used for expressing the polypeptide that comprises alpha-non-natural amino acid includes but not limited to mRNA-peptide integration technology.For example referring to R.Roberts and J.Szostak, Proc.Natl Acad.Sci. (USA) 94:12297-12302 (1997); People such as A.Frankel, Chemistry ﹠amp; Biology 10:1043-1050 (2003).In the method, the mRNA template that will be connected with puromycin (puromycin) on ribosome is translated into peptide.If one or more tRNA molecules are modified, so also alpha-non-natural amino acid can be incorporated in the peptide.After reading last mRNA codon, puromycin is caught the C end of peptide.Have noticeable characteristic if find gained mRNA-peptide concatenator in examining and determine in vitro, can easily disclose its identity so by the mRNA sequence.In this way, can screen the library of the polypeptide that comprises one or more alpha-non-natural amino acids, have the polypeptide of desirable characteristics with discriminating.Recently, reported the in vitro ribosome translation that utilizes purified component, it allows the synthetic peptide that replaces through alpha-non-natural amino acid.For example referring to people such as A.Forster, Proc.Natl Acad.Sci. (USA) 100 (11): 6353-6357 (2003).
Also can use the reconstruct translation system.Successfully use the mixture and the lysate of purified translation factor or be supplemented with purified translation factor (such as, initiation factor-1 (IF-1), IF-2, IF-3, elongation factor T (EF-Tu) or termination factor) the combination of lysate mRNA is translated into protein.Cell free system also can be coupling type and transcribes/translation system, wherein (people such as F.M.Ausubel edits as Current Protocols in Molecular Biology, WileyInterscience, 1993) described in (it is incorporated herein by reference especially), DNA is introduced in the described system, be transcribed into mRNA and translate mRNA.The RNA that transcribes in the eukaryotic transcription system can be heteronuclear RNA (hnRNA) or 5 ' end attach the names of pre-determined candidates (7-methylguanosine) and 3 ' the hold form of the ripe mRNA (it can have advantage in some translation system) that adds poly A tail.For instance, in granulophilocyte lysate system, translate the mRNA that attaches the names of pre-determined candidates with high efficiency.
Can be by any method or technology (including but not limited to chemistry or enzymatic aminoacylization) utilize amino acid needed with the tRNA aminoacylization.
Aminoacylization can realize by aminoacyl tRNA synthetase or other enzyme molecule (including but not limited to ribozyme).Term " ribozyme " can exchange with " catalytic RNA ".Cech and colleague (Cech, 1987, Science.236:1532-1539; People such as McCorkle, 1987, Concepts Biochem.64:221-226) alleged occurrence can serve as the naturally occurring RNA (ribozyme) of catalyzer.Yet, although only confirm these natural RNA catalyzer to the ribonucleic acid substrate-function for cracking and montage, the development of the artificial exploitation of ribozyme recently expands the catalysis pedigree to various chemical reactions.But research has identified the RNA molecule (people such as Illangakekare of catalysis self (2 ') 3 ' terminal aminoacyl RNA key; 1995 Science267:643-647); and can be with amino acid (the people such as Lohse of the RNA molecule from a RNA molecular transfer to another; 1996, Nature 381:442-444).
U.S. Patent Application Publication case 2003/0228593 (it is incorporated herein by reference) describes the ribosomal method of structure and it is in utilizing natural coding and non-naturally encoded amino acid to make the purposes of tRNA aminoacylization.Can make the matrix fixed form of the enzyme molecule (including but not limited to ribozyme) of tRNA aminoacylization make it possible to effective affinity purification aminoacyl product.Suitably the example of matrix comprises agarose, Ago-Gel and magnetic bead.The preparation and the purposes that are used for the matrix fixed form ribozyme of aminoacylization are described in Chemistry and Biology 2003, and in 10:1077-1084 and the U.S. Patent Application Publication case 2003/0228593, it is incorporated herein by reference.
Chemistry aminoacyl method include but not limited to avoid in the aminoacyl process, to use synzyme by the method for introducing in the following document: Hecht and colleague (Hecht, S.M.Acc.Chem.Res.1992,25,545; Heckler, T.G.; Roesser, J.R.; Xu, C; Chang, P.; Hecht, S.M.Biochemistry 1988,27, and 7254; Hecht, S.M.; Alford, B.L.; Kuroda, Y.; Kitano, S.J.Biol.Chem.1978,253,4517); And Schultz, Chamberlin, people such as Dougherty (Cornish, V.W.; Mendel, D.; Schultz, P.G.Angew.Chem.Int.Ed.Engl.1995,34,621; Robertson, S.A.; Ellman, J.A.; Schultz, P.G.J.Am.Chem.Soc.1991,113,2722; Noren, C.J.; Anthony-Cahill, S.J.; Griffith, M.C.; Schultz, P.G.Science1989,244,182; Bain, J.D.; Glabe, C.G.; Dix, T.A.; Chamberlin, A.R.J.Am.Chem.Soc.1989,111,8013; Bain, people Nature such as J.D. 1992,356,537; Gallivan, J.P.; Lester, H.A.; Dougherty, D.A:Chem.Biol.1997,4,740; People J.Biol.Chem.1996 such as Turcatti, 271,19991; Nowak, people Science such as M.W., 1995,268,439; Saks, people J.Biol.Chem.1996 such as M.E., 271,23169; Hohsaka, people J.Am.Chem.Soc.1999 such as T., 121,34).Described method or other chemical aminoacyl method all can be used for making tRNA molecules of ammonia acyl groupization.
The method that produces catalytic RNA can relate to the independent set that produces stochastic kernel carbohydrase sequence; Orthogenic evolution is carried out in described set; At the described set of required aminoacyl screening active ingredients; With the ribozyme sequence of selecting to represent required aminoacyl activity.
Ribozyme can comprise motif and/or the zone that promotes the acyl group activity, such as the zone of GGU motif and enrichment U.For instance, the zone of having reported enrichment U can promote the identification of amino acid substrate, and the GGU motif can form base-pair with 3 of tRNA ' end.The combination in GGU motif and enrichment U zone can promote to discern simultaneously amino acid and tRNA, and therefore promotes the aminoacylization that 3 of tRNA ' is terminal.
Can by use the part that links with tRNAAsnCCCG at random r24mini in vitro select, the concensus sequence that system engineering is subsequently transformed seen in the active clone produces ribozyme.The exemplary ribozyme that obtains by the method is called " Fx3 ribozyme " and is described in No. the 2003/0228593rd, the open application case of the U.S.; its content is incorporated herein by reference, and described ribozyme serves as the synthetic general catalyzer that is loaded with the various aminoacyl tRNA of homology alpha-non-natural amino acid.
On matrix, fixedly can be used for facilitating effective affinity purification of aminoacyl tRNA.Suitably the example of matrix includes but not limited to agarose, Ago-Gel and magnetic bead.Can ribozyme be fixed on the resin by the chemical constitution of utilizing RNA, such as, 3 on the RNA ribose '-suitable-glycol can be through periodate oxidation to obtain corresponding dialdehyde, so that promote RNA fixing on resin.Can use various types of resins, comprise cheap hydrazides resin, wherein reduction amination makes to interact between resin and the ribozyme and forms irreversible key.Can significantly promote synthesizing of aminoacyl tRNA by aminoacyl technology on this post.People Methods 2005 such as Kourouklis; A kind of aminoacyl system based on post is described among the 36:239-4.
The separation of aminoacyl tRNA may be implemented in a variety of ways.A kind of suitable method is to utilize buffer solution (such as the sodium acetate solution that contains 10mM EDTA; The buffer solution that contains 50mM N-(2-ethoxy) piperazine-N '-(3-propane sulfonic acid), 12.5mMKCl (pH 7.0), 10mM EDTA; The perhaps water of edta buffer (pH7.0) simply) elution aminoacyl tRNA from post.
Aminoacyl tRNA can be added in the translation reaction so that the amino acid of tRNA aminoacylization will be incorporated in the selected location in the polypeptide that translation reaction produces.Can use the example of the translation system of aminoacyl tRNA of the present invention to include but not limited to cell lysates.Cell lysates provides by input mRNA and in vitro translates the required reaction component of polypeptide.The example of described reaction component includes but not limited to ribosomal protein, rRNA, amino acid, tRNA, GTP, ATP, translation initiation factor and elongation factor and other factor relevant with translation.In addition, translation system can be translation in batches or separates translation (compartmentalized translation).Translation system makes up the reaction component in the single compartment in batches, and separate translation system the translation reaction assembly is separated with the product that can suppress translation efficiency.These translation systems are on sale on the market.
In addition, can use coupling type to transcribe/translation system.Coupling type transcribes/and translation system allows to import DNA and is transcribed into corresponding mRNA, and mRNA translates through reaction component.The example that commercially available coupling type is transcribed/translated is RapidTranslation System (RTS, Roche Inc.).Described system comprises that the mixture that contains the Escherichia coli lysate is to provide the translation component such as ribosome and translation factor.In addition, comprise that RNA polymerase is transcribed into the mRNA template to be used for translation will import DNA.RTS can separate reaction component via the film that inserts between the reaction compartments (comprise supply/waste compartment and transcribe/translate compartment).
Can carry out the aminoacylization of tRNA by other reagent that includes but not limited to transferase, polymerase, catalytic antibody, multifunctional protein etc.
Stephan describes in Scientist 30-33 page or leaf on October 10th, 2005 and incorporates non-naturally encoded amino acid in the protein other method.People such as Lu are Mol Cell.2001 October; 8 (4): describe a kind of method (engaging) that protein is joined to the synthetic peptide that contains alpha-non-natural amino acid with chemical mode among the 759-69 through marking protein.
X. the posttranslational modification of the alpha-non-natural amino acid component of polypeptide
For simplicity, prevailingly and/or utilize particular instance to describe the posttranslational modification of the alpha-non-natural amino acid component of polypeptide described herein.Yet, the basic description or the particular instance that are provided should do not be not provided in the posttranslational modification of the alpha-non-natural amino acid component of polypeptide described herein, but the posttranslational modification of the alpha-non-natural amino acid component of polypeptide described herein is equally applicable to all compounds in formula I-LXVII scope, is included in any minor or specific compound in this specification, claims and this paper formula I-LXVII scope described in graphic.
Method, composition, technology and the strategy of site specific incorporation of non-natural amino acids during the translated protein have in vivo been developed.By incorporating the alpha-non-natural amino acid that has with the side chain chemistry of naturally occurring amino acid quadrature into, this technology makes may locus specificity ground derivatization recombinant protein.Therefore, the main advantage of methods described herein, composition, technology and strategy is that derivatization protein can be used as now specifies the preparation of homology product.Yet, method as herein described, composition, reactant mixture, technology and strategy are not limited to the non-natural amino acid polypeptides that forms by protein translation technology in vivo, but comprise the non-natural amino acid polypeptides that forms by any technology (comprising only for instance) (for example referring to " expression of alternative system " by name joint) herein through marking protein joint, chemosynthesis, based on the technology of ribozyme.
The ability wide spread that alpha-non-natural amino acid is incorporated in the recombinant protein can be implemented to translate the chemistry of derivatization afterwards, and wherein said derivatization is in vivo or in vitro to take place.More specifically get on very well, utilize the reaction of dicarbapentaborane and diamines to provide some advantages with the polypeptide derivatization that on the alpha-non-natural amino acid part of polypeptide, forms heterocycle (comprising nitrogen heterocyclic ring) key.At first, naturally occurring amino acid (a) do not contain can with the diamines radical reaction to form the dicarbapentaborane of heterocycle (comprising nitrogen heterocyclic ring) key; And (b) do not contain and to react to form two amidos of heterocycle (comprising nitrogen heterocyclic ring) key with dicarbapentaborane, and therefore will be (certain with the reagent that forms described key with the alpha-non-natural amino acid component locus specificity ground reaction of polypeptide through design, suppose that alpha-non-natural amino acid and corresponding reagent have designed to form described key), opposite with the derivatization protein mixture that uses prior art to produce thus, the ability of site selectivity derivatization protein provides single homology product.Secondly, described heterocycle (comprising nitrogen heterocyclic ring) key is stable under biotic factor, shows effective material standed for of using for treatment through the protein of described heterocycle (comprising nitrogen heterocyclic ring) key derivatization.The 3rd, can handle the stability of gained heterocycle (comprising nitrogen heterocyclic ring) key based on the identity (that is, functional group and/or structure) of the alpha-non-natural amino acid that forms heterocycle (comprising nitrogen heterocyclic ring) key.In certain embodiments, the heterocycle of non-natural amino acid polypeptides (comprising nitrogen heterocyclic ring) key has less than about 1 hour half life of decomposition, in other embodiments less than about 1 day, in other embodiments less than about 2 days, in other embodiments less than about 1 week and in other embodiments greater than about 1 week.In other embodiments, gained heterocycle (comprising nitrogen heterocyclic ring) can be stablized at least 2 weeks approximately under the acid condition of gentleness, and in other embodiments, gained heterocycle (comprising nitrogen heterocyclic ring) key can be stablized under the acid condition of gentleness at least 5 days approximately.In other embodiments, non-natural amino acid polypeptides is between under the pH value between about 2 and about 8, in other embodiments under about 2 to about 6 the pH value, can stablize under about 2 to about 4 pH value at least 1 day approximately in other embodiments.In other embodiments, use strategy as herein described, method, composition and technology, one of ordinary skill in the art can synthesize half life of decomposition through regulating (for example to satisfy one of ordinary skill in the art's needs, be used for such as the therapeutical uses that continues release, or diagnostic uses, or industrial use or military use) heterocycle (the comprising nitrogen heterocyclic ring) key of non-natural amino acid polypeptides.
Above-mentioned non-natural amino acid polypeptides can be used for (including but not limited to) novel therapeutic agents, diagnosticum, catalyzing enzyme, industrial enzyme, conjugated protein (including but not limited to antibody and antibody fragment) and (including but not limited to) protein structure and functional study.For example referring to Dougherty, (2000) Unnatural Amino Acids as Probes of ProteinStructure and Function, Current Opinion in Chemical Biology.4:645-652.Only for instance, other purposes of above-mentioned non-natural amino acid polypeptides comprises that purposes, cosmetics, botany, environment, energy based on calibrating produce and/or military use.Yet above-mentioned non-natural amino acid polypeptides can experience further modification, thereby incorporates novel or modified functional group into, comprising: the therapeutic efficiency of controlling polypeptide; Improve the security features of polypeptide; Regulate pharmacokinetics, pharmacology and/or the pharmacodynamics (for example, increase water-soluble, biological usability, increase serum half-life, increase the treatment half life period, regulate immunogenicity, regulate biologically active or prolong circulation timei) of polypeptide; For polypeptide provides other functional group; Label, mark or detectable signal are incorporated in the polypeptide; The stalling characteristic of convenient polypeptide; Any combination with above-mentioned change.
Some embodiment is the stalling characteristic method easily that makes polypeptide, and it comprises to utilize and comprises the homology non-natural amino acid polypeptides that at least one is selected from the alpha-non-natural amino acid of the group that is made up of following each thing: contain the carbonyl alpha-non-natural amino acid, contain the dicarbapentaborane alpha-non-natural amino acid, contain the diamines alpha-non-natural amino acid, contain the ketoamine alpha-non-natural amino acid and contain ketone alkynes alpha-non-natural amino acid.In other embodiments, by biological synthesis method described alpha-non-natural amino acid is incorporated in the polypeptide as described herein.In other or alternate embodiment, described non-natural amino acid polypeptides comprises at least one amino acid whose alpha-non-natural amino acid that is selected from formula I-LXVII.
Method as herein described, composition, strategy and technology are not limited to particular type, kind or the family of polypeptide.In fact, almost any polypeptide all can comprise at least one alpha-non-natural amino acid as herein described.Only for instance, polypeptide can with the therapeutic protein homology that is selected from the group that forms by required polypeptide.Non-natural amino acid polypeptides also can with any polypeptide member homology of somatotropin supergene family.
Described modification comprises to be incorporated other functional group on the alpha-non-natural amino acid component of polypeptide into, and described other functional group includes but not limited to required functional group.
Non-natural amino acid polypeptides as herein described can contain the part that can change into other functional group, and wherein said part includes but not limited to carbonyl, dicarbapentaborane, diamines, ketoamine or ketone alkynes.Described non-natural amino acid polypeptides can be used for or incorporate into be used for preparing, any method, composition, technology and the strategy of purifying, sign and use alpha-non-natural amino acid as herein described, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides.Can use technology as described herein or use as for example March, Advanced Organic Chemistry the 5th edition, (Wiley 2001); And Carey and Sundberg, Advanced Organic Chemistry the 4th edition, (Plenum 2000 for A and B volume, 2001) technology described in (all documents all quote in full mode be incorporated herein) is chemically converted into other functional group (heterocyclic moiety only for instance) with described part.
Therefore, only for instance, can use method and composition as herein described further to modify to contain in the following amino acid any non-natural amino acid polypeptides:
Figure A200680049995D01621
Wherein:
A is optional, and when existing, it is the low-carbon (LC) alkylidene, be substituted low-carbon (LC) alkylidene, low-carbon (LC) cycloalkylidene, be substituted the low-carbon (LC) cycloalkylidene, the low-carbon (LC) alkenylene, be substituted low-carbon (LC) alkenylene, alkynylene, the inferior assorted alkyl of low-carbon (LC), be substituted inferior assorted alkyl, the inferior Heterocyclylalkyl of low-carbon (LC), be substituted low-carbon (LC) inferior Heterocyclylalkyl, arlydene, be substituted arlydene, inferior heteroaryl, be substituted inferior heteroaryl, alkarylene, be substituted alkarylene, inferior aralkyl or be substituted inferior aralkyl;
B is optional, and when existing, its at one end with contain the connection base that two amine moieties are connected, described connection base is selected from the group that is made up of following each group: the low-carbon (LC) alkylidene, be substituted low-carbon (LC) alkylidene, low-carbon (LC) alkenylene, be substituted the low-carbon (LC) alkenylene, the inferior assorted alkyl of low-carbon (LC), be substituted the low-carbon (LC) Asia mix alkyl ,-O-(alkylidene or be substituted alkylidene)-,-S-(alkylidene or be substituted alkylidene)-,-C (O) R " ,-S (O) k(alkylidene or be substituted alkylidene)-(wherein k is 1,2 or 3) ,-C (O)-(alkylidene or be substituted alkylidene)-,-C (S)-(alkylidene or be substituted alkylidene)-,-NR " (alkylidene or be substituted alkylidene)-,-CON (R ")-(alkylidene or be substituted alkylidene)-,-CSN (R ")-(alkylidene or be substituted alkylidene)-and-N (R ") CO-(alkylidene or be substituted alkylidene)-, and each R wherein is " independently for H, alkyl or be substituted alkyl;
J is
Figure A200680049995D01622
Or Wherein:
R 8Be independently selected from H, alkyl, be substituted alkyl, cycloalkyl, be substituted cycloalkyl or amine protecting group;
R 9Be independently selected from H, alkyl, be substituted alkyl, cycloalkyl, be substituted cycloalkyl or amine protecting group;
T 1Be bond, the C that is substituted according to circumstances 1-C 4Alkylidene, the C that is substituted according to circumstances 1-C 4Alkenylene or the assorted alkyl that is substituted according to circumstances;
T 2Be the C that is substituted according to circumstances 1-C 4Alkylidene, the C that is substituted according to circumstances 1-C 4Alkenylene, the assorted alkyl that is substituted according to circumstances, the aryl that is substituted according to circumstances or the heteroaryl that is substituted according to circumstances;
Wherein each optional substituting group is independently selected from low-carbon alkyl, is substituted low-carbon alkyl, low-carbon naphthenic, is substituted low-carbon naphthenic, low-carbon (LC) thiazolinyl, is substituted the low-carbon (LC) thiazolinyl, alkynyl, the assorted alkyl of low-carbon (LC), is substituted assorted alkyl, low-carbon (LC) Heterocyclylalkyl, is substituted the low-carbon (LC) Heterocyclylalkyl, aryl, is substituted aryl, heteroaryl, is substituted heteroaryl, alkaryl, is substituted alkaryl, aralkyl or is substituted 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;
R 3And R 4Be H, halogen, low-carbon alkyl or be substituted low-carbon alkyl, perhaps R independently of one another 3And R 4Or two R 3Group forms cycloalkyl or Heterocyclylalkyl according to circumstances;
Or-the A-B-J-R group form together comprise at least one two amido, through protecting two amidos or dicyclo or tricyclic naphthenes base or Heterocyclylalkyl through covering two amidos;
Or-the B-J-R group form together comprise at least one two amido, through protecting two amidos or dicyclo or tricyclic naphthenes base or cyclophane base or Heterocyclylalkyl through covering two amidos;
Or-the J-R group form together comprise at least one two amido, through protecting two amidos or monocycle or bicyclic cycloalkyl or Heterocyclylalkyl through covering two amidos;
Wherein-last at least one amido of A-B-J-R is the amine through protection according to circumstances;
Figure A200680049995D01631
Wherein:
A is optional, and when existing, it is the low-carbon (LC) alkylidene, be substituted low-carbon (LC) alkylidene, low-carbon (LC) cycloalkylidene, be substituted the low-carbon (LC) cycloalkylidene, the low-carbon (LC) alkenylene, be substituted low-carbon (LC) alkenylene, alkynylene, the inferior assorted alkyl of low-carbon (LC), be substituted inferior assorted alkyl, the inferior Heterocyclylalkyl of low-carbon (LC), be substituted low-carbon (LC) inferior Heterocyclylalkyl, arlydene, be substituted arlydene, inferior heteroaryl, be substituted inferior heteroaryl, alkarylene, be substituted alkarylene, inferior aralkyl or be substituted inferior aralkyl;
B is optional, and when existing, its at one end with contain the connection base that two amine moieties are connected, described connection base is selected from the group that is made up of following each group: the low-carbon (LC) alkylidene, be substituted low-carbon (LC) alkylidene, low-carbon (LC) alkenylene, be substituted the low-carbon (LC) alkenylene, the inferior assorted alkyl of low-carbon (LC), be substituted the low-carbon (LC) Asia mix alkyl ,-O-(alkylidene or be substituted alkylidene)-,-S-(alkylidene or be substituted alkylidene)-,-C (O) R " ,-S (O) k(alkylidene or be substituted alkylidene)-(wherein k is 1,2 or 3) ,-C (O)-(alkylidene or be substituted alkylidene)-,-C (S)-(alkylidene or be substituted alkylidene)-,-NR " (alkylidene or be substituted alkylidene)-,-CON (R ")-(alkylidene or be substituted alkylidene)-,-CSN (R ")-(alkylidene or be substituted alkylidene)-and-N (R ") CO-(alkylidene or be substituted alkylidene)-, and each R wherein is " independently for H, alkyl or be substituted alkyl;
K is
Figure A200680049995D01641
Figure A200680049995D01642
Or
Figure A200680049995D01643
Wherein
T 1Be bond, the C that is substituted according to circumstances 1-C 4Alkylidene, the C that is substituted according to circumstances 1-C 4Alkenylene or the assorted alkyl that is substituted according to circumstances;
Wherein each optional substituting group is independently selected from the low-carbon (LC) alkylidene, be substituted the low-carbon (LC) alkylidene, the low-carbon (LC) cycloalkylidene, be substituted the low-carbon (LC) cycloalkylidene, the low-carbon (LC) alkenylene, be substituted the low-carbon (LC) alkenylene, alkynylene, the inferior assorted alkyl of low-carbon (LC), be substituted inferior assorted alkyl, the inferior Heterocyclylalkyl of low-carbon (LC), be substituted the inferior Heterocyclylalkyl of low-carbon (LC), arlydene, be substituted arlydene, inferior heteroaryl, be substituted inferior heteroaryl, alkylidene aryl, be substituted alkylidene aryl, inferior aralkyl or be substituted inferior aralkyl;
T 2Be selected from the group that forms by following each group: the low-carbon (LC) alkylidene, be substituted low-carbon (LC) alkylidene, low-carbon (LC) alkenylene, be substituted the low-carbon (LC) alkenylene, the inferior assorted alkyl of low-carbon (LC), be substituted the inferior assorted alkyl of low-carbon (LC) ,-O-,-O-(alkylidene or be substituted alkylidene)-,-S-,-S-(alkylidene or be substituted alkylidene)-,-S (O) k-(wherein k is 1,2 or 3) ,-S (O) k(alkylidene or be substituted alkylidene)-,-C (O)-,-C (O)-(alkylidene or be substituted alkylidene)-,-C (S)-,-C (S)-(alkylidene or be substituted alkylidene)-,-N (R ')-,-NR '-(alkylidene or be substituted alkylidene)-,-C (O) N (R ')-,-CON (R ')-(alkylidene or be substituted alkylidene)-,-CSN (R ')-,-CSN (R ')-(alkylidene or be substituted alkylidene)-,-N (R ') CO-(alkylidene or be substituted alkylidene)-,-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 ')-;
T 3For Or
Figure A200680049995D01645
Each X wherein 1Be independently selected from the group that forms by following each group :-O-,-S-,-N (H)-,-N (R)-,-N (Ac)-and-N (OMe)-; X 2For-OR ,-OAc ,-SR ,-N (R) 2,-N (R) (Ac) ,-N (R) (OMe) or N 3, and wherein each R ' is H, alkyl independently or is substituted alkyl;
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;
Or-the A-B-K-R group form together comprise at least one carbonyl (comprising dicarbapentaborane), through protection carbonyl (comprise through protection dicarbapentaborane) or through covering dicyclo or the tricyclic naphthenes base or the Heterocyclylalkyl of carbonyl (comprising) through covering dicarbapentaborane;
Or-the K-R group form together comprise at least one carbonyl (comprising dicarbapentaborane), through protection carbonyl (comprise through protection dicarbapentaborane) or through covering monocycle or the bicyclic cycloalkyl or the Heterocyclylalkyl of carbonyl (comprising) through covering dicarbapentaborane;
Figure A200680049995D01651
Wherein:
A is optional, and when existing, it is the low-carbon (LC) alkylidene, be substituted low-carbon (LC) alkylidene, low-carbon (LC) cycloalkylidene, be substituted the low-carbon (LC) cycloalkylidene, the low-carbon (LC) alkenylene, be substituted low-carbon (LC) alkenylene, alkynylene, the inferior assorted alkyl of low-carbon (LC), be substituted inferior assorted alkyl, the inferior Heterocyclylalkyl of low-carbon (LC), be substituted low-carbon (LC) inferior Heterocyclylalkyl, arlydene, be substituted arlydene, inferior heteroaryl, be substituted inferior heteroaryl, alkarylene, be substituted alkarylene, inferior aralkyl or be substituted inferior aralkyl;
B is optional, and when existing, its at one end with contain the connection base that two amine moieties are connected, described connection base is selected from the group that is made up of following each group: the low-carbon (LC) alkylidene, be substituted low-carbon (LC) alkylidene, low-carbon (LC) alkenylene, be substituted the low-carbon (LC) alkenylene, the inferior assorted alkyl of low-carbon (LC), be substituted the low-carbon (LC) Asia mix alkyl ,-O-(alkylidene or be substituted alkylidene)-,-S-(alkylidene or be substituted alkylidene)-,-C (O) R " ,-S (O) k(alkylidene or be substituted alkylidene)-(wherein k is 1,2 or 3) ,-C (O)-(alkylidene or be substituted alkylidene)-,-C (S)-(alkylidene or be substituted alkylidene)-,-NR " (alkylidene or be substituted alkylidene)-,-CON (R ")-(alkylidene or be substituted alkylidene)-,-CSN (R ")-(alkylidene or be substituted alkylidene)-and-N (R ") CO-(alkylidene or be substituted alkylidene)-, and each R wherein is " independently for H, alkyl or be substituted alkyl;
M 2For
Figure A200680049995D01652
Figure A200680049995D01653
, or
Figure A200680049995D01654
Wherein (a) expression and the bond of B group, and (b) bond of expression and each carbonyl;
T 3For bond, C (R) (R), O or S;
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 3And R 4Be selected from H, halogen, alkyl independently of one another, be substituted alkyl, cycloalkyl or be substituted cycloalkyl, perhaps R 3And R 4Or two R 3Group or two R 4Group forms cycloalkyl or Heterocyclylalkyl according to circumstances;
Figure A200680049995D01661
Wherein:
B is optional, and when existing, it is the connection base that is selected from the group that is made up of following each group: the low-carbon (LC) alkylidene, be substituted low-carbon (LC) alkylidene, low-carbon (LC) alkenylene, be substituted the low-carbon (LC) alkenylene, the inferior assorted alkyl of low-carbon (LC), be substituted the inferior assorted alkyl of low-carbon (LC) ,-O-,-O-(alkylidene or be substituted alkylidene)-,-S-,-S-(alkylidene or be substituted alkylidene)-,-C (O) R " ,-S (O) k-(wherein k is 1,2 or 3) ,-S (O) k(alkylidene or be substituted alkylidene)-,-C (O)-,-NS (O) 2-,-OS (O) 2-,-C (O)-(alkylidene or be substituted alkylidene)-,-C (S)-,-C (S)-(alkylidene or be substituted alkylidene)-,-N (R ')-,-NR '-(alkylidene or be substituted alkylidene)-,-C (O) N (R ')-,-CON (R ')-(alkylidene or be substituted alkylidene)-,-CSN (R ')-,-CSN (R ')-(alkylidene or be substituted alkylidene)-,-N (R ') CO-,-N (R ') CO-(alkylidene or be substituted alkylidene)-,-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 ')-,-C (R ')=N-,-C (R ')=N-N (R ')-,-C (R ') 2-N=N-and-C (R ') 2-N (R ')-N (R ')-, wherein each R ' is H, alkyl independently or is substituted alkyl;
M 1For bond ,-C (R 3) (R 4)-,-O-,-S-,-C (R 3) (R 4)-C (R 3) (R 4)-,-C (R 3) (R 4)-O-,-C (R 3) (R 4)-S-,-O-C (R 3) (R 4)-,-S-C (R 3) (R 4) ,-C (R 3)=C (R 3)-or-C (R 4)=C (R 4)-;
T 3For bond, C (R) (R), O or S;
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 3And R 4Be independently selected from H, halogen, alkyl, be substituted alkyl, cycloalkyl or be substituted cycloalkyl, perhaps R 3And R 4Or two R 3Group or two R 4Group forms cycloalkyl or Heterocyclylalkyl according to circumstances;
Each R aBe independently selected from the group that forms by following each group: H, halogen, alkyl, 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 each R ' is H, alkyl independently or is substituted alkyl; And n is 0 to 8; And
Figure A200680049995D01671
Wherein:
A is optional, and when existing, it is the low-carbon (LC) alkylidene, be substituted low-carbon (LC) alkylidene, low-carbon (LC) cycloalkylidene, be substituted the low-carbon (LC) cycloalkylidene, the low-carbon (LC) alkenylene, be substituted low-carbon (LC) alkenylene, alkynylene, the inferior assorted alkyl of low-carbon (LC), be substituted inferior assorted alkyl, the inferior Heterocyclylalkyl of low-carbon (LC), be substituted low-carbon (LC) inferior Heterocyclylalkyl, arlydene, be substituted arlydene, inferior heteroaryl, be substituted inferior heteroaryl, alkarylene, be substituted alkarylene, inferior aralkyl or be substituted inferior aralkyl;
B is optional, and when existing, its at one end with contain the connection base that two amine moieties are connected, described connection base is selected from the group that is made up of following each group: the low-carbon (LC) alkylidene, be substituted low-carbon (LC) alkylidene, low-carbon (LC) alkenylene, be substituted the low-carbon (LC) alkenylene, the inferior assorted alkyl of low-carbon (LC), be substituted the low-carbon (LC) Asia mix alkyl ,-O-(alkylidene or be substituted alkylidene)-,-S-(alkylidene or be substituted alkylidene)-,-C (O) R " ,-S (O) k(alkylidene or be substituted alkylidene)-(wherein k is 1,2 or 3) ,-C (O)-(alkylidene or be substituted alkylidene)-,-C (S)-(alkylidene or be substituted alkylidene)-,-NR " (alkylidene or be substituted alkylidene)-,-CON (R ")-(alkylidene or be substituted alkylidene)-,-CSN (R ")-(alkylidene or be substituted alkylidene)-and-N (R ") CO-(alkylidene or be substituted alkylidene)-, and each R wherein is " independently for H, alkyl or be substituted alkyl;
G is
Figure A200680049995D01672
Or
Figure A200680049995D01673
T 4For the carbonyl-protection base, include but not limited to
Figure A200680049995D01674
Figure A200680049995D01675
Or
Figure A200680049995D01676
Each X wherein 1Be independently selected from the group that forms by following each group :-O-,-S-,-N (H)-,-N (R)-,-N (Ac)-and-N (OMe)-; X 2For-OR ,-OAc ,-SR ,-N (R) 2,-N (R) (Ac) ,-N (R) (OMe) or N 3, and wherein each R ' is H, alkyl independently or is substituted alkyl;
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 3And R 4Be H, halogen, low-carbon alkyl or be substituted low-carbon alkyl, perhaps R independently of one another 3And R 4Or two R 3Group forms cycloalkyl or Heterocyclylalkyl according to circumstances;
Figure A200680049995D01681
Wherein:
A is optional, and when existing, it is the low-carbon (LC) alkylidene, be substituted low-carbon (LC) alkylidene, low-carbon (LC) cycloalkylidene, be substituted the low-carbon (LC) cycloalkylidene, the low-carbon (LC) alkenylene, be substituted low-carbon (LC) alkenylene, alkynylene, the inferior assorted alkyl of low-carbon (LC), be substituted inferior assorted alkyl, the inferior Heterocyclylalkyl of low-carbon (LC), be substituted low-carbon (LC) inferior Heterocyclylalkyl, arlydene, be substituted arlydene, inferior heteroaryl, be substituted inferior heteroaryl, alkarylene, be substituted alkarylene, inferior aralkyl or be substituted inferior aralkyl;
B is optional, and when existing, its at one end with contain the connection base that two amine moieties are connected, described connection base is selected from the group that is made up of following each group: the low-carbon (LC) alkylidene, be substituted low-carbon (LC) alkylidene, low-carbon (LC) alkenylene, be substituted the low-carbon (LC) alkenylene, the inferior assorted alkyl of low-carbon (LC), be substituted the low-carbon (LC) Asia mix alkyl ,-O-(alkylidene or be substituted alkylidene)-,-S-(alkylidene or be substituted alkylidene)-,-C (O) R " ,-S (O) k(alkylidene or be substituted alkylidene)-(wherein k is 1,2 or 3) ,-C (O)-(alkylidene or be substituted alkylidene)-,-C (S)-(alkylidene or be substituted alkylidene)-,-NR " (alkylidene or be substituted alkylidene)-,-CON (R ")-(alkylidene or be substituted alkylidene)-,-CSN (R ")-(alkylidene or be substituted alkylidene)-and-N (R ") CO-(alkylidene or be substituted alkylidene)-, and each R wherein is " independently for H, alkyl or be substituted alkyl;
G is
Figure A200680049995D01682
Or
T 1Be the C that is substituted according to circumstances 1-C 4Alkylidene, the C that is substituted according to circumstances 1-C 4Alkenylene or the assorted alkyl that is substituted according to circumstances;
T 4For the carbonyl-protection base, include but not limited to
Figure A200680049995D01691
Figure A200680049995D01692
Or
Figure A200680049995D01693
Each X wherein 1Be independently selected from the group that forms by following each group :-O-,-S-,-N (H)-,-N (R)-,-N (Ac)-and-N (OMe)-; X 2For-OR ,-OAc ,-SR ,-N (R) 2,-N (R) (Ac) ,-N (R) (OMe) or N 3, and wherein each R ' is H, alkyl independently or is substituted alkyl;
R is H, halogen, alkyl, is substituted alkyl, cycloalkyl or is substituted cycloalkyl;
Each R ' is H, alkyl independently 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 3And R 4Be H, halogen, low-carbon alkyl or be substituted low-carbon alkyl, perhaps R independently of one another 3And R 4Or two R 3Group forms cycloalkyl or Heterocyclylalkyl according to circumstances.
An aspect of method and composition as herein described is the composition that comprises the polypeptide of at least a alpha-non-natural amino acid with at least one (including but not limited 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 more) posttranslational modification.Alpha-non-natural amino acid through posttranslational modification can be identical or different, include but not limited to can be in protein 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20 or more different loci place comprise 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20 or how different alpha-non-natural amino acids through posttranslational modification.On the other hand, composition comprises that existing at least one (but being less than all) specific amino acids is through the polypeptide of the alpha-non-natural amino acid replacement of posttranslational modification in the polypeptide.Appointment polypeptide for alpha-non-natural amino acid with an above posttranslational modification, the alpha-non-natural amino acid of posttranslational modification can identical or different (include but not limited to that polypeptide can comprise the alpha-non-natural amino acid of the posttranslational modification that two or more are dissimilar, perhaps can comprise the alpha-non-natural amino acid of two identical posttranslational modifications).Appointment polypeptide for alpha-non-natural amino acid with two above posttranslational modifications, the alpha-non-natural amino acid of posttranslational modification can be identical or different, or be the combination of the alpha-non-natural amino acid of the alpha-non-natural amino acid of a plurality of posttranslational modifications of the identical category posttranslational modification different with at least one.
The method of posttranslational modification non-natural amino acid polypeptides
Figure 14 and Figure 17 are for using the illustrative embodiment of modifying non-natural amino acid polypeptides behind method as herein described and the Technical Translator.These and other posttranslational modification is described in hereinafter.
A. the method for posttranslational modification non-natural amino acid polypeptides: contain dicarbapentaborane alpha-non-natural amino acid and the reaction that contains diamines reagent
Naturally occurring amino acid whose side chain lacks high electrophilicity site.Therefore, incorporate into have contain the electrophilic group side chain alpha-non-natural amino acid (only for instance, comprise the amino acid that contains dicarbapentaborane, described dicarbapentaborane such as diketone, keto-aldehyde, ketone ester, ketone acid or ketone thioesters) make and may make described side chain locus specificity derivatization via at least a in the described carbonyl of nucleophillic attack.At aggressive nucleophilic group is under the situation of diamines, and the protein with producing through heterocyclic derivativesization comprises the protein through the nitrogen heterocyclic ring derivatization.Can utilize before the derivatization step or the polypeptide of purifying carries out derivatization and/or further modifies after the derivatization step method.In addition, the method that can utilize synthetic polymer purified before or after described modification, polysaccharide or polynucleotide to carry out derivatization and/or further modify.In addition, the derivatization step can take place under the condition of slight alkalescence in appropriateness acidity, for example comprise the pH value between between about 2 to about 8, the pH value between between about 4 to about 8, the pH value between between about 3 to about 8 or the pH value between about 2 to about 9, or the pH value is between about 4 to about 9, or the pH value is between about 4 to about 10.
The protein derived method that is based upon on the basis that contains dicarbapentaborane protein and the reaction of the molecule that replaces through diamines has unique advantage.The first, diamines in about 5 to about 8 pH value scope (and in other embodiments in about 4 to about 10 pH value scope; And in other embodiments in about 3 to about 8 pH value scope; Or in other embodiments in about 2 to about 9 pH value scope; Or in other embodiments in about 4 to about 9 pH value scope) and contain the condensation of dicarbonyl compound experience to produce heterocycle (comprising nitrogen heterocyclic ring) key.Under these conditions, naturally occurring amino acid whose side chain is not had a reactivity.The second, described selective chemical makes locus specificity derivatization recombinant protein become possibility: derivatization protein now can be prepared as the homology product of appointment.The 3rd, realize that the required temperate condition of diamines as herein described and the reaction that contains the dicarbapentaborane polypeptide as herein described can reversibly destroy the tertiary structure of polypeptide (being to destroy the situation of described tertiary structure except that the reaction purpose certainly) usually.The 4th, reaction at room temperature takes place rapidly, and this allows to use will be at the polypeptide or the reagent of unsettled many types under the higher temperature.The 5th, reaction is easy to take place under aqueous conditions, and this also allows to use the polypeptide and the reagent of incompatible with non-aqueous solution (on any degree).The 6th, even when the ratio of polypeptide or amino acid and reagent is stoichiometry, near-stoichiometric or class stoichiometry, reaction also is easy to take place, thereby does not need to add the product that excessive reagent or polypeptide obtain consumption.The 7th, the design of diamines and dicarbapentaborane part in the visual reactant and regioselectivity and/or regiospecificity ground generation gained heterocycle.At last, diamines is created in heterocycle stable under the biotic factor (comprising nitrogen heterocyclic ring) key with the condensation that contains the dicarbapentaborane molecule.
Only for instance, following alpha-non-natural amino acid for can with as herein described can be used for further modifying contain the dicarbapentaborane non-natural amino acid polypeptides contain the diamines reagent reacting contain the amino acid whose type of dicarbapentaborane:
Wherein:
A is optional, and when existing, it is the low-carbon (LC) alkylidene, be substituted low-carbon (LC) alkylidene, low-carbon (LC) cycloalkylidene, be substituted the low-carbon (LC) cycloalkylidene, the low-carbon (LC) alkenylene, be substituted low-carbon (LC) alkenylene, alkynylene, the inferior assorted alkyl of low-carbon (LC), be substituted inferior assorted alkyl, the inferior Heterocyclylalkyl of low-carbon (LC), be substituted low-carbon (LC) inferior Heterocyclylalkyl, arlydene, be substituted arlydene, inferior heteroaryl, be substituted inferior heteroaryl, alkarylene, be substituted alkarylene, inferior aralkyl or be substituted inferior aralkyl;
B is optional, and when existing, its at one end with contain the connection base that two amine moieties are connected, described connection base is selected from the group that is made up of following each group: the low-carbon (LC) alkylidene, be substituted low-carbon (LC) alkylidene, low-carbon (LC) alkenylene, be substituted the low-carbon (LC) alkenylene, the inferior assorted alkyl of low-carbon (LC), be substituted the low-carbon (LC) Asia mix alkyl ,-O-(alkylidene or be substituted alkylidene)-,-S-(alkylidene or be substituted alkylidene)-,-C (O) R " ,-S (O) k(alkylidene or be substituted alkylidene)-(wherein k is 1,2 or 3) ,-C (O)-(alkylidene or be substituted alkylidene)-,-C (S)-(alkylidene or be substituted alkylidene)-,-NR " (alkylidene or be substituted alkylidene)-,-CON (R ")-(alkylidene or be substituted alkylidene)-,-CSN (R ")-(alkylidene or be substituted alkylidene)-and-N (R ") CO-(alkylidene or be substituted alkylidene)-, and each R wherein is " independently for H, alkyl or be substituted alkyl;
K is
Figure A200680049995D01712
Figure A200680049995D01713
Or Wherein:
T 1Be bond, the C that is substituted according to circumstances 1-C 4Alkylidene, the C that is substituted according to circumstances 1-C 4Alkenylene or the assorted alkyl that is substituted according to circumstances;
Wherein each optional substituting group is independently selected from the low-carbon (LC) alkylidene, be substituted the low-carbon (LC) alkylidene, the low-carbon (LC) cycloalkylidene, be substituted the low-carbon (LC) cycloalkylidene, the low-carbon (LC) alkenylene, be substituted the low-carbon (LC) alkenylene, alkynylene, the inferior assorted alkyl of low-carbon (LC), be substituted inferior assorted alkyl, the inferior Heterocyclylalkyl of low-carbon (LC), be substituted the inferior Heterocyclylalkyl of low-carbon (LC), arlydene, be substituted arlydene, inferior heteroaryl, be substituted inferior heteroaryl, alkylidene aryl, be substituted alkylidene aryl, inferior aralkyl or be substituted inferior aralkyl;
T 2Be selected from the group that forms by following each group: the low-carbon (LC) alkylidene, be substituted low-carbon (LC) alkylidene, low-carbon (LC) alkenylene, be substituted the low-carbon (LC) alkenylene, the inferior assorted alkyl of low-carbon (LC), be substituted the inferior assorted alkyl of low-carbon (LC) ,-O-,-O-(alkylidene or be substituted alkylidene)-,-S-,-S-(alkylidene or be substituted alkylidene)-,-S (O) k-(wherein k is 1,2 or 3),-S (O) k (alkylidene or be substituted alkylidene)-,-C (O)-,-C (O)-(alkylidene or be substituted alkylidene)-,-C (S)-,-C (S)-(alkylidene or be substituted alkylidene)-,-N (R ')-,-NR '-(alkylidene or be substituted alkylidene)-,-C (O) N (R ')-,-CON (R ')-(alkylidene or be substituted alkylidene)-,-CSN (R ')-,-CSN (R ')-(alkylidene or be substituted alkylidene)-,-N (R ') CO-(alkylidene or be substituted alkylidene)-,-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 each R ' is independently selected from H, alkyl or is substituted alkyl;
T 3For
Figure A200680049995D01721
Or
Figure A200680049995D01722
Each X wherein 1Be independently selected from the group that forms by following each group :-O-,-S-,-N (H)-,-N (R)-,-N (Ac)-and-N (OMe)-; X 2For-OR ,-OAc ,-SR ,-N (R) 2,-N (R) (Ac) ,-N (R) (OMe) or N 3, and wherein each R ' is H, alkyl independently or is substituted alkyl;
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;
Or-the A-B-K-R group form together comprise at least one carbonyl (comprising dicarbapentaborane), through protection carbonyl (comprise through protection dicarbapentaborane) or through covering dicyclo or the tricyclic naphthenes base or the Heterocyclylalkyl of carbonyl (comprising) through covering dicarbapentaborane;
Or-the K-R group form together comprise at least one carbonyl (comprising dicarbapentaborane), through protection carbonyl (comprise through protection dicarbapentaborane) or through covering monocycle or the bicyclic cycloalkyl or the Heterocyclylalkyl of carbonyl (comprising) through covering dicarbapentaborane.
In fact, the type that comprises the described polypeptide that contains the dicarbapentaborane alpha-non-natural amino acid is unrestricted, be arranged in polypeptide as long as contain the dicarbapentaborane alpha-non-natural amino acid, thereby make the diamines reagent can be with dicarbapentaborane reaction and do not produce the modified alpha-non-natural amino acid of gained that destroys polypeptide tertiary structure (certainly, except that described destruction is the situation of purpose of reaction) and get final product.
Only for instance, below contain diamines reagent for containing the reaction of dicarbapentaborane alpha-non-natural amino acid and can be used for further modifying the type that contains diamines reagent that contains the dicarbapentaborane non-natural amino acid polypeptides with as herein described:
Wherein:
Each X independently for H, alkyl, be substituted alkyl, thiazolinyl, be substituted thiazolinyl, alkynyl, be substituted alkynyl, alkoxyl, be substituted alkoxyl, alkyl alkoxy, be substituted alkyl alkoxy, polyalkylene oxide, be substituted polyalkylene oxide, aryl, be substituted aryl, heteroaryl, be substituted heteroaryl, alkaryl, be substituted alkaryl, aralkyl, be substituted aralkyl ,-(alkylidene or be substituted alkylidene)-ON (R ") 2,-(alkylidene or be substituted alkylidene)-C (O) SR " ,-(alkylidene or be substituted alkylidene)-S-S-(aryl or be substituted aryl) ,-C (O) R " ,-C (O) 2R " or-C (O) N (R ") 2, each R wherein " independently for hydrogen, alkyl, be substituted alkyl, thiazolinyl, be substituted thiazolinyl, alkoxyl, be substituted alkoxyl, aryl, be substituted aryl, heteroaryl, alkaryl, be substituted alkaryl, aralkyl or be substituted aralkyl;
Or each X is independently selected from the group that is made up of required functional group;
Each L is independently selected from the group that is made up of following each group: alkylidene, be substituted alkylidene, alkenylene, be substituted alkenylene ,-O-,-O-(alkylidene or be substituted alkylidene)-,-S-,-S-(alkylidene or be substituted alkylidene)-,-S (O) k-(wherein k is 1,2 or 3) ,-S (O) k(alkylidene or be substituted alkylidene)-,-C (O)-,-C (O)-(alkylidene or be substituted alkylidene)-,-C (S)-,-C (S)-(alkylidene or be substituted alkylidene)-,-N (R ')-,-NR '-(alkylidene or be substituted alkylidene)-,-C (O) N (R ')-,-CON (R ')-(alkylidene or be substituted alkylidene)-,-(alkylidene or be substituted alkylidene) NR ' C (O) O-(alkylidene or be substituted alkylidene)-,-O-CON (R ')-(alkylidene or be substituted alkylidene)-,-CSN (R ')-,-CSN (R ')-(alkylidene or be substituted alkylidene)-,-N (R ') CO-(alkylidene or be substituted alkylidene)-,-N (R ') C (O) O-,-N (R ") C (O) O-(alkylidene or be substituted alkylidene)-;-S (O) kN (R ')-,-N (R ') C (O) N (R ')-,-N (R ') C (O) N (R ')-(alkylidene or be substituted alkylidene)-,-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, L 1For-C (R ') P-NR '-C (O) O-(alkylidene or be substituted alkylidene)-, wherein p is 0,1 or 2;
Each R ' is H, alkyl independently, is substituted alkyl or amino protecting group;
W is Or
Figure A200680049995D01733
Z 2And Z 3Be independently selected from the group that forms by following each group: bond, the C that is substituted according to circumstances 1-C 4Alkylidene, the C that is substituted according to circumstances 1-C 4Alkenylene, the assorted alkyl that is substituted according to circumstances ,-O-,-S-,-C (O)-,-C (S)-and-N (R ')-; And
N is 1 to 3.
The compound of some embodiment of formula (LVVIII) compound for having formula (LXIX) structure:
Figure A200680049995D01741
Some embodiment of formula (LXIX) compound is the compound that is selected from the group that is made up of following each thing:
Figure A200680049995D01742
With
Figure A200680049995D01744
In other embodiments, described m-PEG or PEG group have the molecular weight that arrives in about 30 kDa scopes about 5.In other embodiments, described m-PEG or PEG group have the molecular weight that arrives in about 50 kDa scopes about 2.In other embodiments, described m-PEG or PEG group have the molecular weight for about 5 kDa.
The compound of some embodiment of formula (LXIX) compound for having formula (LXX) structure:
Figure A200680049995D01745
The compound of some embodiment of formula (LXIX) compound for having formula (LXXI) structure:
Figure A200680049995D01751
In some embodiment of formula (XXII) compound, described m-PEG group has the molecular weight that arrives in about 30kDa scope about 5.In other embodiments, described m-PEG or PEG group have the molecular weight that arrives in about 50kDa scope about 2.In other embodiments, described m-PEG or PEG group have the molecular weight for about 5kDa.
The compound of some embodiment of formula (LXIX) compound for having formula (LXXII) structure:
The compound of some embodiment of formula (LXIX) compound for having formula (LXXIII) structure:
Figure A200680049995D01753
In some embodiment of formula (XXII) compound, described m-PEG group has the molecular weight that arrives in the 30kDa scope 5.
Some embodiment of formula (LXIX) compound is the compound with following structure:
Figure A200680049995D01761
The illustrative embodiment of the method that contains the coupling of dicarbapentaborane alpha-non-natural amino acid on diamines and the polypeptide is provided among Figure 12, Figure 15 and Figure 16.In these illustrative embodiment, the diamines derivatization reagent is added in the buffer solution (the pH value is about 2 to about 9) that contains the dicarbapentaborane non-natural amino acid polypeptides.Reaction is carried out at ambient temperature, and can be by HPLC, FPLC or size exclusion chromatography purifying gained contain the heterocycle non-natural amino acid polypeptides.
In other embodiments, the basic chemicals of a plurality of connections can react with the non-natural amino acid polypeptides locus specificity that replaces through dicarbapentaborane.In one embodiment, connection based method as herein described utilization connects the connection base (single, double or multi-functional) that basic end contains two amine functional groups at least one.Two amine derivatives connect base and produce stable heterocycle (comprising nitrogen heterocyclic ring) key with the protein condensation that replaces through dicarbapentaborane.Two and/or multi-functional connection base (be also referred to as different functionality and connect base) (for example, have one or more other connect the diamines of chemicals) allow locus specificity (for example to connect different molecular, other protein, polymer or little molecule) and non-natural amino acid polypeptides, and simple function connection base (be also referred to as with functionality and connect base) (all replacing through diamines at all ends) promotes the locus specificity dimerization or the oligomerization of non-natural amino acid polypeptides.By this being connected base strategy and in vivo translation technology combination as herein described, makes the three-dimensional structure that to specify through the protein of chemical operation.
B. the method for posttranslational modification non-natural amino acid polypeptides: contain dicarbapentaborane alpha-non-natural amino acid and the reaction that contains ketoamine reagent
Also posttranslational modification technology mentioned above and composition can be used for can with contain the ketoamine reagent reacting contain the dicarbapentaborane alpha-non-natural amino acid to produce modified heterocycle (comprise and the contain nitrogen heterocyclic ring) non-natural amino acid polypeptides that contains.
Only for instance, above described in the A joint contain the dicarbapentaborane alpha-non-natural amino acid also can with as herein described can be used for further modifying contain the dicarbapentaborane non-natural amino acid polypeptides contain the ketoamine reagent reacting.
Only for instance, below contain ketoamine reagent for containing the reaction of dicarbapentaborane alpha-non-natural amino acid and can be used for further modifying the type that contains ketoamine reagent that contains the dicarbapentaborane non-natural amino acid polypeptides with as herein described:
Figure A200680049995D01762
Wherein:
Each X independently for H, alkyl, be substituted alkyl, thiazolinyl, be substituted thiazolinyl, alkynyl, be substituted alkynyl, alkoxyl, be substituted alkoxyl, alkyl alkoxy, be substituted alkyl alkoxy, polyalkylene oxide, be substituted polyalkylene oxide, aryl, be substituted aryl, heteroaryl, be substituted heteroaryl, alkaryl, be substituted alkaryl, aralkyl, be substituted aralkyl ,-(alkylidene or be substituted alkylidene)-ON (R ") 2,-(alkylidene or be substituted alkylidene)-C (O) SR " ,-(alkylidene or be substituted alkylidene)-S-S-(aryl or be substituted aryl) ,-C (O) R " ,-C (O) 2R " or-C (O) N (R ") 2, each R wherein " independently for hydrogen, alkyl, be substituted alkyl, thiazolinyl, be substituted thiazolinyl, alkoxyl, be substituted alkoxyl, aryl, be substituted aryl, heteroaryl, alkaryl, be substituted alkaryl, aralkyl or be substituted aralkyl;
Or each X is independently selected from the group that is made up of required functional group;
Each L is independently selected from the group that is made up of following each group: alkylidene, be substituted alkylidene, alkenylene, be substituted alkenylene ,-O-,-O-(alkylidene or be substituted alkylidene)-,-S-,-S-(alkylidene or be substituted alkylidene)-,-S (O) k-(wherein k is 1,2 or 3) ,-S (O) k(alkylidene or be substituted alkylidene)-,-C (O)-,-C (O)-(alkylidene or be substituted alkylidene)-,-C (S)-,-C (S)-(alkylidene or be substituted alkylidene)-,-N (R ')-,-NR '-(alkylidene or be substituted alkylidene)-,-C (O) N (R ')-,-CON (R ')-(alkylidene or be substituted alkylidene)-,-(alkylidene or be substituted alkylidene) NR ' C (O) O-(alkylidene or be substituted alkylidene)-,-O-CON (R ')-(alkylidene or be substituted alkylidene)-,-CSN (R ')-,-CSN (R ')-(alkylidene or be substituted alkylidene)-,-N (R ') CO-(alkylidene or be substituted alkylidene)-,-N (R ') C (O) O-,-N (R ') C (O) O-(alkylidene or be substituted alkylidene)-,-S (O) kN (R ')-,-N (R ') C (O) N (R ')-,-N (R ') C (O) N (R ')-(alkylidene or be substituted alkylidene)-,-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, L 1For-C (R ') P-NR '-C (O) O-(alkylidene or be substituted alkylidene)-, wherein p is 0,1 or 2, and each R ' independently for H, alkyl, be substituted alkyl or amino protecting group;
W is
Figure A200680049995D01771
G is
Figure A200680049995D01772
Or
Figure A200680049995D01773
T 3For bond, C (R) (R), O or S, and R is H, halogen, alkyl, is substituted alkyl, cycloalkyl or is substituted cycloalkyl;
T 4For the carbonyl-protection base, include but not limited to
Figure A200680049995D01781
Figure A200680049995D01782
Or
Figure A200680049995D01783
Each X wherein 1Be independently selected from the group that forms by following each group :-O-,-S-,-N (H)-,-N (R ")-,-N (Ac)-and-N (OMe)-; X 2For-OR " ,-OAc ,-SR " ,-N (R ") 2,-N (R ") (Ac) ,-N (R ") (OMe) or N 3, and each R wherein is " independently for H, alkyl or be substituted alkyl; And
N is 1 to 3.
In certain embodiments, the basic chemicals of a plurality of connections can react with the non-natural amino acid polypeptides locus specificity that replaces through dicarbapentaborane.In one embodiment, connection based method as herein described utilization connects the connection base (single, double or multi-functional) that basic end contains ketoamine functional group at least one.The connection base of ketoamine derivatization produces stable heterocycle (comprising nitrogen heterocyclic ring) key with the reaction of the protein that replaces through dicarbapentaborane.Two and/or multi-functional connection base (be also referred to as different functionality and connect base) (for example, have one or more other connect the ketoamine of chemicals) allow locus specificity (for example to connect different molecular, other protein, polymer or little molecule) and non-natural amino acid polypeptides, and simple function connection base (be also referred to as with functionality and connect base) (all replacing through ketoamine at all ends) promotes the locus specificity dimerization or the oligomerization of non-natural amino acid polypeptides.By this being connected base strategy and in vivo translation technology combination as herein described, makes the three-dimensional structure that to specify through the protein of chemical operation.
C. the method for posttranslational modification non-natural amino acid polypeptides: contain diamines alpha-non-natural amino acid and the reaction that contains dicarbapentaborane reagent
Also posttranslational modification technology mentioned above and composition can be used for can with contain the dicarbapentaborane reagent reacting contain the diamines alpha-non-natural amino acid to produce modified heterocycle (comprise and the contain nitrogen heterocyclic ring) non-natural amino acid polypeptides that contains.
The protein derived method that is based upon on the basis that contains diamines protein and the reaction of the molecule that replaces through dicarbapentaborane has unique advantage.The first, diamines in about 4 to about 10 pH value scope (and in other embodiments in about 4 to about 10 pH value scope; And in other embodiments in about 3 to about 8 pH value scope; Or in other embodiments in about 2 to about 9 pH value scope; Or in other embodiments in about 4 to about 9 pH value scope) and contain the reaction of dicarbonyl compound experience to produce heterocycle (comprising nitrogen heterocyclic ring) key.Under these conditions, naturally occurring amino acid whose side chain is not had a reactivity.The second, described selective chemical makes locus specificity derivatization recombinant protein become possibility: derivatization protein now can be prepared as the homology product of appointment.The 3rd, realize the tertiary structure (being to destroy the situation of described tertiary structure except that the reaction purpose certainly) that the required temperate condition of dicarbapentaborane reagent and the reaction that contains the diamines polypeptide as herein described can reversibly destroy polypeptide usually that contains as herein described.The 4th, reaction at room temperature takes place rapidly, and this allows to use will be at the polypeptide or the reagent of unsettled many types under the higher temperature.The 5th, reaction is easy to take place under aqueous conditions, and this also allows to use the polypeptide and the reagent of incompatible with non-aqueous solution (on any degree).The 6th, even when the ratio of polypeptide or amino acid and reagent is stoichiometry, near-stoichiometric or class stoichiometry, reaction also is easy to take place, thereby does not need to add the product that excessive reagent or polypeptide obtain consumption.The 7th, the design of diamines and dicarbapentaborane part in the visual reactant and regioselectivity and/or regiospecificity ground generation gained heterocycle.At last, contain dicarbapentaborane reagent and contain the amino acid whose reaction of diamines and be created in heterocycle stable under the biotic factor (comprising nitrogen heterocyclic ring) key.
Only for instance, following alpha-non-natural amino acid for can with as herein described can be used for further modifying contain the diamines non-natural amino acid polypeptides contain the dicarbapentaborane reagent reacting contain the amino acid whose type of diamines:
Figure A200680049995D01791
Wherein:
A is optional, and when existing, it is the low-carbon (LC) alkylidene, be substituted low-carbon (LC) alkylidene, low-carbon (LC) cycloalkylidene, be substituted the low-carbon (LC) cycloalkylidene, the low-carbon (LC) alkenylene, be substituted low-carbon (LC) alkenylene, alkynylene, the inferior assorted alkyl of low-carbon (LC), be substituted inferior assorted alkyl, the inferior Heterocyclylalkyl of low-carbon (LC), be substituted low-carbon (LC) inferior Heterocyclylalkyl, arlydene, be substituted arlydene, inferior heteroaryl, be substituted inferior heteroaryl, alkarylene, be substituted alkarylene, inferior aralkyl or be substituted inferior aralkyl;
B is optional, and when existing, its at one end with contain the connection base that two amine moieties are connected, described connection base is selected from the group that is made up of following each group: the low-carbon (LC) alkylidene, be substituted low-carbon (LC) alkylidene, low-carbon (LC) alkenylene, be substituted the low-carbon (LC) alkenylene, the inferior assorted alkyl of low-carbon (LC), be substituted the low-carbon (LC) Asia mix alkyl ,-O-(alkylidene or be substituted alkylidene)-,-S-(alkylidene or be substituted alkylidene)-,-C (O) R " ,-S (O) k(alkylidene or be substituted alkylidene)-(wherein k is 1,2 or 3) ,-C (O)-(alkylidene or be substituted alkylidene)-,-C (S)-(alkylidene or be substituted alkylidene)-,-NR " (alkylidene or be substituted alkylidene)-,-CON (R ")-(alkylidene or be substituted alkylidene)-,-CSN (R ")-(alkylidene or be substituted alkylidene)-and-N (R ") CO-(alkylidene or be substituted alkylidene)-, and each R wherein is " independently for H, alkyl or be substituted alkyl;
J is
Figure A200680049995D01792
Or
Figure A200680049995D01801
Wherein:
R 8Be independently selected from H, alkyl, be substituted alkyl, cycloalkyl, be substituted cycloalkyl or amine protecting group;
R 9Be independently selected from H, alkyl, be substituted alkyl, cycloalkyl, be substituted cycloalkyl or amine protecting group;
T 1Be bond, the C that is substituted according to circumstances 1-C 4Alkylidene, the C that is substituted according to circumstances 1-C 4Alkenylene or the assorted alkyl that is substituted according to circumstances;
T 2Be the C that is substituted according to circumstances 1-C 4Alkylidene, the C that is substituted according to circumstances 1-C 4Alkenylene, the assorted alkyl that is substituted according to circumstances, the aryl that is substituted according to circumstances or the heteroaryl that is substituted according to circumstances;
Wherein each optional substituting group is independently selected from low-carbon alkyl, is substituted low-carbon alkyl, low-carbon naphthenic, is substituted low-carbon naphthenic, low-carbon (LC) thiazolinyl, is substituted the low-carbon (LC) thiazolinyl, alkynyl, the assorted alkyl of low-carbon (LC), is substituted assorted alkyl, low-carbon (LC) Heterocyclylalkyl, is substituted the low-carbon (LC) Heterocyclylalkyl, aryl, is substituted aryl, heteroaryl, is substituted heteroaryl, alkaryl, is substituted alkaryl, aralkyl or is substituted 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;
R 3And R 4Be H, halogen, low-carbon alkyl or be substituted low-carbon alkyl, perhaps R independently of one another 3And R 4Or two R 3Group forms cycloalkyl or Heterocyclylalkyl according to circumstances;
Or-the A-B-J-R group form together comprise at least one two amido, through protecting two amidos or dicyclo or tricyclic naphthenes base or Heterocyclylalkyl through covering two amidos;
Or-the B-J-R group form together comprise at least one two amido, through protecting two amidos or dicyclo or tricyclic naphthenes base or cyclophane base or Heterocyclylalkyl through covering two amidos;
Or-the J-R group form together comprise at least one two amido, through protecting two amidos or monocycle or bicyclic cycloalkyl or Heterocyclylalkyl through covering two amidos.
In fact, the type that comprises the described polypeptide that contains the diamines alpha-non-natural amino acid is unrestricted, be arranged in polypeptide as long as contain the diamines alpha-non-natural amino acid, thereby make and contain dicarbapentaborane reagent and can and not produce the modified alpha-non-natural amino acid of gained that destroys polypeptide tertiary structure (certainly, except that described destruction is the situation of purpose of reaction) with the diamines radical reaction and get final product.
Only for instance, below contain dicarbapentaborane reagent for containing the reaction of diamines alpha-non-natural amino acid and can be used for further modifying the type that contains dicarbapentaborane reagent that contains the diamines non-natural amino acid polypeptides with as herein described:
Figure A200680049995D01811
Wherein:
Each X independently for H, alkyl, be substituted alkyl, thiazolinyl, be substituted thiazolinyl, alkynyl, be substituted alkynyl, alkoxyl, be substituted alkoxyl, alkyl alkoxy, be substituted alkyl alkoxy, polyalkylene oxide, be substituted polyalkylene oxide, aryl, be substituted aryl, heteroaryl, be substituted heteroaryl, alkaryl, be substituted alkaryl, aralkyl, be substituted aralkyl ,-(alkylidene or be substituted alkylidene)-ON (R ") 2,-(alkylidene or be substituted alkylidene)-C (O) SR " ,-(alkylidene or be substituted alkylidene)-S-S-(aryl or be substituted aryl) ,-C (O) R " ,-C (O) 2R " or-C (O) N (R ") 2, each R wherein " independently for hydrogen, alkyl, be substituted alkyl, thiazolinyl, be substituted thiazolinyl, alkoxyl, be substituted alkoxyl, aryl, be substituted aryl, heteroaryl, alkaryl, be substituted alkaryl, aralkyl or be substituted aralkyl;
Or each X is independently selected from the group that is made up of required functional group;
Each L is independently selected from the group that is made up of following each group: alkylidene, be substituted alkylidene, alkenylene, be substituted alkenylene ,-O-,-O-(alkylidene or be substituted alkylidene)-,-S-,-S-(alkylidene or be substituted alkylidene)-,-S (O) k-(wherein k is 1,2 or 3) ,-S (O) k(alkylidene or be substituted alkylidene)-,-C (O)-,-C (O)-(alkylidene or be substituted alkylidene)-,-C (S)-,-C (S)-(alkylidene or be substituted alkylidene)-,-N (R ')-,-NR '-(alkylidene or be substituted alkylidene)-,-C (O) N (R ')-,-CON (R ')-(alkylidene or be substituted alkylidene)-,-(alkylidene or be substituted alkylidene) NR ' C (O) O-(alkylidene or be substituted alkylidene)-,-O-CON (R ')-(alkylidene or be substituted alkylidene)-,-CSN (R ')-,-CSN (R ')-(alkylidene or be substituted alkylidene)-,-N (R ') CO-(alkylidene or be substituted alkylidene)-,-N (R ') C (O) O-,-N (R ') C (O) O-(alkylidene or be substituted alkylidene)-,-S (O) kN (R ')-,-N (R ') C (O) N (R ')-,-N (R ') C (O) N (R ')-(alkylidene or be substituted alkylidene)-,-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, L 1For-C (R ') P-NR '-C (O) O-(alkylidene or be substituted alkylidene)-, wherein p is 0,1 or 2;
Each R ' is H, alkyl independently, is substituted alkyl or amino protecting group;
W is Or
Figure A200680049995D01822
Wherein each R ' is H independently;
Each G is independently Or
Figure A200680049995D01824
Z 1Be bond, CR 7R 7, O, S, NR ', CR 7R 7-CR 7R 7, CR 7R 7-O, O-CR 7R 7, CR 7R 7-S, S-CR 7R 7, CR 7R 7-NR ', NR '-CR 7R 7
T 3For bond, C (R) (R), O or S; And R is H, halogen, alkyl, is substituted alkyl, cycloalkyl or is substituted cycloalkyl;
T 4For the carbonyl-protection base, include but not limited to
Figure A200680049995D01825
Or Each X wherein 1Be independently selected from the group that forms by following each group :-O-,-S-,-N (H)-,-N (R ")-,-N (Ac)-and-N (OMe)-; X 2For-OR ,-OAc ,-SR " ,-N (R ") 2,-N (R ") (Ac) ,-N (R ") (OMe) or N 3, and each R wherein is " independently for H, alkyl or be substituted alkyl;
M 2For
Figure A200680049995D01829
Or
Figure A200680049995D018210
And
N is 1 to 3.
The illustrative embodiment that contains the method that contains the coupling of diamines alpha-non-natural amino acid on dicarbapentaborane reagent and the polypeptide is provided among Fig. 9 and Figure 10.In this illustrative embodiment, the dicarbapentaborane derivatization reagent is added in the buffer solution (the pH value is about 3 to about 8) that contains the diamines non-natural amino acid polypeptides.Reaction is carried out at ambient temperature, and can be by HPLC, FPLC or size exclusion chromatography purifying gained contain the heterocycle non-natural amino acid polypeptides.
In other embodiments, the basic chemicals of a plurality of connections can react with the non-natural amino acid polypeptides locus specificity that replaces through diamines.In one embodiment, connection based method as herein described utilization connects the connection base (single, double or multi-functional) that basic end contains dicarbapentaborane functional group at least one.The connection base of dicarbapentaborane derivatization produces stable heterocycle (comprising nitrogen heterocyclic ring) key with the condensation of the protein that replaces through diamines.Two and/or multi-functional connection base (for example, have one or more other connect the dicarbapentaborane of chemicals) allow locus specificity (for example to connect different molecular, other protein, polymer or little molecule) and non-natural amino acid polypeptides, and simple function connection base (all replacing through dicarbapentaborane at all ends) promotes the locus specificity dimerization or the oligomerization of non-natural amino acid polypeptides.By this being connected base strategy and in vivo translation technology combination as herein described, makes the three-dimensional structure that to specify through the protein of chemical operation.
D. the method for posttranslational modification non-natural amino acid polypeptides: contain diamines alpha-non-natural amino acid and the reaction that contains ketone alkynes reagent
Also posttranslational modification technology mentioned above and composition can be used for can with contain ketone alkynes reagent reacting contain the diamines alpha-non-natural amino acid to produce modified heterocycle (comprise and the contain nitrogen heterocyclic ring) non-natural amino acid polypeptides that contains.Only for instance, above described in the C joint contain the diamines alpha-non-natural amino acid also can with as herein described can be used for further modifying contain the dicarbapentaborane non-natural amino acid polypeptides contain ketone alkynes reagent reacting.
Only for instance, below contain ketone alkynes reagent for can with containing the reaction of diamines alpha-non-natural amino acid and can be used for further modifying the type that contains ketone alkynes reagent that contains the diamines non-natural amino acid polypeptides described in the C joint:
Figure A200680049995D01831
Wherein:
Each X independently for H, alkyl, be substituted alkyl, thiazolinyl, be substituted thiazolinyl, alkynyl, be substituted alkynyl, alkoxyl, be substituted alkoxyl, alkyl alkoxy, be substituted alkyl alkoxy, polyalkylene oxide, be substituted polyalkylene oxide, aryl, be substituted aryl, heteroaryl, be substituted heteroaryl, alkaryl, be substituted alkaryl, aralkyl, be substituted aralkyl ,-(alkylidene or be substituted alkylidene)-ON (R ") 2,-(alkylidene or be substituted alkylidene)-C (O) SR " ,-(alkylidene or be substituted alkylidene)-S-S-(aryl or be substituted aryl) ,-C (O) R " ,-C (O) 2R " or-C (O) N (R ") 2, each R wherein " independently for hydrogen, alkyl, be substituted alkyl, thiazolinyl, be substituted thiazolinyl, alkoxyl, be substituted alkoxyl, aryl, be substituted aryl, heteroaryl, alkaryl, be substituted alkaryl, aralkyl or be substituted aralkyl;
Or each X is independently selected from the group that is made up of required functional group;
Each L is independently selected from the group that is made up of following each group: alkylidene, be substituted alkylidene, alkenylene, be substituted alkenylene ,-O-,-O-(alkylidene or be substituted alkylidene)-,-S-,-S-(alkylidene or be substituted alkylidene)-,-S (O) k-(wherein k is 1,2 or 3) ,-S (O) k(alkylidene or be substituted alkylidene)-,-C (O)-,-C (O)-(alkylidene or be substituted alkylidene)-,-C (S)-,-C (S)-(alkylidene or be substituted alkylidene)-,-N (R ')-,-NR '-(alkylidene or be substituted alkylidene)-,-C (O) N (R ')-,-CON (R ')-(alkylidene or be substituted alkylidene)-,-(alkylidene or be substituted alkylidene) NR ' C (O) O-(alkylidene or be substituted alkylidene)-,-O-CON (R ')-(alkylidene or be substituted alkylidene)-,-CSN (R ')-,-CSN (R ')-(alkylidene or be substituted alkylidene)-,-N (R ') CO-(alkylidene or be substituted alkylidene)-,-N (R ') C (O) O-,-N (R ') C (O) O-(alkylidene or be substituted alkylidene)-,-S (O) kN (R ')-,-N (R ') C (O) N (R ')-,-N (R ') C (O) N (R ')-(alkylidene or be substituted alkylidene)-,-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, L 1For-C (R ') P-NR '-C (O) O-(alkylidene or be substituted alkylidene)-, wherein p is 0,1 or 2, each R ' is independently for H, alkyl or be substituted alkyl;
W is
Figure A200680049995D01841
G is
Figure A200680049995D01842
Or
Figure A200680049995D01843
T 4For the carbonyl-protection base, include but not limited to Or
Figure A200680049995D01845
Each X wherein 1Be independently selected from the group that forms by following each group :-O-,-S-,-N (H)-,-N (R ")-,-N (Ac)-and-N (OMe)-; X 2For-OR ,-OAc ,-SR ' ,-N (R ") 2,-N (R ") (Ac) ,-N (R ") (OMe) or N 3, and each R wherein " independently for H, alkyl or be substituted alkyl, and n is 1 to 3.
In other embodiments, the basic chemicals of a plurality of connections can react with the non-natural amino acid polypeptides locus specificity that replaces through diamines.In one embodiment, connection based method as herein described utilization connects the connection base (single, double or multi-functional) that basic end contains ketone alkynes functional group at least one.The connection base of ketone alkynes derivatization produces stable heterocycle (comprising nitrogen heterocyclic ring) key with the reaction of the protein that replaces through diamines.Two and/or multi-functional connection base (for example, have one or more other connect the ketone alkynes of chemicals) allow locus specificity (for example to connect different molecular, other protein, polymer or little molecule) and non-natural amino acid polypeptides, and simple function connection base (all replacing through ketone alkynes at all ends) promotes the locus specificity dimerization or the oligomerization of non-natural amino acid polypeptides.By this being connected base strategy and in vivo translation technology combination as herein described, makes the three-dimensional structure that to specify through the protein of chemical operation.
E. the method for posttranslational modification non-natural amino acid polypeptides: contain ketone alkynes alpha-non-natural amino acid and the reaction that contains diamines reagent
Also posttranslational modification technology mentioned above and composition can be used for can with contain the diamines reagent reacting contain ketone alkynes alpha-non-natural amino acid to produce modified heterocycle (comprise and the contain nitrogen heterocyclic ring) non-natural amino acid polypeptides that contains.
The protein derived method that is based upon on the basis that contains ketone alkynes protein and the reaction of the molecule that replaces through diamines has unique advantage.The first, ketone alkynes is in about 4 to about 10 pH value scope and contain the diamine compound reaction to produce heterocycle (comprising nitrogen heterocyclic ring) key.Under these conditions, naturally occurring amino acid whose side chain is not had a reactivity.The second, described selective chemical makes locus specificity derivatization recombinant protein become possibility: derivatization protein now can be prepared as the homology product of appointment.The 3rd, realize the tertiary structure (being to destroy the situation of described tertiary structure except that the reaction purpose certainly) that the required temperate condition of diamines reagent and the reaction that contains ketone alkynes polypeptide as herein described can reversibly destroy polypeptide usually that contains as herein described.The 4th, reaction at room temperature takes place rapidly, and this allows to use will be at the polypeptide or the reagent of unsettled many types under the higher temperature.The 5th, reaction is easy to take place under aqueous conditions, and this also allows to use the polypeptide and the reagent of incompatible with non-aqueous solution (on any degree).The 6th, even when the ratio of polypeptide or amino acid and reagent is stoichiometry, near-stoichiometric or class stoichiometry, reaction also is easy to take place, thereby does not need to add the product that excessive reagent or polypeptide obtain consumption.The 7th, the design of diamines and dicarbapentaborane part in the visual reactant and regioselectivity and/or regiospecificity ground generation gained heterocycle.At last, contain diamines reagent and be created in heterocycle stable under the biotic factor (comprising nitrogen heterocyclic ring) key with the reaction that contains the ketone alkynyl amino acid.
Only for instance, following alpha-non-natural amino acid for containing the diamines reagent reacting and can be used for further modifying the type that contains the ketone alkynyl amino acid that contains ketone alkynes non-natural amino acid polypeptides with as herein described:
Figure A200680049995D01851
Wherein:
A is optional, and when existing, it is the low-carbon (LC) alkylidene, be substituted low-carbon (LC) alkylidene, low-carbon (LC) cycloalkylidene, be substituted the low-carbon (LC) cycloalkylidene, the low-carbon (LC) alkenylene, be substituted low-carbon (LC) alkenylene, alkynylene, the inferior assorted alkyl of low-carbon (LC), be substituted inferior assorted alkyl, the inferior Heterocyclylalkyl of low-carbon (LC), be substituted low-carbon (LC) inferior Heterocyclylalkyl, arlydene, be substituted arlydene, inferior heteroaryl, be substituted inferior heteroaryl, alkarylene, be substituted alkarylene, inferior aralkyl or be substituted inferior aralkyl;
B is optional, and when existing, its at one end with contain the connection base that two amine moieties are connected, described connection base is selected from the group that is made up of following each group: the low-carbon (LC) alkylidene, be substituted low-carbon (LC) alkylidene, low-carbon (LC) alkenylene, be substituted the low-carbon (LC) alkenylene, the inferior assorted alkyl of low-carbon (LC), be substituted the low-carbon (LC) Asia mix alkyl ,-O-(alkylidene or be substituted alkylidene)-,-S-(alkylidene or be substituted alkylidene)-,-C (O) R " ,-S (O) k(alkylidene or be substituted alkylidene)-(wherein k is 1,2 or 3) ,-C (O)-(alkylidene or be substituted alkylidene)-,-C (S)-(alkylidene or be substituted alkylidene)-,-NR " (alkylidene or be substituted alkylidene)-,-CON (R ")-(alkylidene or be substituted alkylidene)-,-CSN (R ")-(alkylidene or be substituted alkylidene)-and-N (R ") CO-(alkylidene or be substituted alkylidene)-, and each R wherein is " independently for H, alkyl or be substituted alkyl;
G is
Figure A200680049995D01861
Or
Figure A200680049995D01862
T 4For the carbonyl-protection base, include but not limited to
Figure A200680049995D01863
Or
Figure A200680049995D01865
Each X wherein 1Be independently selected from the group that forms by following each group :-O-,-S-,-N (H)-,-N (R)-,-N (Ac)-and-N (OMe)-; X 2For-OR ,-OAc ,-SR ,-N (R) 2,-N (R) (Ac) ,-N (R) (OMe) or N 3, and wherein each R ' is H, alkyl independently or is substituted alkyl;
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 3And R 4Be H, halogen, low-carbon alkyl or be substituted low-carbon alkyl, perhaps R independently of one another 3And R 4Or two R 3Group forms cycloalkyl or Heterocyclylalkyl according to circumstances.
In one embodiment, the basic chemicals of a plurality of connections can react with the non-natural amino acid polypeptides locus specificity that replaces through ketone alkynes.In one embodiment, connection based method as herein described utilization connects the connection base (single, double or multi-functional) that basic end contains two amine functional groups at least one.The connection base of two amine derivatives produces stable heterocycle (comprising nitrogen heterocyclic ring) key with the reaction of the protein that replaces through ketone alkynes.Two and/or multi-functional connection base (for example, have one or more other connect the diamines of chemicals) allow locus specificity (for example to connect different molecular, other protein, polymer or little molecule) and non-natural amino acid polypeptides, and simple function connection base (all replacing through diamines at all ends) promotes the locus specificity dimerization or the oligomerization of non-natural amino acid polypeptides.By this being connected base strategy and in vivo translation technology combination as herein described, makes the three-dimensional structure that to specify through the protein of chemical operation.
F. the method for posttranslational modification non-natural amino acid polypeptides: contain ketoamine alpha-non-natural amino acid and the reaction that contains dicarbapentaborane reagent
Also posttranslational modification technology mentioned above and composition can be used for can with contain the dicarbapentaborane reagent reacting contain the ketoamine alpha-non-natural amino acid to produce modified heterocycle (comprise and the contain nitrogen heterocyclic ring) non-natural amino acid polypeptides that contains.
The protein derived method that is based upon on the basis that contains ketoamine protein and the reaction of the molecule that replaces through dicarbapentaborane has unique advantage.The first, ketoamine in about 4 to about 10 pH value scope (and in other embodiments in about 4 to about 10 pH value scope; And in other embodiments in about 3 to about 8 pH value scope; Or in other embodiments in about 2 to about 9 pH value scope; Or in other embodiments in about 4 to about 9 pH value scope) and contain the reaction of dicarbonyl compound experience to produce heterocycle (comprising nitrogen heterocyclic ring) key.Under these conditions, naturally occurring amino acid whose side chain is not had a reactivity.The second, described selective chemical makes locus specificity derivatization recombinant protein become possibility: derivatization protein now can be prepared as the homology product of appointment.The 3rd, realize the tertiary structure (being to destroy the situation of described tertiary structure except that the reaction purpose certainly) that the required temperate condition of dicarbapentaborane reagent and the reaction that contains the ketoamine polypeptide as herein described can reversibly destroy polypeptide usually that contains as herein described.The 4th, reaction at room temperature takes place rapidly, and this allows to use will be at the polypeptide or the reagent of unsettled many types under the higher temperature.The 5th, reaction is easy to take place under aqueous conditions, and this also allows to use the polypeptide and the reagent of incompatible with non-aqueous solution (on any degree).The 6th, even when the ratio of polypeptide or amino acid and reagent during for about 1:1 or roughly near 1:1, reaction also is easy to generation, thereby does not need to add the product that excessive reagent or polypeptide obtain consumption.The 7th, the design of ketoamine and dicarbapentaborane part in the visual reactant and regioselectivity and/or regiospecificity ground generation gained heterocycle.At last, contain dicarbapentaborane reagent and contain the amino acid whose reaction of ketoamine and be created in heterocycle stable under the biotic factor (comprising nitrogen heterocyclic ring) key.
Only for instance, following alpha-non-natural amino acid for can with as herein described contain the dicarbapentaborane reagent reacting and can be used for further modifying contain the ketoamine non-natural amino acid polypeptides contain the amino acid whose type of ketoamine:
Figure A200680049995D01871
Wherein:
A is optional, and when existing, it is the low-carbon (LC) alkylidene, be substituted low-carbon (LC) alkylidene, low-carbon (LC) cycloalkylidene, be substituted the low-carbon (LC) cycloalkylidene, the low-carbon (LC) alkenylene, be substituted low-carbon (LC) alkenylene, alkynylene, the inferior assorted alkyl of low-carbon (LC), be substituted inferior assorted alkyl, the inferior Heterocyclylalkyl of low-carbon (LC), be substituted low-carbon (LC) inferior Heterocyclylalkyl, arlydene, be substituted arlydene, inferior heteroaryl, be substituted inferior heteroaryl, alkarylene, be substituted alkarylene, inferior aralkyl or be substituted inferior aralkyl;
B is optional, and when existing, its at one end with contain the connection base that two amine moieties are connected, described connection base is selected from the group that is made up of following each group: the low-carbon (LC) alkylidene, be substituted low-carbon (LC) alkylidene, low-carbon (LC) alkenylene, be substituted the low-carbon (LC) alkenylene, the inferior assorted alkyl of low-carbon (LC), be substituted the low-carbon (LC) Asia mix alkyl ,-O-(alkylidene or be substituted alkylidene)-,-S-(alkylidene or be substituted alkylidene)-,-C (O) R " ,-S (O) k(alkylidene or be substituted alkylidene)-(wherein k is 1,2 or 3) ,-C (O)-(alkylidene or be substituted alkylidene)-,-C (S)-(alkylidene or be substituted alkylidene)-,-NR " (alkylidene or be substituted alkylidene)-,-CON (R ")-(alkylidene or be substituted alkylidene)-,-CSN (R ")-(alkylidene or be substituted alkylidene)-and-N (R ") CO-(alkylidene or be substituted alkylidene)-, and each R wherein is " independently for H, alkyl or be substituted alkyl;
G is
Figure A200680049995D01881
Or
Figure A200680049995D01882
T 1Be the C that is substituted according to circumstances 1-C 4Alkylidene, the C that is substituted according to circumstances 1-C 4Alkenylene or the assorted alkyl that is substituted according to circumstances;
T 4For the carbonyl-protection base, include but not limited to
Figure A200680049995D01883
Figure A200680049995D01884
Or
Figure A200680049995D01885
Each X wherein 1Be independently selected from the group that forms by following each group :-O-,-S-,-N (H)-,-N (R)-,-N (Ac)-and-N (OMe)-; X 2For-OR ,-OAc ,-SR ,-N (R) 2,-N (R) (Ac) ,-N (R) (OMe) or N 3, and wherein each R ' is H, alkyl independently or is substituted alkyl;
R is H, halogen, alkyl, is substituted alkyl, cycloalkyl or is substituted cycloalkyl;
Each R ' is H, alkyl independently 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; And
R 3And R 4Be H, halogen, low-carbon alkyl or be substituted low-carbon alkyl, perhaps R independently of one another 3And R 4Or two R 3Group forms cycloalkyl or Heterocyclylalkyl according to circumstances.
In one embodiment, the basic chemicals of a plurality of connections can react with the non-natural amino acid polypeptides locus specificity that replaces through ketoamine.In one embodiment, connection based method as herein described utilization connects the connection base (single, double or multi-functional) that basic end contains dicarbapentaborane functional group at least one.The connection base of dicarbapentaborane derivatization produces stable heterocycle (comprising nitrogen heterocyclic ring) key with the reaction of the protein that replaces through ketoamine.Two and/or multi-functional connection base (for example, have one or more other connect the dicarbapentaborane of chemicals) allow locus specificity (for example to connect different molecular, other protein, polymer or little molecule) and non-natural amino acid polypeptides, and simple function connection base (all replacing through dicarbapentaborane at all ends) promotes the locus specificity dimerization or the oligomerization of non-natural amino acid polypeptides.By this being connected base strategy and in vivo translation technology combination as herein described, makes the three-dimensional structure that to specify through the protein of chemical operation.
G. add the example of functional group: with the macromolecule polyalcohol of non-natural amino acid polypeptides coupling
Can use composition as herein described, method, technology and strategy realization various modifications to non-natural amino acid polypeptides as herein described.Described modification comprises to be incorporated other functional group on the alpha-non-natural amino acid component of polypeptide into, and described functional group includes but not limited to required functional group.Illustrative limiting examples as composition as herein described, method, technology and strategy, below describe and macromolecule polyalcohol is added in the non-natural amino acid polypeptides concentrating on, should be appreciated that simultaneously compositions related, method, technology and strategy are applicable to that also (utilize in case of necessity suitably modify and one of ordinary skill in the art can utilize the disclosure of this paper to carry out) add other functional group, include but not limited to functional group mentioned above.
Can provide novel biological nature with the biological nature of adjusting non-natural amino acid polypeptides (or corresponding natural amino acid polypeptide) and/or to non-natural amino acid polypeptides (or corresponding natural amino acid polypeptide) with multiple macromolecule polyalcohol and other molecule and non-natural amino acid polypeptides coupling as herein described.Can be with these macromolecule polyalcohols via any sense substituent of alpha-non-natural amino acid or alpha-non-natural amino acid or add any substituting group or the functional group and the non-natural amino acid polypeptides coupling of alpha-non-natural amino acid to.
Can be with water-soluble polymer and the alpha-non-natural amino acid coupling of incorporating in polypeptide as herein described (natural or synthetic), polynucleotide, polysaccharide or the synthetic polymer.Can be by any functional group or the substituting group of incorporating alpha-non-natural amino acid in the polypeptide or alpha-non-natural amino acid into or any functional group or the substituting group coupling water-soluble polymer that adds alpha-non-natural amino acid to.In some cases, non-natural amino acid polypeptides as herein described comprises the naturally occurring amino acid of the alpha-non-natural amino acid of one or more and water-soluble polymer coupling and one or more and water-soluble polymer coupling.Hydrophilic polymer and bioactive molecule covalently bound representing increase bioactive molecule (comprise protein, peptide and especially hydrophobic molecule) water-soluble (such as, under physiological environment), biological usability; Increase serum half-life; Increase the treatment half life period; Regulate immunogenicity; Regulate biologically active; Or a kind of method that prolongs circulation timei.Other key character of described hydrophilic polymer comprises bio-compatible, avirulence and non-immunogenicity.For the therapeutical uses of final products preparation, polymer preferably will be for pharmaceutically acceptable.
The example of hydrophilic polymer (for example includes but not limited to poly alkyl ether and its alkoxy end-capped analog, polyoxyethylene glycol, polyoxyethylene/propane diols and its analog through the methoxy or ethoxy end-blocking, especially polyoxyethylene glycol, the latter is also referred to as polyethylene glycol or PEG); Polyvinylpyrrolidone; Polyethylene alkyl ether; Ju oxazoline, Ju Wan oxazolin and Ju Qiang base Wan oxazolin; Polyacrylamide, poly-alkyl acrylamide and poly-hydroxy alkyl acrylamide (for example, poly-hydroxypropyl Methacrylamide and its derivative); Poly-hydroxy alkyl acrylate; Poly sialic acid and its analog; The hydrophilic peptide sequence; Polysaccharide and its derivative comprise glucan and glucan derivative, for example Sensor Chip CM 5, dextran sulfate, GAG, cellulose and its derivative, for example carboxymethyl cellulose, hydroxy alkyl cellulose; Chitin and its derivative, for example shitosan, succinyl group shitosan, carboxymethyl chitosan, CMC; Hyaluronic acid and its derivative; Starch; Alginic acid ester; Chondroitin sulfate; Albumin; Amylopectin (pullulan) and carboxymethyl amylopectin; Polyaminoacid and its derivative, for example polyglutamic acid, polylysine, poly-aspartate, poly-asparagine; Copolymer-maleic anhydride is such as styrene maleic anhydride copolymer, divinyl ethyl ether copolymer-maleic anhydride, polyvinyl alcohol; Its copolymer; Its trimer; Its mixture; Derivative with above-mentioned substance.Water-soluble polymer can be any structure form, includes but not limited to linearity, forked or branch form.In certain embodiments, have about 2 particularly useful to the water-soluble polymer main chain of about 300 ends.Multifunctional polymer derivant includes but not limited to have the linear polymer of two ends, each functional group's bond terminal and can be identical or different.In certain embodiments, water-soluble polymer comprises poly-(ethylene glycol) part.The molecular weight of polymer can be in broad range, include but not limited between about 100Da and about 100,000Da or higher between.The molecular weight of polymer can between the 000Da, include but not limited to about 100 between about 100Da and about 100,000Da, about 95,000Da, about 90,000Da, about 85,000Da, about 80,000Da, about 75,000Da, about 70,000Da, about 65,000Da, about 60,000Da, about 55,000Da, about 50,000Da, about 45,000Da, about 40,000Da, about 35,000Da, about 30,000Da, about 25,000Da, about 20,000Da, about 15,000Da, about 10,000Da, about 9,000Da, about 8,000Da, about 7,000Da, about 6,000Da, about 5,000Da, about 4,000Da, about 3,000Da, about 2,000Da, about 1,000Da, about 900Da, about 800Da, about 700Da, about 600Da, about 500Da, about 400Da, about 300Da, about 200Da and about 100Da.In certain embodiments, the molecular weight of polymer is between about 100Da and about 50, between the 000Da.In certain embodiments, the molecular weight of polymer is between about 100Da and about 40, between the 000Da.In certain embodiments, the molecular weight of polymer is between about 1, and 000Da and about 40 is between the 000Da.In certain embodiments, the molecular weight of polymer is between about 2, and 000Da is to about 50, between the 000Da.In certain embodiments, the molecular weight of polymer is between about 5, and 000Da and about 40 is between the 000Da.In certain embodiments, the molecular weight of polymer is between about 10, and 000Da and about 40 is between the 000Da.In certain embodiments, peg molecule is a branched polymers.The molecular weight of side chain PEG can be between about 1, and 000Da and about 100 between the 000Da, includes but not limited to about 100,000Da, about 95,000Da, about 90,000Da, about 85,000Da, about 80,000Da, about 75,000Da, about 70,000Da, about 65,000Da, about 60,000Da, about 55,000Da, about 50,000Da, about 45,000Da, about 40,000Da, about 35,000Da, about 30,000Da, about 25,000Da, about 20,000Da, about 15,000Da, about 10,000Da, about 9,000Da, about 8,000Da, about 7,000Da, about 6,000Da, about 5,000Da, about 4,000Da, about 3,000Da, about 2,000Da and about 1,000Da.In certain embodiments, the molecular weight of side chain PEG is between about 1, and 000Da and about 50 is between the 000Da.In certain embodiments, the molecular weight of side chain PEG is between about 1, and 000Da and about 40 is between the 000Da.In certain embodiments, the molecular weight of side chain PEG is between about 5, and 000Da and about 40 is between the 000Da.In certain embodiments, the molecular weight of side chain PEG is between about 5, and 000Da and about 20 is between the 000Da.In certain embodiments, the molecular weight of side chain PEG is between about 2, and 000Da is to about 50, between the 000Da.One of ordinary skill in the art will recognize, the above-mentioned tabulation of water-soluble in fact main chain is never detailed and only be illustrative, and all polymeric materials that expection has an above-mentioned quality all are applicable in the method and composition as herein described.
As indicated above, an example of hydrophilic polymer is a polyethylene glycol, is abbreviated as PEG, and it has been widely used in medicine or the artificial graft's thing and during considerable other of bio-compatible, avirulence and non-immunogenicity use.Polymer as herein described: polypeptide embodiment should be appreciated that the example of PEG as hydrophilic polymer simultaneously and can similarly other hydrophilic polymer be used for described embodiment.
PEG is well-known water-soluble polymer, and it can be on sale or can prepare (Sandler and Karo, Polymer Synthesis by making the ethylene glycol ring-opening polymerisation according to the well-known method in affiliated field on the market, Academic Press, New York, the 3rd volume, 138-161 page or leaf).PEG clarifies usually, and is colourless, odorless, and water soluble to thermally-stabilised, is inertia to many chemical agents, not hydrolysis or rotten and nontoxic usually.Think that poly-(ethylene glycol) for bio-compatible, it is reported that PEG can and not cause harm with living tissue or organism coexistence.More particularly, PEG is essentially non-immunogenic, it is reported that PEG is not inclined to produce immune response in vivo.When with body in have certain required function molecule (such as bioactivator) when being connected, PEG tends to cover reagent and can reduce or eliminate any immune response, thereby makes organism can stand the existence of described reagent.The PEG concatenator is not inclined to produce the essence immune response or cause and solidifies or other undesirable effect.
Term " PEG " is widely used in contains any peg molecule (not considering the size of PEG or the modification of its end), and can be expressed from the next to being connected with non-natural amino acid polypeptides:
XO-(CH 2CH 2O) n-CH 2CH 2-Y，
Wherein n be about 2 to about 10,000 and X be H or end modified, include but not limited to C 1-4Alkyl, protecting group or end modified base.Term PEG includes but not limited to any type of polyethylene glycol, comprise difunctionality PEG, multi-arm PEG, derivatization PEG, forked PEG, branch PEG (each chain has about 1kDa and arrives the molecular weight that about 50kDa or about 1kDa arrive about 20kDa to about 100kDa, about 1kDa), side joint PEG (that is the PEG or the related polymer that, have the functional group of one or more and main polymer chain side joint) or have the PEG of degradable linkage.In one embodiment, n is that about 20 to about 2000 PEG is applicable in the method and composition as herein described.In certain embodiments, water-soluble polymer comprises polyalkylene glycol moiety.The molecular weight of PEG polymer can be in broad range, include but not limited between about 100Da and about 100,000Da or higher between.The molecular weight of polymer can between the 000Da, include but not limited to about 100 between about 100Da and about 100,000Da, about 95,000Da, about 90,000Da, about 85,000Da, about 80,000Da, about 75,000Da, about 70,000Da, about 65,000Da, about 60,000Da, about 55,000Da, about 50,000Da, about 45,000Da, about 40,000Da, about 35,000Da, about 30,000Da, about 25,000Da, about 20,000Da, about 15,000Da, about 10,000Da, about 9,000Da, about 8,000Da, about 7,000Da, about 6,000Da, about 5,000Da, about 4,000Da, about 3,000Da, about 2,000Da, about 1,000Da, about 900Da, about 800Da, about 700Da, about 600Da, about 500Da, about 400Da, about 300Da, about 200Da and about 100Da.In certain embodiments, the molecular weight of polymer is between about 100Da and about 50, between the 000Da.In certain embodiments, the molecular weight of polymer is between about 100Da and about 40, between the 000Da.In certain embodiments, the molecular weight of polymer is between about 1, and 000Da and about 40 is between the 000Da.In certain embodiments, the molecular weight of polymer is between about 2, and 000Da is to about 50, between the 000Da.In certain embodiments, the molecular weight of polymer is between about 5, and 000Da and about 40 is between the 000Da.In certain embodiments, the molecular weight of polymer is between about 10, and 000Da and about 40 is between the 000Da.In certain embodiments, peg molecule is a branched polymers.The molecular weight of side chain PEG can be between about 1, and 000Da and about 100 between the 000Da, includes but not limited to about 100,000Da, about 95,000Da, about 90,000Da, about 85,000Da, about 80,000Da, about 75,000Da, about 70,000Da, about 65,000Da, about 60,000Da, about 55,000Da, about 50,000Da, about 45,000Da, about 40,000Da, about 35,000Da, about 30,000Da, about 25,000Da, about 20,000Da, about 15,000Da, about 10,000Da, about 9,000Da, about 8,000Da, about 7,000Da, about 6,000Da, about 5,000Da, about 4,000Da, about 3,000Da, about 2,000Da and about 1,000Da.In certain embodiments, the molecular weight of side chain PEG is between about 1, and 000Da and about 50 is between the 000Da.In certain embodiments, the molecular weight of side chain PEG is between about 1, and 000Da and about 40 is between the 000Da.In certain embodiments, the molecular weight of side chain PEG is between about 5, and 000Da and about 40 is between the 000Da.In certain embodiments, the molecular weight of side chain PEG is between about 5, and 000Da and about 20 is between the 000Da.In other embodiments, the molecular weight of side chain PEG is between about 2, and 000Da is to about 50, between the 000Da.Multiple PEG molecule is described in (including but not limited to) ShearwaterPolymers, and in Inc. catalogue, the Nektar Therapeutics catalogue, it is incorporated herein with way of reference.
The particular instance of functional end-group includes but not limited to that carbonic acid N-succinimido ester is (for example referring to United States Patent (USP) the 5th in the document, 281, No. 698, the 5th, 468, No. 478), amine is (for example referring to, people Makromol.Chem.182:1379 (1981) such as Buckmann; People Eur.Polym.J.19:1177 (1983) such as Zalipsky), hydrazides (for example referring to, people Makromol.Chem.179:301 (1978) such as Andresz), propionic acid succinimide ester and butyric acid succinimide ester are (for example referring to, people Polyethylene glycol Chemistry ﹠amp such as Olson; Biological Applications, 170-181 page or leaf, Harris ﹠amp; Zalipsky compiles, ACS, Washington, D.C., 1997; Also referring to United States Patent (USP) the 5th, 672, No. 662), the succinic acid succinimide ester (for example referring to, people Makromol.Chem.180:1381 (1979) such as people Cancer Biochem.Biophys.7:175 (1984) such as Abuchowski and Joppich), succinimide ester (for example referring to, United States Patent (USP) the 4th, 670, No. 417), the BTA carbonic ester (for example referring to, United States Patent (USP) the 5th, 650, No. 234), glycidyl ether (for example referring to, people Eur.J Biochem.94:11 (1979) such as Pitha, people such as Elling, Biotech.Appl.Biochem.13:354 (1991)), oxygen base carbonylic imidazole (for example referring to, people such as Beauchamp, Anal.Biochem.131:25 (1983); People J.Controlled Release 1:251 (1985) such as Tondelli), p-nitrophenyl carbonate ester (for example referring to, Veronese waits the people, Appl.Biochem.Biotech., 11:141 (1985); With people such as Sartore, Appl.Biochem.Biotech., 27:45 (1991)), aldehyde (for example referring to, people J.Polym.Sci.Chem. such as Harris compile 22:341 (1984); United States Patent (USP) the 5th, 824, No. the 5th, 252,714, No. 784, United States Patent (USP)), maleimide is (for example referring to, people BioTechnology 8:343 (1990) such as Goodson; People Chemistry of Peptides andProteins 2:29 (1984) such as Romani) and Kogan, Synthetic Comm.22:2417 (1992)), former pyridyl disulfide (for example referring to, people Bioconj.Chem.4:314 (1993) such as Woghiren), propenyl (for example referring to, people such as Sawhney, Macromolecules, 26:581 (1993)), vinyl sulfoxide is (for example referring to, United States Patent (USP) the 5th, 900, No. 461).The mode that all above-mentioned lists of references and patent are all quoted in full is incorporated herein.
In some cases, with hydroxyl or methoxyl group end-blocking, that is, X is H or CH to PEG at an end 3(" methoxyl group PEG ").Perhaps, PEG can the reactive group end-blocking, thereby forms the double functional copolymer.The typical reaction group can comprise that the reactive group that is generally used for functional group reactions seen in 20 kinds of common amino acids (includes but not limited to dimaleoyl imino, activated carbon acid esters (including but not limited to p-nitrophenyl ester), active ester (includes but not limited to N-hydroxy-succinamide, p-nitrophenyl ester) and aldehyde) and to 20 kinds of common amino acids be inertia but with alpha-non-natural amino acid in functional group's (including but not limited to diamines and dicarbapentaborane) of existing complementary functional groups specific reaction.
Another end that it should be noted that the PEG that represents with Y in following formula will directly or via alpha-non-natural amino acid be connected to polypeptide (synthetic or natural), polynucleotide, polysaccharide or synthetic polymer indirectly.When Y is two amidos, contain so diamines PEG reagent can with polypeptide in contain the alpha-non-natural amino acid reaction of dicarbapentaborane to form via heterocycle (comprising nitrogen heterocyclic ring) the PEG group that key is connected with polypeptide.When Y is two amidos, the PEG reagent that contains diamines so also can with polypeptide in contain the alpha-non-natural amino acid reaction of ketone alkynes to form via heterocycle (comprising nitrogen heterocyclic ring) the PEG group that key is connected with polypeptide.When Y is dicarbapentaborane, contain so dicarbapentaborane PEG reagent can with polypeptide in contain the alpha-non-natural amino acid reaction of diamines to form via heterocycle (comprising nitrogen heterocyclic ring) the PEG group that key is connected with polypeptide.When Y is dicarbapentaborane, the PEG reagent that contains dicarbapentaborane so also can with polypeptide in contain the alpha-non-natural amino acid reaction of ketoamine to form via heterocycle (comprising nitrogen heterocyclic ring) the PEG group that key is connected with polypeptide.When Y is the ketone alkynyl, contain so the ketone alkynyl PEG reagent can with polypeptide in contain the alpha-non-natural amino acid reaction of diamines to form via heterocycle (comprising nitrogen heterocyclic ring) the PEG group that key is connected with polypeptide.When Y is the ketoamine base, contain so ketoamine PEG reagent can with polypeptide in contain the alpha-non-natural amino acid reaction of dicarbapentaborane to form via heterocycle (comprising nitrogen heterocyclic ring) the PEG group that key is connected with polypeptide.The case description of appropriate reaction condition, purification process and reagent is in this specification and accompanying drawing.Figure 17 provides following example: i) contain the dicarbapentaborane non-natural amino acid polypeptides and form the reaction that contains the heterocycle non-natural amino acid polypeptides that is connected with the PEG group with the PEG reagent that contains diamines; Ii) contain the diamines non-natural amino acid polypeptides and form the reaction that contains the heterocycle non-natural amino acid polypeptides that is connected with the PEG group with the PEG reagent that contains dicarbapentaborane; Iii) contain ketone alkynes non-natural amino acid polypeptides and form the reaction that contains the heterocycle non-natural amino acid polypeptides that is connected with the PEG group with the PEG reagent that contains diamines.In addition, Figure 23 provides the limiting examples of protein Pegylation, and the PEG reagent that wherein contains diamines reacts with the dicarbapentaborane alpha-non-natural amino acid of incorporating in the protein that contains, thereby forms heterocyclic bond.
Only plan to limit the type or the classification of the PEG reagent that can be used for composition as herein described, method, technology and strategy for instance and not, Figure 18 provide form the PEG reagent that contains diamines or through the protection form contain diamines PEG reagent or through the illustrative example of the synthetic method that contains diamines PEG reagent of the form of covering.In addition, Figure 19 provide form contain dicarbapentaborane PEG reagent or through the protection form contain dicarbapentaborane PEG reagent or through the illustrative example of the synthetic method that contains dicarbapentaborane PEG reagent of the form of covering.In addition; Figure 20 provides and forms difunctionality PEG reagent or through the difunctionality PEG reagent of protection form or through the illustrative example of the synthetic method of the difunctionality PEG of the form of covering reagent, and Figure 21 provides and forms difunctionality and connect base or connect base or connect the illustrative example of the synthetic method of base through the difunctionality of the form of covering through the difunctionality of protection form.In addition, Figure 22 provides and forms trifunctional PEG reagent or through the trifunctional PEG reagent of protection form or through the illustrative example of the synthetic method of the trifunctional PEG of the form of covering reagent.
When needs were connected each end of different molecular and polymer, the Heterobifunctional derivative was also particularly useful.For instance, the terminal molecule that is connected and will has an acetenyl of ω-N-amino-N-azido PEG will allow to have active electrophilic group molecule and PEG one of (such as aldehyde, ketone, active ester, activated carbon acid esters etc.) is connected with another end of PEG.
In certain embodiments, nucleophilic group (including but not limited to diamines) can react to form heterocycle (comprising nitrogen heterocyclic ring) with existing dicarbapentaborane in the alpha-non-natural amino acid, and it in some cases can be by experiencing further reaction with suitable agent treated.Perhaps, can incorporate into nucleophilic group in the polypeptide and use it for that existing dicarbapentaborane reacts in preferential and the water-soluble polymer via alpha-non-natural amino acid.In general, at least one end of PEG molecule can be used for reacting with alpha-non-natural amino acid.
Therefore, in certain embodiments, will comprise the polypeptide of alpha-non-natural amino acid and water-soluble polymer (such as, polyethylene glycol (PEG)) via the alpha-non-natural amino acid side chain and connect.Alpha-non-natural amino acid as herein described, method and composition provide the special effective method that utilizes PEG derivatives selectively modifying protein, it relates to response and selects codon that alpha-non-natural amino acid (include but not limited to contain do not see 20 kinds of functional group or substituent amino acid in the natural amino acid of incorporating into) selectivity is incorporated in the protein, and utilized suitable reactive PEG derivative to modify described amino acid subsequently.Various known chemical methodes are applicable to that all alpha-non-natural amino acid method and composition as herein described is to incorporate water-soluble polymer in the protein into.
Main polymer chain can be linearity or branch.The general known branched polymers main chain in affiliated field.Usually, branched polymers has central branch core and a plurality of linear polymer chain that are connected with central branch core.PEG uses with the branch form, described branch form can by with oxirane and various polyalcohol (such as, glycerine, glycerine oligomer, pentaerythrite and sorbitol) addition prepares.Central authorities' branch part also can be derived from some amino acid (such as, lysine).Branch poly-(ethylene glycol) can general formula R (PEG-OH) mExpression, wherein R derives from the core, and such as glycerine, glycerine oligomer or pentaerythrite, and m represents the quantity of arm.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; Molecule described in U.S. patent application case 2003/0143596, WO 96/21469 and the WO 93/21259, the mode that each patent is quoted in full is incorporated herein) also can be used as main polymer chain.
Branch PEG also can be (the YCHZ with PEG 2) nThe form of forked PEG of expression, wherein Y is for connecting base, and the reactive terminal group that is connected with CH for the atomic link by designated length of Z.Another branch form side joint PEG has along the PEG main chain rather than at the reactive group (such as carboxyl) of PEG chain end.
Except that the PEG of these forms, polymer also can have weak bond or degradable linkage through being prepared in main chain.For instance, PEG can be through being prepared into the ester bond that has the hydrolysis of being easy in main polymer chain.As shown here, this hydrolysis causes polymer cracking to become to have the fragment of lower molecular weight:
-PEG-CO 2-PEG-+H 2O→PEG-CO 2H+HO-PEG-。
One of ordinary skill in the art should be appreciated that term polyethylene glycol or PEG represent or comprise the known form of ownership in affiliated field, include but not limited to form disclosed herein.The molecular weight of polymer can between the 000Da, include but not limited to about 100 between about 100Da and about 100,000Da, about 95,000Da, about 90,000Da, about 85,000Da, about 80,000Da, about 75,000Da, about 70,000Da, about 65,000Da, about 60,000Da, about 55,000Da, about 50,000Da, about 45,000Da, about 40,000Da, about 35,000Da, about 30,000Da, about 25,000Da, about 20,000Da, about 15,000Da, about 10,000Da, about 9,000Da, about 8,000Da, about 7,000Da, about 6,000Da, about 5,000Da, about 4,000Da, about 3,000Da, about 2,000Da, about 1,000Da, about 900Da, about 800Da, about 700Da, about 600Da, about 500Da, about 400Da, about 300Da, about 200Da and about 100Da.In certain embodiments, the molecular weight of polymer is between about 100Da and about 50, between the 000Da.In certain embodiments, the molecular weight of polymer is between about 100Da and about 40, between the 000Da.In certain embodiments, the molecular weight of polymer is between about 1, and 000Da and about 40 is between the 000Da.In certain embodiments, the molecular weight of polymer is between about 2, and 000Da is to about 50, between the 000Da.In certain embodiments, the molecular weight of polymer is between about 5, and 000Da and about 40 is between the 000Da.In certain embodiments, the molecular weight of polymer is between about 10, and 000Da and about 40 is between the 000Da.
For making the desirable characteristics maximization of PEG, the total molecular weight of the PEG polymer that is connected with bioactive molecule and hydration status must be enough high to give the favorable characteristics of common PEG polymer join dependency, such as the water-soluble and circulating half-life that increases, and can influence the biologically active of parent molecule sharply.
Method and composition as herein described can be used for producing the polymer of homogeneous in fact: the protein conjugates preparation.As used herein, " homogeneous in fact " meaning refers to polymer according to observations: the protein conjugates molecule is greater than half of gross protein.Polymer: the Pegylation polypeptide formulations of " homogeneous in fact " of the present invention that protein conjugates biologically active and this paper are provided is enough homogeneous with the polypeptide formulations of the advantage that represents homogeneous preparation (for example, in the convenient clinical practice between every batch and every batch the predictability of pharmacokinetics).
Also can select to prepare polymer: the mixture of protein conjugates molecule, and the advantage that this paper provided is to select to desire to be included in the single polymers in the mixture: the ratio of protein conjugates.Therefore, in case of necessity, the polymer moieties that is connected that can prepare range protein and various quantity (promptly, two, three mixture, the fourth class) and with described concatenator and the single polymers that uses method preparation as herein described: protein conjugates combination, and obtain having predetermined single polymers: the mixture of protein conjugates ratio.
The ratio of peg molecule and protein molecule will change, and their concentration in reactant mixture also will change.In general, can determine best ratio (with regard to reaction efficiency, because have minute quantity excessive unreacted protein or polymer) by the molecular weight of selected polyethylene glycol and the quantity available of available reactive group.With regard to molecular weight, the molecular weight of polymer is high more usually, and the quantity of the polymer molecule that can be connected with protein is just few more so.Similarly, when optimizing these parameters, should consider the branch situation of polymer.In general, molecular weight high more (or branch is many more), polymer so: the ratio of protein is just high more.
As used herein and when containing hydrophilic polymer: during the polypeptides concatenator, the amount that term " treatment effective dose " further instigates the required benefit to the patient to increase.Described amount will be different with the difference of individuality, and will decide on multiple factor (comprising patient's the overall physical condition and the potential cause of disease of disease, illness or symptom that desire is treated).Can use by one of ordinary skill in the art and disclose the treatment effective dose that available material and program come easily to determine the present composition.
The quantity of the water-soluble polymer that described and the modified or not modified non-natural amino acid polypeptides of adjustable abridged edition literary composition is connected (promptly, Pegylation or glycosylated degree) so that pharmacology, pharmacokinetics or the pharmacodynamic profile of change (include but not limited to increase or reduce) to be provided, such as half life period in vivo.In certain embodiments, the half life period of polypeptide than increase without modified polypeptides at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 2 times, about 5 times, about 10 times, about 50 times or at least about 100 times.
In one embodiment, comprise the polypeptide that contains carbonyl or dicarbapentaborane alpha-non-natural amino acid with the PEG derivative modification that contains end two amine moieties that directly are connected with the PEG main chain.In another embodiment, comprise the polypeptide that contains ketone alkynes alpha-non-natural amino acid with the PEG derivative modification that contains end two amine moieties that directly are connected with the PEG main chain.
In certain embodiments, the terminal PEG derivative of diamines will have following structure:
RO-(CH 2CH 2O) n-O-(CH 2) m-CH 2-NH-NH 2，
Wherein R is simple alkyl (methyl, ethyl, a propyl group etc.), m be about 2 to about 10 and n be about 100 to about 1,000 (that is, mean molecule quantity is between about 5 to about 40kDa).The molecular weight of polymer can between the 000Da, include but not limited to about 100 between about 100Da and about 100,000Da, about 95,000Da, about 90,000Da, about 85,000Da, about 80,000Da, about 75,000Da, about 70,000Da, about 65,000Da, about 60,000Da, about 55,000Da, about 50,000Da, about 45,000Da, about 40,000Da, about 35,000Da, about 30,000Da, about 25,000Da, about 20,000Da, about 15,000Da, about 10,000Da, about 9,000Da, about 8,000Da, about 7,000Da, about 6,000Da, about 5,000Da, about 4,000Da, about 3,000Da, about 2,000Da, about 1,000Da, about 900Da, about 800Da, about 700Da, about 600Da, about 500Da, about 400Da, about 300Da, about 200Da and about 100Da.In certain embodiments, the molecular weight of polymer is between about 100Da and about 50, between the 000Da.In certain embodiments, the molecular weight of polymer is between about 100Da and about 40, between the 000Da.In certain embodiments, the molecular weight of polymer is between about 1, and 000Da and about 40 is between the 000Da.In certain embodiments, the molecular weight of polymer is between about 2, and 000Da is to about 50, between the 000Da.In certain embodiments, the molecular weight of polymer is between about 5, and 000Da and about 40 is between the 000Da.In certain embodiments, the molecular weight of polymer is between about 10, and 000Da and about 40 is between the 000Da.
In one embodiment, comprise the polypeptide that contains the dicarbapentaborane alpha-non-natural amino acid with the PEG derivative modification that contains the terminal ketoamine part that directly is connected with the PEG main chain.
In certain embodiments, the terminal PEG derivative of ketoamine will have following structure:
RO-(CH 2CH 2O) n-O-(CH 2) m-C(O)-CH 2-NH 2，
Wherein R is simple alkyl (methyl, ethyl, a propyl group etc.), m be about 2 to 10 and n be about 100 to about 1,000 (that is, mean molecule quantity is between about 5 to about 40kDa).The molecular weight of polymer can between the 000Da, include but not limited to about 100 between about 100Da and about 100,000Da, about 95,000Da, about 90,000Da, about 85,000Da, about 80,000Da, about 75,000Da, about 70,000Da, about 65,000Da, about 60,000Da, about 55,000Da, about 50,000Da, about 45,000Da, about 40,000Da, about 35,000Da, about 30,000Da, about 25,000Da, about 20,000Da, about 15,000Da, about 10,000Da, about 9,000Da, about 8,000Da, about 7,000Da, about 6,000Da, about 5,000Da, about 4,000Da, about 3,000Da, about 2,000Da, about 1,000Da, about 900Da, about 800Da, about 700Da, about 600Da, about 500Da, about 400Da, about 300Da, about 200Da and about 100Da.In certain embodiments, the molecular weight of polymer is between about 100Da and about 50, between the 000Da.In certain embodiments, the molecular weight of polymer is between about 100Da and about 40, between the 000Da.In certain embodiments, the molecular weight of polymer is between about 1, and 000Da and about 40 is between the 000Da.In certain embodiments, the molecular weight of polymer is between about 2, and 000Da is to about 50, between the 000Da.In certain embodiments, the molecular weight of polymer is between about 5, and 000Da and about 40 is between the 000Da.In certain embodiments, the molecular weight of polymer is between about 10, and 000Da and about 40 is between the 000Da.
In another embodiment, use the PEG derivative that contains the terminal dicarbapentaborane part that directly is connected to modify to comprise and contain the amino acid whose polypeptide of diamines with the PEG main chain.In another embodiment, use the PEG derivative that contains the terminal dicarbapentaborane part that directly is connected to modify to comprise and contain the amino acid whose polypeptide of ketoamine with the PEG main chain.
In certain embodiments, the terminal PEG derivative of dicarbapentaborane will have following structure:
RO-(CH 2CH 2O) n-O-(CH 2) 2-NH-C(O)(CH 2) m-C(O)-CH 2-C(O)-R，
Wherein R is simple alkyl (methyl, ethyl, a propyl group etc.), m be about 2 to about 10 and n be about 100 to about 1,000 (that is, mean molecule quantity is between about 5 to about 40kDa).The molecular weight of polymer can between the 000Da, include but not limited to about 100 between about 100Da and about 100,000Da, about 95,000Da, about 90,000Da, about 85,000Da, about 80,000Da, about 75,000Da, about 70,000Da, about 65,000Da, about 60,000Da, about 55,000Da, about 50,000Da, about 45,000Da, about 40,000Da, about 35,000Da, about 30,000Da, about 25,000Da, about 20,000Da, about 15,000Da, about 10,000Da, about 9,000Da, about 8,000Da, about 7,000Da, about 6,000Da, about 5,000Da, about 4,000Da, about 3,000Da, about 2,000Da, about 1,000Da, about 900Da, about 800Da, about 700Da, about 600Da, about 500Da, about 400Da, about 300Da, about 200Da and about 100Da.In certain embodiments, the molecular weight of polymer is between about 100Da and about 50, between the 000Da.In certain embodiments, the molecular weight of polymer is between about 100Da and about 40, between the 000Da.In certain embodiments, the molecular weight of polymer is between about 1, and 000Da and about 40 is between the 000Da.In certain embodiments, the molecular weight of polymer is between about 2, and 000Da is to about 50, between the 000Da.In certain embodiments, the molecular weight of polymer is between about 5, and 000Da and about 40 is between the 000Da.In certain embodiments, the molecular weight of polymer is between about 10, and 000Da and about 40 is between the 000Da.
In another embodiment, use the PEG derivative that contains the terminal ketone alkynyl moiety that directly is connected to modify to comprise and contain the amino acid whose polypeptide of diamines with the PEG main chain.
In certain embodiments, the terminal PEG derivative of ketone alkynes has following structure:
RO-(CH 2CH 2O) n-O-(CH 2) 2-NH-C(O)(CH 2) m-C(O)-C≡C-R ，
Wherein R is simple alkyl (methyl, ethyl, a propyl group etc.), m be about 2 to about 10 and n be about 100 to about 1,000 (that is, mean molecule quantity is between about 5 to about 40kDa).The molecular weight of polymer can between the 000Da, include but not limited to about 100 between about 100Da and about 100,000Da, about 95,000Da, about 90,000Da, about 85,000Da, about 80,000Da, about 75,000Da, about 70,000Da, about 65,000Da, about 60,000Da, about 55,000Da, about 50,000Da, about 45,000Da, about 40,000Da, about 35,000Da, about 30,000Da, about 25,000Da, about 20,000Da, about 15,000Da, about 10,000Da, about 9,000Da, about 8,000Da, about 7,000Da, about 6,000Da, about 5,000Da, about 4,000Da, about 3,000Da, about 2,000Da, about 1,000Da, about 900Da, about 800Da, about 700Da, about 600Da, about 500Da, about 400Da, about 300Da, about 200Da and about 100Da.In certain embodiments, the molecular weight of polymer is between about 100Da and about 50, between the 000Da.In certain embodiments, the molecular weight of polymer is between about 100Da and about 40, between the 000Da.In certain embodiments, the molecular weight of polymer is between about 1, and 000Da and about 40 is between the 000Da.In certain embodiments, the molecular weight of polymer is between about 2, and 000Da is to about 50, between the 000Da.In certain embodiments, the molecular weight of polymer is between about 5, and 000Da and about 40 is between the 000Da.In certain embodiments, the molecular weight of polymer is between about 10, and 000Da and about 40 is between the 000Da.
In another embodiment, modify to comprise with the branch PEG derivative that contains terminal two amine moieties and contain carbonyl or the amino acid whose polypeptide of dicarbapentaborane, wherein the molecular weight of each bar chain of branch PEG about 10 in the scope of about 40kDa and be about 5 to arrive about 20kDa in other embodiments.The molecular weight of branched polymers can be in broad range, include but not limited between about 100Da and about 100,000Da or higher between.The molecular weight of polymer can between the 000Da, include but not limited to about 100 between about 100Da and about 100,000Da, about 95,000Da, about 90,000Da, about 85,000Da, about 80,000Da, about 75,000Da, about 70,000Da, about 65,000Da, about 60,000Da, about 55,000Da, about 50,000Da, about 45,000Da, about 40,000Da, about 35,000Da, about 30,000Da, about 25,000Da, about 20,000Da, about 15,000Da, about 10,000Da, about 9,000Da, about 8,000Da, about 7,000Da, about 6,000Da, about 5,000Da, about 4,000Da, about 3,000Da, about 2,000Da, about 1,000Da, about 900Da, about 800 Da, about 700Da, about 600Da, about 500Da, about 400Da, about 300Da, about 200Da and about 100Da.In certain embodiments, the molecular weight of polymer is between about 100Da and about 50, between the 000Da.In certain embodiments, the molecular weight of polymer is between about 100Da and about 40, between the 000Da.In certain embodiments, the molecular weight of polymer is between about 1, and 000Da and about 40 is between the 000Da.In certain embodiments, the molecular weight of polymer is between about 2, and 000Da is to about 50, between the 000Da.In certain embodiments, the molecular weight of polymer is between about 5, and 000Da and about 40 is between the 000Da.In certain embodiments, the molecular weight of polymer is between about 10, and 000Da and about 40 is between the 000Da.
In another embodiment, comprise the polypeptide that contains the diamines alpha-non-natural amino acid with the branch PEG derivative modification that contains terminal dicarbapentaborane part.The molecular weight of branched polymers can be in broad range, include but not limited between about 100Da and about 100,000Da or higher between.The molecular weight of polymer can between the 000Da, include but not limited to about 100 between about 100Da and about 100,000Da, about 95,000Da, about 90,000Da, about 85,000Da, about 80,000Da, about 75,000Da, about 70,000Da, about 65,000Da, about 60,000Da, about 55,000Da, about 50,000Da, about 45,000Da, about 40,000Da, about 35,000Da, about 30,000Da, about 25,000Da, about 20,000Da, about 15,000Da, about 10,000Da, about 9,000Da, about 8,000Da, about 7,000Da, about 6,000Da, about 5,000Da, about 4,000Da, about 3,000Da, about 2,000Da, about 1,000Da, about 900Da, about 800Da, about 700Da, about 600Da, about 500Da, about 400Da, about 300Da, about 200Da and about 100Da.In certain embodiments, the molecular weight of polymer is between about 100Da and about 50, between the 000Da.In certain embodiments, the molecular weight of polymer is between about 100Da and about 40, between the 000Da.In certain embodiments, the molecular weight of polymer is between about 1, and 000Da and about 40 is between the 000Da.In certain embodiments, the molecular weight of polymer is between about 2, and 000Da is to about 50, between the 000Da.In certain embodiments, the molecular weight of polymer is between about 5, and 000Da and about 40 is between the 000Da.In certain embodiments, the molecular weight of polymer is between about 10, and 000Da and about 40 is between the 000Da.
In another embodiment, comprise the polypeptide that contains the ketoamine alpha-non-natural amino acid with the branch PEG derivative modification that contains terminal dicarbapentaborane part.The molecular weight of branched polymers can be in broad range, include but not limited between about 100Da and about 100,000Da or higher between.The molecular weight of polymer can between the 000Da, include but not limited to about 100 between about 100Da and about 100,000Da, about 95,000Da, about 90,000Da, about 85,000Da, about 80,000Da, about 75,000Da, about 70,000Da, about 65,000Da, about 60,000Da, about 55,000Da, about 50,000Da, about 45,000Da, about 40,000Da, about 35,000Da, about 30,000Da, about 25,000Da, about 20,000Da, about 15,000Da, about 10,000Da, about 9,000Da, about 8,000Da, about 7,000Da, about 6,000Da, about 5,000Da, about 4,000Da, about 3,000Da, about 2,000Da, about 1,000Da, about 900Da, about 800Da, about 700Da, about 600Da, about 500Da, about 400Da, about 300Da, about 200Da and about 100Da.In certain embodiments, the molecular weight of polymer is between about 100Da and about 50, between the 000Da.In certain embodiments, the molecular weight of polymer is between about 100Da and about 40, between the 000Da.In certain embodiments, the molecular weight of polymer is between about 1, and 000Da and about 40 is between the 000Da.In certain embodiments, the molecular weight of polymer is between about 2, and 000Da is to about 50, between the 000Da.In certain embodiments, the molecular weight of polymer is between about 5, and 000Da and about 40 is between the 000Da.In certain embodiments, the molecular weight of polymer is between about 10, and 000Da and about 40 is between the 000Da.
In another embodiment, comprise the polypeptide that contains ketone alkyl alpha-non-natural amino acid with the branch PEG derivative modification that contains terminal two amine moieties.The molecular weight of branched polymers can be in broad range, include but not limited between about 100Da and about 100,000Da or higher between.The molecular weight of polymer can between the 000Da, include but not limited to about 100 between about 100Da and about 100,000Da, about 95,000Da, about 90,000Da, about 85,000Da, about 80,000Da, about 75,000Da, about 70,000Da, about 65,000Da, about 60,000Da, about 55,000Da, about 50,000Da, about 45,000Da, about 40,000Da, about 35,000Da, about 30,000Da, about 25,000Da, about 20,000Da, about 15,000Da, about 10,000Da, about 9,000Da, about 8,000Da, about 7,000Da, about 6,000Da, about 5,000Da, about 4,000Da, about 3,000Da, about 2,000Da, about 1,000Da, about 900Da, about 800Da, about 700Da, about 600Da, about 500Da, about 400Da, about 300Da, about 200Da and about 100Da.In certain embodiments, the molecular weight of polymer is between about 100Da and about 50, between the 000Da.In certain embodiments, the molecular weight of polymer is between about 100Da and about 40, between the 000Da.In certain embodiments, the molecular weight of polymer is between about 1, and 000Da and about 40 is between the 000Da.In certain embodiments, the molecular weight of polymer is between about 2, and 000Da is to about 50, between the 000Da.In certain embodiments, the molecular weight of polymer is between about 5, and 000Da and about 40 is between the 000Da.In certain embodiments, the molecular weight of polymer is between about 10, and 000Da and about 40 is between the 000Da.
In another embodiment, comprise the polypeptide that contains the dicarbapentaborane alpha-non-natural amino acid with at least a PEG derivative modification with apparatus derivatorius.In certain embodiments, the PEG derivative that contains two amidos will have following structure:
RO-(CH 2CH 2O) n-O-(CH 2) m-CH 2-NH-NH 2，
Wherein R is simple alkyl (methyl, ethyl, a propyl group etc.), and X is NH, O, S, C (O) or do not exist according to circumstances, m be about 2 to about 10 and n be about 100 to about 1,000.
In another embodiment, comprise the polypeptide that contains the dicarbapentaborane alpha-non-natural amino acid with at least a PEG derivative modification with apparatus derivatorius.In certain embodiments, the PEG derivative that contains the ketoamine base will have following structure:
RO-(CH 2CH 2O) n-O-(CH 2) m-C(O)-CH 2-NH 2，
Wherein R is simple alkyl (methyl, ethyl, a propyl group etc.), and X is NH, O, S, C (O) or do not exist according to circumstances, m be about 2 to about 10 and n be about 100 to about 1,000.
In another embodiment, comprise the polypeptide that contains the diamines alpha-non-natural amino acid with at least a PEG derivative modification with apparatus derivatorius.In certain embodiments, the PEG derivative that contains dicarbapentaborane will have following structure:
RO-(CH 2CH 2O) n-O-(CH 2) 2-N H-C(O)(CH 2) m-C(O)-CH 2-C(O)-R
Wherein R is simple alkyl (methyl, ethyl, a propyl group etc.), and X is NH, O, S, C (O) or do not exist according to circumstances, m be about 2 to about 10 and n be about 100 to about 1,000.
In another embodiment, comprise the polypeptide that contains the diamines alpha-non-natural amino acid with at least a PEG derivative modification with apparatus derivatorius.In certain embodiments, the PEG derivative that contains the ketone alkynyl will have following structure:
RO-(CH 2CH 2O) n-O-(CH 2) 2-NH-C(O)(CH 2) m-C(O)-C≡C-R，
Wherein R is simple alkyl (methyl, ethyl, a propyl group etc.), and X is NH, O, S, C (O) or do not exist according to circumstances, m be about 2 to about 10 and n be about 100 to about 1,000.
In another embodiment, comprise the polypeptide that contains the ketoamine alpha-non-natural amino acid with at least a PEG derivative modification with apparatus derivatorius.In certain embodiments, the PEG derivative that contains dicarbapentaborane will have following structure:
RO-(CH 2CH 2O) n-O-(CH 2) 2-NH-C(O)(CH 2) m-C(O)-CH 2-C(O)-R ，
Wherein R is simple alkyl (methyl, ethyl, a propyl group etc.), and X is NH, O, S, C (O) or do not exist according to circumstances, m be about 2 to about 10 and n be about 100 to about 1,000.
In another embodiment, comprise the polypeptide that contains ketone alkyl alpha-non-natural amino acid with at least a PEG derivative modification with apparatus derivatorius.In certain embodiments, the PEG derivative that contains two amidos will have following structure:
RO-(CH 2CH 2O) n-O-(CH 2) m-CH 2-NH-NH 2，
Wherein R is simple alkyl (methyl, ethyl, a propyl group etc.), and X is NH, O, S, C (O) or do not exist according to circumstances, m be about 2 to about 10 and n be about 100 to about 1,000.
Can use relevant PEG is functionalized and link some comments and monograph.For example referring to 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 Reviews inTherapeutic.Drug Carrier Systems 9:249-304 (1992); Zalipsky, Bioconjugate Chem.6:150-165 (1995).
The method of polymer activation also is found in WO 94/17039, United States Patent (USP) the 5th, 324, No. 844, WO 94/18247, WO 94/04193, United States Patent (USP) the 5th, 219, No. 564, United States Patent (USP) the 5th, 122, No. 614, WO 90/13540, United States Patent (USP) the 5th, 281, No. 698 and WO 93/15189, and about the binding between living polymer and the enzyme, described enzyme includes but not limited to coagulation factor VIII (WO 94/15625), haemoglobin (WO 94/09027), oxygen carrier molecule (United States Patent (USP) the 4th, 412, No. 989), ribalgilase and superoxide dismutase (people such as Veronese, App.Biochem.Biotech.11:141-152 (1985)), the mode that all documents are all quoted in full is incorporated herein.
In case of necessity, can be further purified the Pegylation non-natural amino acid polypeptides as herein described that is obtained by the hydrophobicity chromatogram by the known program of one or more one of ordinary skill in the art, described program includes but not limited to affinity chromatography, anion or cation-exchange chromatography (including but not limited to use DEAE SEPHAROSE), silica gel chromatograph, reversed-phase HPLC, gel filtration (including but not limited to use SEPHADEX G-75), hydrophobic interaction chromatograph, size exclusion chromatography, immobilized metal ion afinity chromatography, ultrafiltration/saturating filter, precipitation with alcohol, ammonium sulfate precipitation, chromatofocusing, displcement chromatography, electrophoretic procedures (including but not limited to the isoelectric focusing of preparation type), differential dissolving (including but not limited to ammonium sulfate precipitation) or extraction.Can by GPC via with the globulin reference material relatively estimate apparent molecular weight (Preneta AZ,
Figure A200680049995D0203173441QIETU
Figure A200680049995D0203173458QIETU
(Harris ﹠amp; Angal compiles) IRL Press 1989,293-306).Can evaluate non-natural amino acid polypeptides by proteolytic degradation (including but not limited to the trypsase cracking) mass spectral analysis subsequently: the purity of PEG concatenator.People such as Pepinsky R.B., J. Pharmcol.﹠amp; Exp.Ther.297 (3): 1059-66 (2001).
The water-soluble polymer that is connected with the alpha-non-natural amino acid of polypeptide described herein can be in the case of unrestricted through further derivatization or replacement.
G. strengthen sero-abluminous affinity
Also various molecules and non-natural amino acid polypeptides as herein described can be merged to regulate serum half-life.In certain embodiments, with molecule with as herein described modified or is connected or merges with enhancing for modified non-natural amino acid polypeptides to the endogenous sero-abluminous affinity in the animal body.
For instance, in some cases, the reorganization fusion of preparation polypeptide and albumin binding sequence.Exemplary albumin binding sequence include but not limited to albumin from streptococcus protein G in conjunction with the territory (for example referring to people such as Makrides, people such as J.Pharmacol.Exp.Ther.277 (1): 534-542 (1996) and Sjolander, J, Immunol.Methods201:115-123 (1997)) or albumin binding peptide (such as people such as Dennis, the peptide described in J.Biol.Chem.277 (38): the 35035-35043 (2002)).
In other embodiments, utilize fatty acid with modified or not modified non-natural amino acid polypeptides acidylate as herein described.In some cases, fatty acid promotes to combine with sero-abluminous.For example referring to people such as Kurtzhals, Biochem.J.312:725-731 (1995).
In other embodiments, modified or not modified non-natural amino acid polypeptides as herein described and seralbumin (including but not limited to human serum albumins) are directly merged.One of ordinary skill in the art will recognize, also multiple other molecule can be connected combining with adjusting and seralbumin or other serum component with modified or not modified non-natural amino acid polypeptides as described herein.
H. the glycosylation of non-natural amino acid polypeptides as herein described
Method and composition as herein described comprises incorporates the polypeptide that one or more have the alpha-non-natural amino acid of saccharide residue into.Saccharide residue can be natural (including but not limited to the N-acetyl glucosamine) or non-natural (including but not limited to 3-fluorine galactose).Can by N-or O-connect glycosidic inkage (including but not limited to N-acetyl group galactose-L-serine) or non-natural key (include but not limited to heterocycle (comprising nitrogen heterocyclic ring) key or corresponding C-or S-connect glucosides) sugar and alpha-non-natural amino acid are linked together.
Sugar (including but not limited to glycosyl) part in vivo or in vitro can be added in the non-natural amino acid polypeptides.In certain embodiments, use through two amido derivatizations sugar-modified comprise contain the dicarbapentaborane alpha-non-natural amino acid polypeptide to produce the corresponding glycosylated polypeptides that connects via heterocycle (comprising nitrogen heterocyclic ring) key.In other embodiments, use through dicarbapentaborane derivatization sugar-modified comprise contain the diamines alpha-non-natural amino acid polypeptide to produce the corresponding glycosylated polypeptides that connects via heterocycle (comprising nitrogen heterocyclic ring) key.After alpha-non-natural amino acid is connected, can further modify sugar to produce the oligosaccharides that combines with non-natural amino acid polypeptides by handling with glycosyl transferase and other enzyme.For example referring to people such as H.Liu, J.Am.Chem.Soc.125:1702-1703 (2003).
I. connect the purposes and the application of base, comprise polypeptide dimer and polymer
Except that functional group is directly added to the non-natural amino acid polypeptides, also can at first connect the alpha-non-natural amino acid part of basic molecular modification polypeptide with multi-functional (for example, two, three, four), further modify subsequently.That is to say that at least one end of the basic molecule of multi-functional connection reacts with at least one alpha-non-natural amino acid in the polypeptide and multi-functional another end at least that is connected base can be used for further functionalized.If all ends of multi-functional connection base are identical, (decide) to form the same polymer of non-natural amino acid polypeptides so on stoichiometric condition.If the multi-functional end that connects base has different chemical reactivities, at least one end of so much functionality connection base will combine with non-natural amino acid polypeptides and another end can react with different functional groups subsequently, only for instance, described functional group comprises required functional group.
Multi-functional connection base has following universal architecture:
Figure A200680049995D02041
Wherein:
Each X is independently-J-R ,-K-R ,-G-C ≡-R or-C (O)-CH 2-NR 2Wherein,
J is
Figure A200680049995D02042
Or
Figure A200680049995D02051
Wherein:
R 8Be independently selected from H, alkyl, be substituted alkyl, cycloalkyl, be substituted cycloalkyl or amine protecting group;
R 9Be independently selected from H, alkyl, be substituted alkyl, cycloalkyl, be substituted cycloalkyl or amine protecting group;
T 1Be bond, the C that is substituted according to circumstances 1-C 4Alkylidene, the C that is substituted according to circumstances 1-C 4Alkenylene or the assorted alkyl that is substituted according to circumstances;
T 2Be the C that is substituted according to circumstances 1-C 4Alkylidene, the C that is substituted according to circumstances 1-C 4Alkenylene, the assorted alkyl that is substituted according to circumstances, the aryl that is substituted according to circumstances or the heteroaryl that is substituted according to circumstances;
Wherein each optional substituting group is independently selected from the low-carbon (LC) alkylidene, is substituted the low-carbon (LC) alkylidene, the low-carbon (LC) cycloalkylidene, is substituted low-carbon (LC) cycloalkylidene, low-carbon (LC) alkenylene, is substituted the low-carbon (LC) alkenylene, alkynylene, the inferior assorted alkyl of low-carbon (LC), is substituted inferior assorted alkyl, the inferior Heterocyclylalkyl of low-carbon (LC), is substituted low-carbon (LC) inferior Heterocyclylalkyl, arlydene, is substituted arlydene, inferior heteroaryl, is substituted inferior heteroaryl, alkarylene, is substituted alkarylene, inferior aralkyl or is substituted inferior aralkyl;
R is H, alkyl, is substituted alkyl, cycloalkyl or is substituted cycloalkyl;
K is
Figure A200680049995D02052
Figure A200680049995D02053
Figure A200680049995D02054
Or
Figure A200680049995D02055
G is
Figure A200680049995D02056
Or
Figure A200680049995D02057
Each R ' is H, alkyl independently or is substituted alkyl;
T 1And T 2Independently for the low-carbon (LC) alkylidene, be substituted low-carbon (LC) alkylidene, low-carbon (LC) cycloalkylidene, be substituted the low-carbon (LC) cycloalkylidene, the low-carbon (LC) alkenylene, be substituted low-carbon (LC) alkenylene, alkynylene, the inferior assorted alkyl of low-carbon (LC), be substituted inferior assorted alkyl, the inferior Heterocyclylalkyl of low-carbon (LC), be substituted low-carbon (LC) inferior Heterocyclylalkyl, arlydene, be substituted arlydene, inferior heteroaryl, be substituted inferior heteroaryl, alkarylene, be substituted alkarylene, inferior aralkyl or be substituted inferior aralkyl;
L and M independently for bond, H, low-carbon (LC) alkylidene, be substituted low-carbon (LC) alkylidene, low-carbon (LC) cycloalkylidene, be substituted the low-carbon (LC) cycloalkylidene, the low-carbon (LC) alkenylene, be substituted low-carbon (LC) alkenylene, alkynylene, the inferior assorted alkyl of low-carbon (LC), be substituted inferior assorted alkyl, the inferior Heterocyclylalkyl of low-carbon (LC), be substituted low-carbon (LC) inferior Heterocyclylalkyl, arlydene, be substituted arlydene, inferior heteroaryl, be substituted inferior heteroaryl, alkarylene, be substituted alkarylene, inferior aralkyl or be substituted inferior aralkyl, perhaps L and M can form aryl, heteroaryl, cycloalkyl or Heterocyclylalkyl together;
T 3For bond, C (R) (R), O or S;
T 4For
Figure A200680049995D02061
Or
Figure A200680049995D02062
Each X wherein 1Be independently selected from the group that forms by following each group :-O-,-S-,-N (H)-,-N (R)-,-N (Ac)-and-N (OMe)-; X 2For-OR ' ,-OAc ,-SR ,-N (R ') 2,-N (R ') (Ac) ,-N (R ') (OMe) or N 3
Each L is independently selected from the group that is made up of following each group: alkylidene, be substituted alkylidene, alkenylene, be substituted alkenylene ,-O-,-O-(alkylidene or be substituted alkylidene)-,-S-,-S-(alkylidene or be substituted alkylidene)-,-S (O) k-(wherein k is 1,2 or 3) ,-S (O) k(alkylidene or be substituted alkylidene)-,-C (O)-,-C (O)-(alkylidene or be substituted alkylidene)-,-C (S)-,-C (S)-(alkylidene or be substituted alkylidene)-,-N (R ')-,-NR '-(alkylidene or be substituted alkylidene)-,-C (O) N (R ')-,-CON (R ')-(alkylidene or be substituted alkylidene)-,-(alkylidene or be substituted alkylidene) NR ' C (O) O-(alkylidene or be substituted alkylidene)-,-O-CON (R ')-(alkylidene or be substituted alkylidene)-,-CSN (R ')-,-CSN (R ')-(alkylidene or be substituted alkylidene)-,-N (R ') CO-(alkylidene or be substituted alkylidene)-,-N (R ') C (O) O-,-N (R ") C (O) O-(alkylidene or be substituted alkylidene)-;-S (O) kN (R ')-,-N (R ') C (O) N (R ')-,-N (R ') C (O) N (R ')-(alkylidene or be substituted alkylidene)-,-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, L 1For-C (R ') P-NR '-C (O) O-(alkylidene or be substituted alkylidene)-, wherein p is 0,1 or 2;
W is-J-R ,-K-R ,-G-C ≡ C-R or-C (O)-CH 2-NR 2And n is 1 to 3.
Figure 20 provides synthetic difunctionality homotype to connect the illustrative example of base (homolinker), and wherein said connection base has two identical ends, i.e. two amidos.Described connect base can be used for forming contain the dicarbapentaborane non-natural amino acid polypeptides homodimer to form two heterocyclic bonds.Perhaps; if the described end of base that connects is through protection; can use described connection base through part protection will combining with containing the dicarbapentaborane non-natural amino acid polypeptides without the diamines of protection is terminal so via heterocyclic bond, thus stay other coupled reaction of being used in after protecting another through protecting end.In addition, although the result that required heterodimer will be polluted by some homodimers probably can occur, the stoichiometric reagent of handled can provide similar results (heterodimer).
Figure 24 provides by two kinds of protein are connected the illustrative example that protein derivedization is carried out in basic coupling via the difunctionality homotype, and wherein only for instance, described connection base connects base for PEG.
Figure 21 provides synthetic Heterobifunctional to connect the illustrative example of base, and wherein said connection base has two different ends, only for instance, and two amidos and azanol base.In addition, Figure 25 and Figure 27 are provided at multistep and use Heterobifunctional to be connected the basic illustrative example that the PEG group is connected with non-natural amino acid polypeptides in synthetic suddenly.In first step, so described in the illustrative drawings, contain the carbonyl non-natural amino acid polypeptides and contain the azanol difunctionality and be connected radical reaction and contain the oxime non-natural amino acid polypeptides with formation.Yet difunctionality connects base and still keeps two amine functional groups, its in second step can with the PEG reagent reacting that contains dicarbapentaborane to form the non-natural amino acid polypeptides of Pegylation via heterocyclic bond.
Figure 22 provides synthetic trifunctional to connect the illustrative example of base, and wherein said connection base has three different functional groups, only for instance, and two amidos and two azanol bases.In addition, Figure 26 is provided at the synthetic suddenly middle illustrative example of using trifunctional connection base that the PEG group is connected with the non-natural amino acid polypeptides dimer of multistep.In first step, so described in the illustrative drawings, contain the carbonyl non-natural amino acid polypeptides and be connected basic azanol partial reaction with trifunctional and contain oxime non-natural amino acid polypeptides dimer with formation.Yet trifunctional connects base and still keeps two amine functional groups, its in second step can with the PEG reagent reacting that contains dicarbapentaborane to form the non-natural amino acid polypeptides dimer of Pegylation via heterocyclic bond.
Method and composition as herein described also provides polypeptides in combination, such as homodimer, heterodimer, with polymer or heteromultimeric (that is, tripolymer, the tetramer etc.).Only for instance, below describe concentrating on GH supergene family member, yet the method described in this section, technology and composition can be applicable to provide almost any other polypeptide of the benefit of dimer and polymer form, only for instance, comprise required polypeptide.
Therefore, contain the GH supergene family member of containing one or more alpha-non-natural amino acids in method as herein described, technology and the composition, its with another GH supergene family member or its variant or for the polypeptide main chain of any other polypeptide of non-GH supergene family member or its variant directly in conjunction with or via being connected basic combination.Owing to compare with monomer, the molecular weight of GH supergene family member's dimer or polymer concatenator increases to some extent, so it can represent novel or required characteristic, includes but not limited to pharmacology, pharmacokinetics, the pharmacodynamics different with respect to monomer GH supergene family member; The treatment half life period through regulating; Or plasma half-life through regulating.In certain embodiments, GH supergene family member as herein described will regulate the dimerization of GH supergene family member acceptor.Plant in other is implemented, GH supergene family member's dimer as herein described or polymer will serve as GH supergene family member receptor antagonist, activator or conditioning agent.
In certain embodiments, GH supergene family member polypeptide is direct connection, includes but not limited to via Asn-Lys amido link or Cys-Cys disulfide bond.In certain embodiments, the GH supergene family member's polypeptide that connects and/or the non-GH supergene family member of connection will comprise different alpha-non-natural amino acids to promote dimerization, the non-GH supergene family member polypeptide that includes but not limited to comprise the GH supergene family member that contains the dicarbapentaborane alpha-non-natural amino acid and/or connection with comprise the 2nd GH supergene family member polypeptide that contains the diamines alpha-non-natural amino acid and link, and described polypeptide reacts via the formation of corresponding heterocycle (comprising nitrogen heterocyclic ring).
Perhaps, the non-GH supergene family member of two GH supergene family member's polypeptide and/or connection connects via connecting base.Can use any Heterobifunctional connection base or connect the non-GH supergene family member polypeptide that base connects two GH supergene family members and/or connection with difunctionality, it can have identical or different primary sequence.In some cases, be used for the connection base that the non-GH supergene family member polypeptide chain with GH supergene family member and/or connection is connected together and can be difunctionality PEG reagent.
In certain embodiments, method and composition as herein described provides the water-soluble difunctionality with dumbbell structure to connect base, and it comprises: a) contain azido, alkynes, hydrazine, diamines, hydrazides, azanol or carbonyl (comprising dicarbapentaborane) part on main polymer chain at least the first end; And b) at least the second functional group on main polymer chain second end.Second functional group can be identical or different with first functional group.In certain embodiments, second functional group can not with first functional group reactions.In certain embodiments, method and composition as herein described provides the water soluble compound of at least one arm that comprises the branch molecular structure.For instance, the branch molecular structure can be the dendron shape.
In certain embodiments, method and composition as herein described provides the polymer that comprises one or more GH supergene family members, and it is by forming with the reaction of water-soluble active polymer, and it has following structure:
R-(CH 2CH 2O) n-O-(CH 2) m-X，
Wherein n is about 5 to about 3,000, and m is about 2 to about 10, and X can be and contains azido, alkynes, hydrazine, diamines, hydrazides, azanol, acetyl group or carbonyl (comprising dicarbapentaborane) part, and R be can be identical or different with X END CAPPED GROUP, functional group or leaving group.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; the N-hydroxy-succinamide ester; 1-BTA ester; carbonic acid N-hydroxy-succinamide ester; carbonic acid 1-BTA ester; acetal; aldehyde; aldehydrol; thiazolinyl; acrylate; methacrylate; acrylamide; active sulfone; amine; aminooxy group; through protection amine; hydrazides; through the protection hydrazides; through protection mercaptan; carboxylic acid; through the protection carboxylic acid; isocyanates; different thiocyanic ester; maleimide; vinyl sulfone; two sulfo-pyridines; vinylpyridine; iodo-acetamide; epoxides; glyoxal; diketone; methanesulfonates; tosylate and triflate; alkene and ketone.In another embodiment, can use the connection base to connect transcription factor.The expression that gene needs multiple transcription factor to come the effectively start encoding proteins.Can connect the artificial activation that utilizes the synthetic transcription factor of alpha-non-natural amino acid and be used for the intensifier target gene via connection base as indicated above.Transcription factor through connecting can be under the situation that does not have normal activation signals cascade in conjunction with target DNA and promote raising of RNA polymerase, thereby under the situation of no desired signal expressing gene.In another embodiment, can connect the part of cell receptor with effective activated receptor.The growth factor (PDGF) in blood platelet source forms dimer so that in conjunction with its acceptor.Can connect the PDGF that contains alpha-non-natural amino acid via connection base as indicated above in dimer forms also offers medicine so that effective combination of pdgf receptor to be provided.Other embodiment of the protein that connects comprises the antibody of connection.Can connect stimulation, combination or the neutralization of two kinds of different antibody (separately to the unique epi-position tool specificity on identical or the adjacent target) to be used to strengthen.For instance, can connect two kinds of specific antibody of different epi-position tools being found on the gp120 of HIV and the gp40 that is associated to provide the more efficiently neutralization of target.Similarly, the antibody of connection can be used for the irritation cell surface receptor.For instance, can connect antibody at the CD3 of TXi Baoshouti and CD4 so that the necessary stimulus of receptor activation to be provided.Another embodiment comprises the peptide that is connected with nucleic acid.For instance, can will be connected with the therapeutic nucleic acids of required target with throwing in conjunction with the cell receptor of cell surface or the part of protein.The part of described connection promotes the picked-up of nucleic acid, thereby described subsequently nucleic acid is brought into play its therapeutic action at cell inner expression.Similarly, peptide can be connected with nucleic acid to promote the packing or the condensation of nucleic acid.
The functional group that connects on the base needn't be identical, also needn't be two amidos.The chemistry that uses this specification to describe in detail in the whole text, one of ordinary skill in the art can design a kind of connection base, and wherein at least one functional group can form heterocycle (comprising nitrogen heterocycle) with non-natural amino acid polypeptides; Another functional group that connects on the base can utilize other known chemistry, comprises the well-known chemistry based on nucleophilic/electrophilic reagent of organic chemistry filed.
J. add the example of functional group: the stalling characteristic of convenient polypeptide
Natural existence or non-natural amino acid polypeptides may be for a variety of reasons (include but not limited to the dissolubility of polypeptide or in conjunction with feature) and be difficult to separate from sample.For instance, in the polypeptide of preparation for treatment usefulness, described polypeptide can be from separating the engineered recombination system with excessive generation polypeptide.Yet,, confirm usually to be difficult to so reach required purity level owing to the dissolubility of polypeptide or in conjunction with feature.Method as herein described, composition, technology and strategy provide the solution of relevant this situation.
Use method as herein described, composition, technology and strategy, one of ordinary skill in the art can make with required homologous peptide contain the heterocycle non-natural amino acid polypeptides of (comprising nitrogen heterocyclic ring), the non-natural amino acid polypeptides that wherein contains heterocycle (comprising nitrogen heterocyclic ring) has uncertified separation characteristic.In one embodiment, produce the non-natural amino acid polypeptides of homology by biological synthesis method.In other or extra embodiment, alpha-non-natural amino acid is incorporated in the structure of one of alpha-non-natural amino acid as herein described.In other or extra embodiment, incorporate alpha-non-natural amino acid into end or interior location place and further incorporate into through locus specificity ground.
In one embodiment, had as the gained alpha-non-natural amino acid that produces by biological synthesis method required through improving separation characteristic.In other or extra embodiment, alpha-non-natural amino acid comprises and heterocycle (the comprising nitrogen heterocyclic ring) key that the group of improved separation characteristic is provided.In other or extra embodiment, alpha-non-natural amino acid is further modified to form modified heterocycle (the comprising nitrogen heterocyclic ring) non-natural amino acid polypeptides that contains, and wherein said modification provides and provides heterocycle (the comprising nitrogen heterocyclic ring) key of the group of improved separation characteristic.In certain embodiments, described group directly is connected with alpha-non-natural amino acid, and in other embodiments, described group is connected with alpha-non-natural amino acid via connecting base.In certain embodiments, described group is connected with alpha-non-natural amino acid, in other embodiments, needs series of chemical that described group is connected with alpha-non-natural amino acid by single chemical reaction.Preferably will give the group of improved separation characteristic and be connected, and under the reaction condition that is utilized, not be connected with naturally occurring amino acid with alpha-non-natural amino acid locus specificity ground in the non-natural amino acid polypeptides.
Be a kind of method that detects the existence of polypeptide in patient's body on the other hand, described method comprise throw with effective dose have the formula (XXXVIII) or (XXXIX) homology non-natural amino acid polypeptides of structure:
Figure A200680049995D02101
Wherein:
A is optional, and when existing, it is the low-carbon (LC) alkylidene, be substituted low-carbon (LC) alkylidene, low-carbon (LC) cycloalkylidene, be substituted the low-carbon (LC) cycloalkylidene, the low-carbon (LC) alkenylene, be substituted low-carbon (LC) alkenylene, alkynylene, the inferior assorted alkyl of low-carbon (LC), be substituted inferior assorted alkyl, the inferior Heterocyclylalkyl of low-carbon (LC), be substituted low-carbon (LC) inferior Heterocyclylalkyl, arlydene, be substituted arlydene, inferior heteroaryl, be substituted inferior heteroaryl, alkarylene, be substituted alkarylene, inferior aralkyl or be substituted inferior aralkyl;
B is optional, and when existing, its at one end with contain the connection base that two amine moieties are connected, described connection base is selected from the group that is made up of following each group: the low-carbon (LC) alkylidene, be substituted low-carbon (LC) alkylidene, low-carbon (LC) alkenylene, be substituted the low-carbon (LC) alkenylene, the inferior assorted alkyl of low-carbon (LC), be substituted the low-carbon (LC) Asia mix alkyl ,-O-(alkylidene or be substituted alkylidene)-,-S-(alkylidene or be substituted alkylidene)-,-C (O) R " ,-S (O) k(alkylidene or be substituted alkylidene)-(wherein k is 1,2 or 3) ,-C (O)-(alkylidene or be substituted alkylidene)-,-C (S)-(alkylidene or be substituted alkylidene)-,-NR " (alkylidene or be substituted alkylidene)-,-CON (R ")-(alkylidene or be substituted alkylidene)-,-CSN (R ")-(alkylidene or be substituted alkylidene)-and-N (R ") CO-(alkylidene or be substituted alkylidene)-, and each R wherein is " independently for H, alkyl or be 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 3And R 4Be H, halogen, low-carbon alkyl or be substituted low-carbon alkyl, perhaps R independently of one another 3And R 4Or two R 3Group forms cycloalkyl or Heterocyclylalkyl according to circumstances;
Z 1Be bond, CR 7R 7, O, S, NR ', CR 7R 7-CR 7R 7, CR 7R 7-O, O-CR 7R 7, CR 7R 7-S, S-CR 7R 7, CR 7R 7-NR ', NR '-CR 7R 7
Z 2Be selected from the group that forms by following each group: bond ,-C (O)-,-C (S)-, the C that is substituted according to circumstances 1-C 3Alkylidene, the C that is substituted according to circumstances 1-C 3Alkenylene and the assorted alkyl that is substituted according to circumstances;
R 6With each R 7Be independently selected from the group that forms by following each group: H, alkyl, be substituted alkyl, thiazolinyl, be substituted thiazolinyl, alkynyl, be substituted alkynyl, alkoxyl, be substituted alkoxyl, alkyl alkoxy, be substituted alkyl alkoxy, polyalkylene oxide, be substituted polyalkylene oxide, aryl, be substituted aryl, heteroaryl, be substituted heteroaryl, alkaryl, be substituted alkaryl, aralkyl, be substituted aralkyl ,-(alkylidene or be substituted alkylidene)-ON (R ") 2,-(alkylidene or be substituted alkylidene)-C (O) SR " ,-(alkylidene or be substituted alkylidene)-S-S-(aryl or be substituted aryl) ,-C (O) R " ,-C (O) 2R " or-C (O) N (R ") 2, each R wherein " independently for hydrogen, alkyl, be substituted alkyl, thiazolinyl, be substituted thiazolinyl, alkoxyl, be substituted alkoxyl, aryl, be substituted aryl, heteroaryl, alkaryl, be substituted alkaryl, aralkyl or be substituted aralkyl;
Or any two adjacent R 7Group forms 5 yuan to 8 yuan heterocycles, cycloalkyl or aromatic rings that are substituted according to circumstances together; Wherein said optional substituting group is selected from halogen, OH, C 1-6Alkyl, C 1-6Alkoxyl, halogen-C 1-6Alkyl, halogen-C 1-6Alkoxyl, aryl, halogen aryl and heteroaryl;
Condition is Z 1Add Z 2Provide and be no more than 3 annular atomses;
R 5For H, alkyl, be substituted alkyl, thiazolinyl, be substituted thiazolinyl, alkynyl, be substituted alkynyl, alkoxyl, be substituted alkoxyl, alkyl alkoxy, be substituted alkyl alkoxy, polyalkylene oxide, be substituted polyalkylene oxide, aryl, be substituted aryl, heteroaryl, be substituted heteroaryl, alkaryl, be substituted alkaryl, aralkyl, be substituted aralkyl ,-(alkylidene or be substituted alkylidene)-ON (R ") 2,-(alkylidene or be substituted alkylidene)-C (O) SR " ,-(alkylidene or be substituted alkylidene)-S-S-(aryl or be substituted aryl) ,-C (O) R " ,-C (O) 2R " or-C (O) N (R ") 2, each R wherein " independently for hydrogen, alkyl, be substituted alkyl, thiazolinyl, be substituted thiazolinyl, alkoxyl, be substituted alkoxyl, aryl, be substituted aryl, heteroaryl, alkaryl, be substituted alkaryl, aralkyl, be substituted aralkyl;
Or R 5Be L-X, wherein X is selected from the group that is made up of required functional group; And L is optional, and when existing, it is the connection base that is selected from the group that is made up of following each group: alkylidene, be substituted alkylidene, alkenylene, be substituted alkenylene ,-O-,-O-(alkylidene or be substituted alkylidene)-,-S-,-S-(alkylidene or be substituted alkylidene)-,-S (O) k-(wherein k is 1,2 or 3) ,-S (O) k(alkylidene or be substituted alkylidene)-,-C (O)-,-C (O)-(alkylidene or be substituted alkylidene)-,-C (S)-,-C (S)-(alkylidene or be substituted alkylidene)-,-N (R ')-,-NR '-(alkylidene or be substituted alkylidene)-,-C (O) N (R ')-,-CON (R ')-(alkylidene or be substituted alkylidene)-,-CSN (R ')-,-CSN (R ')-(alkylidene or be substituted alkylidene)-,-N (R ') CO-(alkylidene or be substituted alkylidene)-,-N (R ') C (O) O-,-(alkylidene or be substituted alkylidene)-O-N=CR '-,-(alkylidene or be substituted alkylidene)-C (O) NR '-(alkylidene or be substituted alkylidene)-,-(alkylidene or be substituted alkylidene)-S (O) k-(alkylidene or be substituted alkylidene)-S-,-(alkylidene or be substituted alkylidene)-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 each R ' is H, alkyl independently or is substituted alkyl;
Or its active metabolite, salt or pharmaceutically acceptable prodrug or solvate.
Another embodiment is a kind of method that detects the existence of polypeptide in patient's body, and described method comprises the homology non-natural amino acid polypeptides of throwing with effective dose, and wherein said alpha-non-natural amino acid is the specific site place that incorporates in the polypeptide.Another embodiment is a kind of method that detects the existence of polypeptide in patient's body, and described method comprises the homology non-natural amino acid polypeptides of throwing with effective dose, and wherein said alpha-non-natural amino acid is to use translation system to incorporate in the polypeptide.
Another embodiment is a kind of method that detects the existence of polypeptide in patient's body, and described method comprises the homology non-natural amino acid polypeptides of throwing with effective dose, and wherein said alpha-non-natural amino acid is to use the posttranslational modification system to incorporate in the polypeptide.Another embodiment is a kind of method that detects the existence of polypeptide in patient's body, and described method comprises the homology non-natural amino acid polypeptides of throwing with effective dose, and wherein said translation system comprises:
(i) polynucleotide of coded polypeptide, wherein said polynucleotide comprise the also corresponding selection codon in angle of striking that designs in advance with alpha-non-natural amino acid; With
The tRNA that (ii) comprises alpha-non-natural amino acid, wherein said tRNA is to described selection codon tool specificity.
Another embodiment is a kind of method that detects the existence of polypeptide in patient's body, and described method comprises the homology non-natural amino acid polypeptides of throwing with effective dose, and wherein said polynucleotide is the mRNA that is produced in the translation system.Another embodiment is a kind of method that detects the existence of polypeptide in patient's body, and described method comprises the homology non-natural amino acid polypeptides of throwing with effective dose, and wherein said translation system comprises plasmid DNA or phage DNA or the genomic DNA that comprises described polynucleotide.Other or extra embodiment are for detecting the method for the existence of polypeptide in patient's body, and described method comprises the homology non-natural amino acid polypeptides of throwing with effective dose, and wherein said polynucleotide stable integration is in genomic DNA.
Another embodiment is a kind of method that detects the existence of polypeptide in patient's body, described method comprises the homology non-natural amino acid polypeptides of throwing with effective dose, and wherein said translation system comprises being selected from the specific tRNA of selection codon tool by the molecular group of following password: amber codon, ochre codon, opal codon, unique codon, rare codon, non-natural codon, five base codons and four base codons.
Another embodiment is a kind of method that detects the existence of polypeptide in patient's body, and described method comprises the homology non-natural amino acid polypeptides of throwing with effective dose, and wherein said translation system comprises quadrature tRNA and quadrature aminoacyl tRNA synthetase.
Another embodiment is a kind of method that detects the existence of polypeptide in patient's body, and described method comprises the homology non-natural amino acid polypeptides of throwing with effective dose, and wherein said polynucleotide is synthetic by ribosome.Another embodiment is a kind of method that detects the existence of polypeptide in patient's body, described method comprises the homology non-natural amino acid polypeptides of throwing with effective dose, and wherein said translation system is the in vivo translation system that comprises the cell that is selected from following organism group: prokaryotes, eucaryote, mammal, Escherichia coli, pseudomonas, fungi, yeast, archeobacteria, eubacteria, plant, insect and protist.
Another embodiment is a kind of method that detects the existence of polypeptide in patient's body, described method comprises the homology non-natural amino acid polypeptides of throwing with effective dose, and wherein said translation system is the in vitro translation system that comprises from bacterial cell, archeobacteria cell or eukaryotic cell extract.Another embodiment is a kind of method that detects the existence of polypeptide in patient's body, described method comprises the homology non-natural amino acid polypeptides of throwing with effective dose, and the alpha-non-natural amino acid of wherein said polypeptide can stablized in the aqueous solution about 1 month between the pH value of about 2 pH value and about 8.Another embodiment is a kind of method that detects the existence of polypeptide in patient's body, and described method comprises the homology non-natural amino acid polypeptides of throwing with effective dose, and wherein said alpha-non-natural amino acid can be stablized at least 2 weeks approximately.Another embodiment is a kind of method that detects the existence of polypeptide in patient's body, and described method comprises the homology non-natural amino acid polypeptides of throwing with effective dose, and wherein said alpha-non-natural amino acid can be stablized at least 5 days approximately.
Another embodiment is a kind of method that detects the existence of polypeptide in patient's body, and described method comprises the homology non-natural amino acid polypeptides of throwing with effective dose, wherein said polypeptide for the protein of the therapeutic protein homology that is selected from the group that forms by required polypeptide.
Another embodiment is a kind of method that detects the existence of polypeptide in patient's body, and described method comprises the homology non-natural amino acid polypeptides of throwing with effective dose, and wherein said alpha-non-natural amino acid has formula (XLI) or (XLII) structure:
Figure A200680049995D02131
Each R wherein aBe independently selected from the group that forms by following each group: H, halogen, alkyl, be substituted alkyl ,-N (R ') 2,-C (O) N (R ') 2,-OR ', C (O) R ' and-S (O) kR ', wherein k is 1,2 or 3, and wherein each R ' is H, alkyl independently or is substituted alkyl.
Another embodiment is a kind of method that detects the existence of polypeptide in patient's body, and described method comprises the homology non-natural amino acid polypeptides of throwing with effective dose, and described alpha-non-natural amino acid has following structure:
Or
Figure A200680049995D02142
In other or extra embodiment, gained non-natural amino acid polypeptides and GH supergene family member homology, however the method described in this section, technology and composition can be applicable to can be from almost any other polypeptide of improved separation characteristic benefit, only for instance, it comprises required polypeptide.
In other or extra embodiment, the group of giving improved separation characteristic improves the water-soluble of polypeptide; In other embodiments, described group improves the binding characteristic of polypeptide; In other embodiments, described group provides new binding characteristic (only for instance, comprising vitamin h group or vitamin h conjugated group) for polypeptide.Improve among the water miscible embodiment of polypeptide at described group, described group is selected from water-soluble polymer as herein described, only for instance, comprises any PEG polymeric groups as herein described.
K. add the example of functional group: detect the existence of polypeptide
Natural existence or non-natural amino acid polypeptides may be for a variety of reasons (include but not limited to lack be easy to combine with polypeptide reagent or mark) and be difficult to detect in sample (comprising in vivo sample and in vitro sample).Method as herein described, composition, technology and strategy provide the solution for this situation.
Use method as herein described, composition, technology and strategy, one of ordinary skill in the art can make with required homologous peptide contain the heterocycle non-natural amino acid polypeptides of (comprising nitrogen heterocyclic ring), the non-natural amino acid polypeptides that wherein contains heterocycle (comprising nitrogen heterocyclic ring) allows to detect sample in vivo and the polypeptide in the sample in vitro.In one embodiment, produce the non-natural amino acid polypeptides of homology by biological synthesis method.In other or extra embodiment, alpha-non-natural amino acid is incorporated in the structure of one of alpha-non-natural amino acid as herein described.In other or extra embodiment, incorporate alpha-non-natural amino acid into end or interior location place and further incorporate into through locus specificity ground.
In one embodiment, had required detected characteristics as the gained non-natural amino acid polypeptides that produces by biological synthesis method.In other or extra embodiment, non-natural amino acid polypeptides comprises at least one alpha-non-natural amino acid that is selected from the group that is made up of following each thing so that improved detected characteristics to be provided: contain the carbonyl alpha-non-natural amino acid, contain the dicarbapentaborane alpha-non-natural amino acid, contain the diamines alpha-non-natural amino acid, contain the ketoamine alpha-non-natural amino acid, contain ketone alkynes alpha-non-natural amino acid and contain heterocycle (comprising nitrogen heterocyclic ring) amino acid.In other embodiments, by biological synthesis method described alpha-non-natural amino acid is incorporated in the polypeptide as described herein.In other or alternate embodiment, non-natural amino acid polypeptides comprises at least one amino acid whose alpha-non-natural amino acid that is selected from formula I-LXVII.In other or extra embodiment, alpha-non-natural amino acid comprises and the heterocyclic bond that the group of improved detected characteristics is provided.In other or extra embodiment, alpha-non-natural amino acid is further modified to form modified heterocycle (the comprising nitrogen heterocyclic ring) non-natural amino acid polypeptides that contains, and wherein said modification provides and provides heterocycle (the comprising nitrogen heterocyclic ring) key of the group of improved detected characteristics.In certain embodiments, described group directly is connected with alpha-non-natural amino acid, and in other embodiments, described group is connected with alpha-non-natural amino acid via connecting base.In certain embodiments, described group is connected with alpha-non-natural amino acid, in other embodiments, needs series of chemical that described group is connected with alpha-non-natural amino acid by single chemical reaction.Preferably will give the group of improved detected characteristics and be connected, and under the reaction condition that is utilized, not be connected with naturally occurring amino acid with alpha-non-natural amino acid locus specificity ground in the non-natural amino acid polypeptides.
In other or extra embodiment, gained non-natural amino acid polypeptides and GH supergene family member homology, yet, almost any other polypeptide that method described in this section, technology and composition can be applicable to sample in vivo and in vitro detect in the sample, only for instance, it comprises required polypeptide.
In other or extra embodiment, the group of giving improved detected characteristics is selected from the group that is made up of following each group: mark, dyestuff, affinity labeling, photoaffinity labeling, spin labeling, fluorogen, radioactive segment also have the part of heavy atom, isotope-labeled part, biophysics probe, phosphorescence group, chemiluminescent groups, electron dense group, magnetic group, chromophore, energy transfer agent, detectable label and its any combination.
In one embodiment, antagonist carries out engineered so that it contains the unique antigen on radioactive label and the antibody recognition cancer cell.Radioactive label is connected with the alpha-non-natural amino acid that is positioned at antibody.Utilize radioactive label via alpha-non-natural amino acid labelled antibody and purifying after labelled antibody, it thrown and suspect that suffer from can be by the individuality through the cancer of labelled antibody identification.Throwing with after labelled antibody the existence that can determine cancerous tissue through the existence and the position of labelled antibody in patient's body.One of ordinary skill in the art can utilize this system to determine that suitable antigen and cancer cell type are for detection.Similarly, one of ordinary skill in the art can determine suitable detection technique according to the radiolabeled type that is connected with antibody via alpha-non-natural amino acid.Throw with allow to detect through labelled antibody cancer in patient's body, in the individual body transfer and/or to individual body in the effect of treatment for cancer.
In another embodiment, to the peptide that combines with antigen on the cell surface carry out engineered so that its contain be used in to individuality throw with peptide after follow the trail of the dyestuff of described peptide, include but not limited to fluorescent dye.Described dyestuff is connected with peptide via the alpha-non-natural amino acid that is positioned at peptide, and described peptide is thrown with individual.Utilize imaging that one of ordinary skill in the art are easy to differentiate or detection technique to realize the location of peptide and its part or combine.
In another embodiment, via the alpha-non-natural amino acid that is positioned at peptide, polypeptide or protein metal group or containing metal part are connected with peptide, polypeptide or protein.With the peptide of suitable mark, polypeptide or protein throw with required individuality with via one of ordinary skill in the art known technology for detection and imaging.Through mark peptide, polypeptide or protein, can make various diseases, metabolic pathway, physiological structure or cellular component imaging by these.One of ordinary skill in the art can be identified for suitable target and the detection or the formation method of mark.For instance, can use magnetic resonance imaging (MRI) to detect the interior existence of individual body through mark peptide, polypeptide or protein.
L. add the example of functional group: the treatment characteristic of improving polypeptide
Natural existence or non-natural amino acid polypeptides can provide the particular treatment benefit to the patient who suffers from particular disorder, disease or symptom.Described treatment benefit will be decided on multiple factor, only comprise for instance: the safety profile of polypeptide seek peace pharmacokinetics, pharmacology and/or the pharmacodynamics (for example, water-soluble, biological usability, serum half-life, treatment half life period, immunogenicity, biologically active or circulation timei) of polypeptide.In addition, maybe advantageously provide other functional group, such as the cytotoxic compound or the medicine that connect to polypeptide; It is as herein described with polymer and heteromultimeric to form maybe may to need to connect other polypeptide.Described modification preferably can not destroy the activity and/or the tertiary structure of original polypeptide.Method as herein described, composition, technology and strategy provide at these ways to solve the problem.
Use method as herein described, composition, technology and strategy, one of ordinary skill in the art can make with required homologous peptide contain the heterocycle non-natural amino acid polypeptides of (comprising nitrogen heterocyclic ring), the non-natural amino acid polypeptides that wherein contains heterocycle (comprising nitrogen heterocyclic ring) has improved treatment feature.In one embodiment, produce the non-natural amino acid polypeptides of homology by biological synthesis method.In other or extra embodiment, alpha-non-natural amino acid is incorporated in the structure of one of alpha-non-natural amino acid as herein described.In other or extra embodiment, incorporate alpha-non-natural amino acid into end or interior location place and further incorporate into through locus specificity ground.
In one embodiment, had as the gained alpha-non-natural amino acid that produces by biological synthesis method required through improving the treatment feature.In other or extra embodiment, alpha-non-natural amino acid comprises and heterocycle (the comprising nitrogen heterocyclic ring) key that the group of improved treatment feature is provided.In other or extra embodiment, alpha-non-natural amino acid is further modified to form modified heterocycle (the comprising nitrogen heterocyclic ring) non-natural amino acid polypeptides that contains, and wherein said modification provides and provides heterocycle (the comprising nitrogen heterocyclic ring) key of the group of improved treatment feature.In certain embodiments, described group directly is connected with alpha-non-natural amino acid, and in other embodiments, described group is connected with alpha-non-natural amino acid via connecting base.In certain embodiments, described group is connected with alpha-non-natural amino acid, in other embodiments, needs series of chemical that described group is connected with alpha-non-natural amino acid by single chemical reaction.Preferably will give the group of improved treatment feature and be connected, and under the reaction condition that is utilized, not be connected with naturally occurring amino acid with alpha-non-natural amino acid locus specificity ground in the non-natural amino acid polypeptides.
In other or extra embodiment, gained non-natural amino acid polypeptides and GH supergene family member homology, however the method described in this section, technology and composition can be applicable to can be from almost any other polypeptide of improved treatment feature benefit, only for instance, it comprises required polypeptide.
In other or extra embodiment, the group of giving improved treatment feature improves the water-soluble of polypeptide; In other embodiments, described group improves the binding characteristic of polypeptide; In other embodiments, described group provides new binding characteristic (only for instance, comprising vitamin h group or vitamin h conjugated group) for polypeptide.Improve among the water miscible embodiment of polypeptide at described group, described group is selected from water-soluble polymer as herein described, only for instance, comprises the 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, medicine that can connect from the non-natural amino acid polypeptides cracking or cytotoxic compound are so that be delivered to required treatment position with medicine or cytotoxic compound.In other embodiments, described group is second polypeptide, for example comprises containing the heterocycle non-natural amino acid polypeptides of (comprising nitrogen heterocyclic ring), comprises the polypeptide that for example has the amino acid structure identical with first non-natural amino acid polypeptides in addition.
In other or extra embodiment, the non-natural amino acid polypeptides that contains heterocycle (comprising nitrogen heterocyclic ring) is the modified heterocycle non-natural amino acid polypeptides of (comprising nitrogen heterocyclic ring) that contains.In other or extra embodiment, with respect to the naturally occurring amino acid polypeptide of homology, the non-natural amino acid polypeptides that contains heterocycle (comprising nitrogen heterocyclic ring) increases the biological usability of polypeptide.In other or extra embodiment, with respect to the naturally occurring amino acid polypeptide of homology, the non-natural amino acid polypeptides that contains heterocycle (comprising nitrogen heterocyclic ring) increases the security features of polypeptide.In other or extra embodiment, with respect to the naturally occurring amino acid polypeptide of homology, the non-natural amino acid polypeptides that contains heterocycle (comprising nitrogen heterocyclic ring) makes the water-soluble increase of polypeptide.In other or extra embodiment, with respect to the naturally occurring amino acid polypeptide of homology, the non-natural amino acid polypeptides that contains heterocycle (comprising nitrogen heterocyclic ring) increases the treatment half life period of polypeptide.In other or extra embodiment, with respect to the naturally occurring amino acid polypeptide of homology, the non-natural amino acid polypeptides that contains heterocycle (comprising nitrogen heterocyclic ring) increases the serum half-life of polypeptide.In other or extra embodiment, with respect to the naturally occurring amino acid polypeptide of homology, the non-natural amino acid polypeptides that contains heterocycle (comprising nitrogen heterocyclic ring) prolongs the circulation timei of polypeptide.In other or extra embodiment,, contain the biologically active that the non-natural amino acid polypeptides of heterocycle (comprising nitrogen heterocyclic ring) is regulated polypeptide with respect to the naturally occurring amino acid polypeptide of homology.In other or extra embodiment,, contain the immunogenicity that the non-natural amino acid polypeptides of heterocycle (comprising nitrogen heterocyclic ring) is regulated polypeptide with respect to the naturally occurring amino acid polypeptide of homology.
XI. the therapeutical uses of modified polypeptide
For simplicity, prevailingly and/or utilize particular instance to describe the modified or not modified non-natural amino acid polypeptides described in this section.Yet, universal description or particular instance that modified or not modified non-natural amino acid polypeptides described in this section should not only limit in this section to be provided, but the modified or not modified non-natural amino acid polypeptides described in this section is equally applicable to comprise all modified or not modified non-natural amino acid polypeptides of at least one alpha-non-natural amino acid in formula I-LXVII scope, comprise this specification, claims and graphic described in any minor or specific compound in formula I-LXVII scope.
Modified or not modified non-natural amino acid polypeptides as herein described (comprising that it is with polymer and heteromultimeric) serves many purposes, and includes but not limited to: the treatment, the diagnosis, based on calibrating, industry, cosmetics, botany, environment, energy generation, the consumer goods and/or military use.As non-limitative illustration, provide the following therapeutical uses of modified or not modified non-natural amino acid polypeptides.
Modified or not modified non-natural amino acid polypeptides as herein described can be used for treating various disease conditions, symptom or disease.Throw with modified or not modified non-natural amino acid polypeptides product as herein described and can in human body, cause in the activity that commercially available polypeptide formulations represents any.The average magnitude of modified or not modified non-natural amino acid polypeptides product can change and especially should be based upon on the basis of medical practitioner's recommendation and prescription.The exact amount of modified or not modified non-natural amino acid polypeptides is the problem of different people, different views, and it is to be foundation with the factor such as other composition in the definite type of treated symptom, the patient's that treated situation and the composition.One of ordinary skill in the art can easily determine the amount desiring to give according to the therapy of utilizing modified or not modified non-natural amino acid polypeptides.
A. offer medicine and medical composition
Modified or not modified non-natural amino acid polypeptides as described herein (include but not limited to synzyme, comprise the protein of one or more alpha-non-natural amino acids etc.) (includes but not limited to) be used for the treatment of purposes with suitable medical supporting agent combination according to circumstances.For instance, described composition comprises the modified or not modified non-natural amino acid polypeptides as described herein for the treatment of effective dose and pharmaceutically acceptable supporting agent or excipient.Described supporting agent or excipient include but not limited to physiological saline, buffer saline, dextrose, water, glycerine, ethanol and/or its combination.Composite adapts through being prepared into the dispensing pattern.In general, well-known throwing in affiliated field and method of protein and its can be applicable to throw and modified or not modified non-natural amino acid polypeptides as described herein.
According to the well-known method in affiliated field, test comprises the therapeutic composition of one or more modified or not modified non-natural amino acid polypeptides as described herein in one or more suitable in vitro and/or in vivo animal disease models according to circumstances, to determine effect, tissue metabolism and to assess dosage.Specifically, activity that at first can be by non-natural and natural amino acid homologue, stability or other are suitably measured (including but not limited to more modified polypeptide and natural amino acid polypeptide to comprise one or more alpha-non-natural amino acids) (promptly in relevant calibrating) and are determined dosage.
Dispensing can be introduced molecule so that it carries out with blood or the tight any approach that contacts of histocyte by being generally used for.Modified or not modified non-natural amino acid polypeptides as described herein be in any suitable manner according to circumstances with one or more pharmaceutically acceptable supporting agents throw with.Throw with the proper method of modified or not modified non-natural amino acid polypeptides as described herein all available to the patient, although and can use more than one approach to throw and particular composition, particular approach can provide faster and more effective effect or reaction than another approach usually.
Pharmaceutically acceptable supporting agent be by throw and particular composition and the ad hoc approach that is used to throw with composition partly determine.Therefore, the suitable composite that has multiple medical composition as herein described.
Can throw and non-natural amino acid polypeptides as herein described and the composition that comprises described polypeptide by any conventional route of protein or peptide that is applicable to, described approach includes but not limited to without the intestines approach, for example includes but not limited to injection or any other injection or the infusion form of subcutaneous or intravenous injection.Polypeptide medical composition (comprising various non-natural amino acid polypeptides as herein described) can by number of ways throw with, include but not limited in oral, intravenous, the peritonaeum, in the muscle, transdermal, subcutaneous, local, hypogloeeis or rectal.Also can throw and the composition that comprises modified or not modified non-natural amino acid polypeptides as described herein via liposome.One of ordinary skill in the art are known described dosing way and suitable composite usually.Non-natural amino acid polypeptides as herein described can use separately or be used in combination with other suitable component (including but not limited to medical supporting agent).
Separately or with the modified or not modified non-natural amino acid polypeptides as described herein of other suitable combination of components also can be made into aerosol composite (that is, it can " nebulize ") with via suck throw with.The aerosol composite can be put into pressurization and can accept propellant, such as dicholorodifluoromethane, propane, nitrogen etc.
Be suitable for without intestines dispensings (such as by in (in the joint), intravenous, the muscle in the joint, in the intracutaneous, peritonaeum and subcutaneous route) composite comprise water-based and the aseptic parenteral solution of opening such as non-aqueous, it can contain antioxidant, buffer, bacteriostatic agent and make composite and solute that the blood of predetermined acceptor etc. is opened, and the water-based and the non-aqueous sterile suspensions that can comprise suspending agent, solubilizer, thickener, stabilizing agent and preservative.Composite through packing nucleic acid can be provided in unit dose or multiple dose airtight container (such as ampoule and bottle).
Without intestines dispensing and intravenous dispensing is preferred medication administration method.Specifically, the dosing way (including but not limited to be generally used for EPO, IFN, GH, G-CSF, GM-CSF, IFN, interleukins, antibody and/or any other approach of transferrin matter pharmaceutically) and the composite of current use that has been used for natural amino acid homologue therapeutic agent provides preferred dosing way modified or not modified non-natural amino acid polypeptides and composite as described herein.
In the situation of composition as herein described and method, throwing is enough to cause in time useful therapeutic response with patient's dosage in patient's body.Activity, stability or the serum half-life of the effect by specific composite, employed modified or not modified non-natural amino acid polypeptides and patient's symptom and desire treatment patient's body weight or surface area are determined dosage.Dosage size also existence, the nature and extent of any adverse side effect of the dispensing by following particular composition in particular patient waits to determine.
In the treatment of determining disease (including but not limited to cancer, genetic disease, diabetes, AIDS etc.) or prevention desire throw and the effective dose of composite the time, doctor's evaluation cycle plasma content, composite toxicity, progression of disease and/or when relevant () anti-non-natural amino acid polypeptides production of antibodies.
For example throw with 70 kilograms of patients' dosage usually in the scope of the dosage of the therapeutic protein that equals current use, described scope can be adjusted at the change of compositions related activity or serum half-life.Pharmaceutical formulation as herein described can be come the supplementary therapy condition by any known routine treatment, comprise antibody throw with, vaccine is thrown and, throwing and cytotoxic agent, natural amino acid polypeptide, nucleic acid, nucleotide analog, biological response modifier etc.
For dispensing, pharmaceutical formulation as herein described be with the speed of determining by the LD-50 of relative allocation thing or ED-50 throw with, and/or include but not limited to when quality that is applied to the patient and general health, observe any side effect of the modified of various concentration or not modified non-natural amino acid polypeptides.Dispensing can single dose or divided dose realization.
If fever, shiver with cold or myalgia appear in the patient of experience composite infusion, it receives aspirin (aspirin), brufen (ibuprofen), paracetamol (acetaminophen) or other pain/fever control medicine of suitable dosage so.Before infusion will be carried out 30 minutes, give aspirin, brufen, paracetamol or (including but not limited to) diphenhydramine (diphenhydramine) in advance to the patient of experience infusion reaction (such as fever, myalgia and shiver with cold).Pethidine (Meperidine) is used for can not be to antipyretic and even more serious shiver with cold and the myalgia of antihistaminicum rapid-action.The order of severity of visual response is slowed down or is interrupted cell infusion.
Modified or not modified non-natural amino acid polypeptides as described herein can directly be thrown and mammalian subject.Dispensing can be by being generally used for that polypeptide is introduced individual any approach.Modified or not modified non-natural amino acid polypeptides as described herein comprises and is suitable for per os, per rectum, part, suction (including but not limited to via aerosol), oral cavity (including but not limited to the hypogloeeis), transvaginal, without intestines (include but not limited in subcutaneous, the muscle, in the intracutaneous, joint, in the pleura, in the peritonaeum, in the brain, intra-arterial or intravenous), part (promptly, skin and mucomembranous surface, comprise airway surface) and the transdermal dispensing, but optimal approach will be decided on the character and the order of severity of treatment symptom under any particular cases.Dispensing can be local or whole body.Can in unit dose or multiple dose airtight container (such as ampoule and bottle), provide composite.Modified or not modified non-natural amino acid polypeptides as described herein can be prepared into the mixture of unit dose injectable forms (including but not limited to solution, suspension or emulsion) with pharmaceutically acceptable supporting agent.Modified or not modified non-natural amino acid polypeptides as described herein also can by continuous infusion (including but not limited to use micropump), single group such as osmotic pump annotate or slowly-releasing storage tank formula composite throw with.
The composite that is suitable for offeing medicine comprise water-based and non-aqueous solution, etc. open sterile solution, the solute that it can contain antioxidant, buffer, bacteriostatic agent and composite etc. is opened; And water-based and non-aqueous sterile suspensions, it can comprise suspending agent, solubilizer, thickener, stabilizing agent and preservative.Solution and suspension can be by aseptic powdery, particle and the preparation tablets of previous described kind.
Freeze drying is to be used for the common technology that protein is provided that water is removed from the protein formulation of being paid close attention to.Freeze drying or freeze-drying are that at first will to desire dry material freezing and remove the process of ice or chilled solvent subsequently by distillation in vacuum environment.The stability that can comprise lyophilized products when excipient stores to strengthen stability and/or improvement in freezing dry process in the composite of freeze-drying in advance.Pikal, people Pharm.Res.8 (3): 285-291 (1991) such as M.Biopharm.3 (9) 26-30 (1990) and Arakawa.
One of ordinary skill in the art are the atomized drying of known drug also.For instance, referring to Drug Dev.Ind.Pharm, 18 (11 ﹠amp; 12), Broadhead among the 1169-1206 (1992), people such as J., " The Spray Drying of Pharmaceuticals ".Except that small-molecule drug, also multiple biomaterial atomized drying and these materials are comprised: enzyme, serum, blood plasma, microorganism and yeast.Because atomized drying can change into no dust or reunion fine powder with single stage technology with liquid pharmaceutical preparation, so atomized drying is useful technology.Basic fundamental comprises following four steps: a) make feedstock solution be atomized into spraying; B) spraying-air contact; C) dry spraying; With d) desciccate is separated with dry air.United States Patent (USP) the 6th, 235, No. 710 and the 6th, 001, description prepares recombinant erythropoietin by atomized drying in No. 800 (its mode of quoting in full is incorporated herein).
Medical composition as herein described can comprise pharmaceutically acceptable supporting agent, excipient or stabilizing agent.Pharmaceutically acceptable supporting agent be by throw and particular composition and the ad hoc approach that is used to throw with composition partly determine.Therefore, the multiple suitable composite (comprising optionally pharmaceutically acceptable supporting agent, excipient or stabilizing agent) that has medical composition modified or not modified non-natural amino acid polypeptides as herein described is (for example referring to Remington:TheScience 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, Marcel Decker, New York, N.Y., 1980; With Pharmaceutical Dosage Forms and Drug DeliverySystems, the 7th edition (Lippincott Williams ﹠amp; Wilkins, 1999)).Suitably supporting agent comprises buffer solution, and it contains succinate, phosphate, borate, HEPES, citrate, imidazoles, acetate, bicarbonate and other organic acid; Antioxidant includes but not limited to ascorbic acid; Low molecular weight polypeptide includes but not limited to the polypeptide less than about 10 residues; Protein includes but not limited to seralbumin, gelatin or immunoglobulin; Hydrophilic polymer includes but not limited to polyvinylpyrrolidone; Amino acid includes but not limited to glycine, glutamine, asparagine, arginine, histidine or histidine derivative, methionine, glutamate or lysine; Monose, disaccharides and other carbohydrate include but not limited to trehalose, sucrose, glucose, mannose or dextrin; Chelating agent includes but not limited to EDTA and natrium adetate (edentate disodium); Bivalent metal ion includes but not limited to zinc, cobalt or copper; Sugar alcohol includes but not limited to mannitol or sorbitol; The salify equilibrium ion includes but not limited to sodium; And/or non-ionic surface active agent, include but not limited to Tween TM(including but not limited to Tween 80 (polysorbate80) and Tween 20 (polysorbate20)), Pluronics TM(include but not limited to general stream niacin F68 (poloxamer 188 (poloxamer 188)) or PEG with other general stream niacin (pluronic acid).Suitable surfactant for example includes but not limited to poly-(oxirane)-poly-(expoxy propane)-poly-(oxirane) (promptly, (PEO-PPO-PEO)) or poly-(expoxy propane)-poly-(oxirane)-poly-(expoxy propane) (that is, (PPO-PEO-PPO)) or its be combined as the polyethers on basis.PEO-PPO-PEO and PPO-PEO-PPO are on the market with trade name Pluronics TM, R-Pluronics TM, Tetronics TMAnd R-Tetronics TM(BASF Wyandotte Corp., Wyandotte Mich.) sell and are further described in United States Patent (USP) the 4th, 820, and in No. 352, the mode that described patent is quoted in full is incorporated herein.Other ethylene/polypropylene block polymers can be suitable surfactant.The combination of surfactant or surfactant can be used for making the Pegylation non-natural amino acid polypeptides stable to one or more stress (including but not limited to by the stress that stirs generation).More above-mentioned materials can be described as " increasing long-pending agent (bulking agent) ".Some also can be described as " tension change agent (tonicitymodifier) ".Antibiotic antiseptic also can be applicable to product stability and antibiotic effect; Suitably preservative includes but not limited to phenmethylol, benzalkonium chloride (benzalkonium chloride), metacresol, methyl p-hydroxybenzoate/propylparaben, cresols and phenol or its combination.
Modified or not modified non-natural amino acid polypeptides as described herein (comprising the polypeptide that is connected with water-soluble polymer) such as PEG can also by sustained release system or as the part of sustained release system throw with.Sustained-release composition comprises the semipermeability polymer substrate of (including but not limited to) shaping article form, includes but not limited to film or micro-capsule.Lasting release matrix comprises the bio-compatible material, such as poly-(2-hydroxyethyl methacrylate) (people such as Langer, J.Biomed.Mater.Res., 15:267-277 (1981); Langer, Chem.Tech., 12:98-105 (1982)), ethylene vinyl acetate (people such as Langer, with above) or poly-D-(-)-3-hydroxybutyric acid (EP 133,988), polylactide (PLA) (United States Patent (USP) the 3rd, 773, No. 919; EP 58,481), poly-glycolide (glycolic acid polymer), polylactide copolymerization glycolide (copolymer of lactic acid and glycolic), polyanhydride, the copolymer of L-glutamic acid and γ-ethyl-L-glutamate (people such as U.Sidman, Biopolymers, 22,547-556 (1983)), poly-(ortho acid) ester, polypeptide, hyaluronic acid, collagen, chondroitin sulfate, carboxylic acid, fatty acid, phosphatide, polysaccharide, nucleic acid, polyaminoacid, amino acid is (such as phenyl alanine, tyrosine, isoleucine), polynucleotide, polyethylene propylene, polyvinylpyrrolidone and silicone.Sustained-release composition also comprises liposome embedded compound.The liposome that can contain compound by following known method preparation own: DE 3,218, and 121; People such as Eppstein, 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; EP36,676; EP 143,949; Japanese patent application case 83-118008; United States Patent (USP) the 4th, 485, No. 045, the 4th, 619, No. 794, the 5th, 021, No. 234 and the 4th, 544, No. 545; And EP 102,324.
Can be by for example DE 3,218,121; 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., 11:4030-4034 (1980); EP 52,322; EP36,676; EP 143,949; Japanese patent application case 83-118008; United States Patent (USP) the 4th, 485, No. 045, the 4th, 619, No. 794, the 5th, 021, No. 234 and the 4th, 544, No. 545; And the method described in the EP 102,324 prepares liposome embedded polypeptide.The composition of liposome and size have been known for people or can easily have been determined with one of ordinary skill in the art's experience.Some case descriptions of liposome are in people such as for example Park JW, Proc.Natl.Acad.Sci.USA92:1327-1331 (1995); Lasic D and Papahadjopoulos D (volume):
Figure A200680049995D0222174914QIETU
Figure A200680049995D0223174929QIETU
(1998); People such as Drummond DC, Liposomal drug delivery systems for cancer therapy, Teicher B (volume):
Figure A200680049995D0223174950QIETU
(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 are among the Cancer Res.63:3154-3161 (2003).
In the situation of composition as herein described, composite and method, throwing should be enough to cause in time useful reaction with patient's dosage in individual body.In general, every dose throw without intestines and the total medical effective dose of modified or not modified non-natural amino acid polypeptides as described herein in about 0.01 microgram of per kilogram weight in patients every day to about 100 micrograms of per kilogram weight in patients, perhaps every day per kilogram of body weight about 0.05 milligram in the about 1 milligram scope of per kilogram weight in patients, but this is to be judged as foundation with treatment.Administration frequency also is to be judged as foundation with treatment, and it is higher or lower to be used for human commercially available prod frequency than approval.In general, polymer as described herein: polypeptide concatenator (only comprising the Pegylation polypeptide for instance) can by in the above-mentioned dosing way any throw with.
XII. structure-the functional relationship of modified polypeptide
Modified or not modified non-natural amino acid polypeptides as described herein (include but not limited to synzyme, comprise the protein of one or more alpha-non-natural amino acids etc.) will be given different physics of its residing polypeptide and chemical feature.The validity of described feature will be decided on the structure of modifying on the structure of alpha-non-natural amino acid, the alpha-non-natural amino acid or the two, and can be assessed via the structure-emic experimental model of evaluation test polypeptide.
In the experimental model of any appointment, replace natural amino acid in required polypeptide or the protein with alpha-non-natural amino acid.Contain the peptide or protein of alpha-non-natural amino acid in expression after, utilize alternative R group library to make protein derivedization.These R groups can with contained alpha-non-natural amino acid reaction in polypeptide or the protein.By selecting R group library with structure or chemical similarity through the R of replacement amino acid group.After in the alpha-non-natural amino acid that novel R group is added in the protein, then screen protein at function in the suitable test macro or activity.For instance, in protein, replace phenyl alanine with alpha-non-natural amino acid.Add in the alpha-non-natural amino acid in the substituting R group library that subsequently the R group with phenyl alanine is had similar features.Single substituting R group is added in the non-natural R group, and the R group that is added comprises ring, heterocycle, conjugate ring or other chemical part of giving (but being not limited to) similar chemistry and architectural feature.Screen derivatization protein and the interpolation function associated alpha-non-natural amino acid that replaces recently by test in the suitable experimental model of determining easily one of ordinary skill in the art subsequently.The example of experimental model includes but not limited to basis calibrating (based assay), acellular calibrating, the calibrating based on cell, tissue culture object model and animal model.
In another embodiment, indoles is replaced on the alpha-non-natural amino acid with the pharmacophore that is used for drug discovery active or in detection as useful fluorescent core.For promoting described interpolation, utilize two step reaction optimized to form based on the R group of indoles or be applicable to that the R group that indoles synthesizes adds in the alpha-non-natural amino acid by at room temperature in aqueous buffer solution, carrying out indoles.After this reaction, the required activity of screening derivatization protein.
For instance, can assess the middle alpha-non-natural amino acid of sour α glucosidase (GAA) in Pang Beishi disease (Pompe disease) mouse model replaces alleviating the effect of Pang Beishi disease.Can produce and be expressed in the GAA molecular library that selected site in the enzyme contains various aminoacid replacement via present invention disclosed herein.Subsequently can be in the mouse model of Pang Beishi disease (through breeding into the mouse of GAA genetic defect (GAA-/-)) assessment as disclosed herein not modified or through the activity of the enzyme that contains alpha-non-natural amino acid of posttranslational modification form.Can or allow any other dosing way of effective protein delivery and absorption to throw and the enzyme that contains alpha-non-natural amino acid through intravenous, per os.Can be by the measurement of degraded of the glycogen in the mouse body and/or clearance rate; The evaluation of the serum content of GAA; Cardiomegaly, cardiomyopathy, bone myopathy or other one of ordinary skill in the art are easy to the change of the sign differentiating and monitor or reduce and assess alleviating of dispensing effect, half life of enzyme and Pang Beishi disease.
Modified or not modified non-natural amino acid polypeptides as herein described can be used in the multiple commercial Application.Use in modified or not modified non-natural amino acid polypeptides product as herein described can cause that commercially available polypeptide formulations represented in commercial Application the activity any.
For instance, available alpha-non-natural amino acid is modified the change that is used to produce the enzyme of ethanol and examines and determine its function.Can produce and express via present invention disclosed herein and contain alcohol dehydrogenase II and the pyruvate decarboxylase library that various alpha-non-natural amino acids replace.Can replace a change of the alcohol of giving or causing generation efficient at alpha-non-natural amino acid subsequently and screen the enzyme of modifying through alpha-non-natural amino acid.Can easily screen during (but being not limited to) make to the increase of the affinity of substrate and conversion ratio and with the industry that described increase is applied to ethanol by well-known technology in the affiliated field.
Other example of the commercial Application of present invention disclosed herein comprises the environment removing of weed killer herbicide and insecticide.Promote from contaminated soil, to remove atrazine with the enzyme that makes weed killer herbicide atrazine (atrazine) metabolism commonly used, thereby make soil nontoxic.Can produce and express via present invention disclosed herein and contain the modified atrazine chlorine hydrolase library that alpha-non-natural amino acid replaces.The change of the ability of the atrazine dechlorination that can find at making in the environment and alpha-non-natural amino acid replace a library that any new model of the atrazine metabolism of giving or causing is screened the atrazine chlorine hydrolase of modifying through alpha-non-natural amino acid subsequently.As mentioned before, can include but not limited to the increase of atrazine or intermediate metabolism via the change of well-known technical evaluation enzyme efficient in the affiliated field.
Example
Example 1
Figure A200680049995D02241
Synthetic
Employed synthetic being described in the following reaction scheme:
Figure A200680049995D02251
A)
Figure A200680049995D02252
Synthetic
(5.0g 31mmol) adds Ac in the solution in pyridine (50mL) to matulane to 1-under 0 ℃ 2O (30mL, 318mmol).At room temperature with the mixture stirred overnight and with MeOH (100mL) stopped reaction.After removing solvent in a vacuum, by flash chromatography (silica, 20-50% EtOAc/ hexane) purifying residue to obtain colorless oil (6.72g, 87%): 1HNMR (500MHz, CDCl 3) δ 7.28 (d, J=8.4Hz, 2H), 7.24 (d, J=8.4Hz, 2H), 2.47 (s, 6H), 2.40 (s, 3H), 2.14 (s, 3H); 13C NMR (125 MHz, CDCl3) δ 171.8,169.5, and 139.1,138.8,130.4,126.4,25.4,22.3,21.3.
B)
Figure A200680049995D02253
Synthetic
To N ', (6.4g is 25.8mmol) in CCl for N '-diacetyl-N-p-methylphenyl acethydrazide 4Add in the solution (300mL) N-bromosuccinimide (5.1g, 28.7mmol).Heating blends under refluxing.Adding 2,2 '-azobis isobutyronitrile (AIBN, 0.2g, 1.2mmol).Under refluxing, the gained mixture was stirred 36 hours and cool to room temperature.With mixture H 2Anhydrous Na is used in O and salt water washing 2SO 4The bromide (8.62g) to obtain being brown oil is filtered and concentrated to drying.The not purified crude product that is about to is directly used in the next step.
C)
Figure A200680049995D02254
Synthetic
To EtONa (2.3g, 32.1mmol) add in the solution in EtOH (80mL) the 2-diethyl acetamido (6.3g, 29.0mmol).Under 0 ℃, the gained mixture was stirred 20 minutes.Disposable adding N ', and N '-diacetyl-N-(4-(bromomethyl) phenyl) acethydrazide (8.62g, 26.4mmol).Under 80 ℃, mixture is heated whole night and cool to room temperature.(10g 50mmol) is added in the reactant mixture with citric acid.After removing most of solvent, with EtOAc (500mL) dilution residue.With mixture H 2Anhydrous Na is used in O and salt water washing 2SO 4Drying is filtered and is concentrated.By 2-(4-(acetamido) the benzyl)-2-diethyl acetamido (4.17g, two steps 35%) of flash chromatography (silica, 15-80% EtOAc/ hexane) purifying residue to obtain being yellow oil: 1HNMR (500MHz, CDCl 3) δ 7.23 (d, J=8.0Hz, 2H), 7.03 (d, J=8.0Hz, 2H), 6.57 (s, 1H), 4.29-4.20 (m, 4H), 3.65 (m, 2H), 2.41 (s, 6H), 2.08 (s, 3H), 2.01 (s, 3H), 1.27 (t, J=3.6Hz, 6H); 13C NMR (125 MHz, CDCl 3) δ 171.7,169.3,169.2,167.4,140.3,136.4,131.3,126.2,67.2,63.0,37.4,25.3,23.2,22.3,14.2.
D)
Figure A200680049995D02261
Synthetic
To 2-(4-(acetamido) benzyl)-2-diethyl acetamido (572mg, 1.24mmol) add in the solution in the Yu diox (15mL) HCl (12N, 15mL).Under refluxing, the heating of gained mixture is concentrated whole night and in vacuum.In residue, add MeOH (1mL).Adding ether (200mL) is the product (231mg, 81%) of solid, shaped with precipitation: 1H NMR (500MHz, D 2O) δ 7.28 (d, J=8.5Hz, 2H), 7.00 (d, J=8.5Hz, 2H), 4.21 (dd, J=7.4,5.7Hz, 1H), 3.26 (dd, J=9.2,5.7Hz, 1H), 3.15 (dd, J=14.7,7.4Hz, 1H); 13C NMR (125MHz, D 2O) δ 171.5,142.9, and 130.3,129.0,115.7,54.1,34.7.
Example 2
Figure A200680049995D02262
Synthetic
Employed synthetic being described in the following reaction scheme:
Figure A200680049995D02271
A)
Figure A200680049995D02272
Synthetic
In NaOH solution (40mL, 25 volume %), add ether (60mL) under 0 ℃.Anti-riot shielding is placed on the reaction bulb front.Through 3 parts of 3 minutes branches in the gained mixture, add N-nitroso-N-methyl ureas (6.0g, 57.9mmol).Under 0 ℃, will react and stir 10 minutes.Ether is separated with the sodium hydroxide layer.(7.5g is 25.4mmol) in the solution in anhydrous THF (20mL), up to raw material complete obiteration (by the TLC monitoring) through 5 minutes organic layer to be added the N-Boc-4-hydroxymethyl phenylalanine by part (about 6 interpolations).Add 5 glacial acetic acids subsequently with stopped reaction.Behind rotary evaporation removal organic solvent, add ethyl acetate.With the saturated NaHCO of organic layer 3Solution, H 2O and salt solution continuous washing are used anhydrous MgSO subsequently 4Drying, filter and concentrate to obtain the pulverous product (5.9g, 75%) that is white in color: 1H NMR (500MHz, CDCl 3) δ 7.27 (d, J=8.0Hz, 2H), 7.09 (d, J=8.0Hz, 2H), 5.01 (d, J=7.9Hz, 1H), 4.63 (s, 2H), 4.55 (dt, J=7.7,6.2Hz, 1H), 3.69 (s, 3H), 3.10 (dd, J=13.8,5.7Hz, 1H), 3.02 (dd, J=13.8,6.0Hz, 1H), 2.02 (br s, 1H), 1.40 (s, 9H); 13C NMR (125MHz, CDCl 3) δ 172.5,155.3,139.9,135.5,129.6,127.4,80.1,65.0,54.6,52.4,38.1,28.4.
B)
Figure A200680049995D02281
Synthetic
Under 0 ℃ to alcohol (6.0g, 19.4mmol) and pyridine (12mL is 150mmol) in CH 2Cl 2(400mL) in agitating solution, add the high iodine alkane of Dai Si-Martin (14.2g, 33.4mmol).Under the room temperature with the mixture stirred overnight.Subsequently by adding saturated Na 2S 2O 3-NaHCO 3The aqueous solution (1:1,300mL) stopped reaction and use CH 2Cl 2Extraction.Organic layer merged and use H 2Anhydrous Na is used in O and salt water washing subsequently 2SO 4Drying is filtered and is concentrated in a vacuum.By flash chromatography (silica, 1:100-1:1 hexane: EtOAc) the purifying residue aldehyde product (5.48g, 92%) of solid, shaped that obtains being white in color: 1H NMR (500MHz, CDCl 3) δ 9.98 (s, 1H), 7.81 (d, J=7.8Hz, 2H), 7.30 (d, J=7.8Hz, 2H), 5.04 (d, J=7.8Hz, 1H), 4.62 (dt, J=7.2,6.2Hz, 1H), 3.71 (s, 3H), 3.21 (dd, J=13.7,5.7Hz, 1H), 3.10 (dd, J=13.7,6.4Hz, 1H), 1.40 (s, 9H); 13C NMR (125MHz, CDCl 3) δ 192.1,172.1,155.2,143.7,135.5,130.3,130.1,80.4,54.4,52.6,38.9,28.5.
C)
Figure A200680049995D02282
Synthetic
(3.07g 10mmol) adds tert-butyl carbazate in the solution in hexane (150mL) to above-mentioned aldehyde.Under refluxing with gained mixture heating 1 hour and concentrate.In residue, add BH 3THF (1M, 10mL, 10mmol).At room temperature with mixture stirring 15 minutes and by adding saturated NaHCO 3The solution stopped reaction.Extract mixture with EtOAc.With organic layer H 2Anhydrous Na is used in O and salt water washing subsequently 2SO 4Drying is filtered and is concentrated in a vacuum.By flash chromatography (silica, 2:1-1:2 hexane: EtOAc) the purifying residue product (3.1g, 73%) of solid, shaped that obtains being white in color.
D)
Figure A200680049995D02283
Synthetic
(1.3g 3.1mmol) adds LiOH (10mL, 1 N) in the solution in diox (10mL) to above-mentioned methyl esters under 0 ℃.Under uniform temp, mixture stirred 1 hour and by add citric acid solution (5%, 200mL) stopped reaction.Extract mixture with EtOAc.With organic layer H 2Anhydrous Na is used in O and salt water washing subsequently 2SO 4The acid (1.08g, 86%) to obtain being solid, shaped is filtered and concentrated to drying.
E)
Figure A200680049995D02291
Synthetic
(1.0g is 2.4mmol) in CH to above-mentioned acid under 0 ℃ 2Cl 2Add trifluoroacetic acid (20mL) in the solution (10mL).Under 0 ℃, reactant mixture was stirred 2 hours and concentrated in a vacuum.In residue, add MeOH (1mL), add HCl (2.0mL is in the 4N Yu diox) subsequently.Adding ether (200mL) is the product (0.4g, 80%) of solid, shaped with precipitation.
Example 3
Provided among this example in detail Fig. 5 to contain dicarbapentaborane amino acid whose synthetic.Preparation as shown in Figure 5 contains the alpha-non-natural amino acid of dicarbapentaborane.
Example 4
Provided among this example in detail Fig. 6 to contain dicarbapentaborane amino acid whose synthetic.Preparation as shown in Figure 6 contains the alpha-non-natural amino acid of dicarbapentaborane.
Example 5
Provided among this example in detail Fig. 7 to contain diamines amino acid whose synthetic.Preparation as shown in Figure 7 contains the alpha-non-natural amino acid of diamines.
Example 6
Provided among this example in detail Fig. 8 to contain diamines amino acid whose synthetic.Preparation as shown in Figure 8 contains the alpha-non-natural amino acid of diamines.
Example 7 self-contained dicarbapentaborane amino acid and contain diamines reagent and form pyrazoles.
Figure A200680049995D02292
(pH8.5 10mM) adds diketone in the solution in the tris buffer solution to methyl hydrazine (0.15mL).At room temperature with mixture stirring 3 hours and by adding citric acid solution (5%) stopped reaction.With EtOAc extraction gained mixture.With organic layer H 2Anhydrous Na is used in O and salt water washing subsequently 2SO 4Drying is filtered and is concentrated.(silica, the 10:1-1:1 hexane: EtOAc) the purifying residue is with the product (isomer ratio of about 3:1,117mg, 81%) of the solid, shaped that obtains being white in color by flash chromatography.
Example 8
This example in detail contains diamines amino acid as provide among Fig. 9 with containing the modification of dicarbapentaborane reagent.
Example 9
This example in detail contains diamines amino acid as provide among Figure 10 with containing the modification of dicarbapentaborane reagent.
Example 10
This example in detail contains dicarbapentaborane amino acid as provide among Figure 12 with containing the modification of diamines reagent.
Example 11
This example in detail connects the synthetic of base as the two amine-functionalized PEG that provided among Figure 18.
Example 12
This example in detail connects the synthetic of base as the functionalized PEG of the dicarbapentaborane that is provided among Figure 19.
Example 13
This example in detail connects the synthetic of base as the Bifunctionalized PEG of the diamines that is provided among Figure 20.
Example 14
This example in detail is synthetic as the Heterobifunctional connection base that provided among Figure 21.
Example 15
This example in detail trifunctional as shown in Figure 22 connects the synthetic of base.
Example 16
This example in detail makes the hGH Pegylation as provide among Figure 23 with containing diamines PEG reagent.
Example 17
This example in detail makes two hGH polypeptide dimerizations as provide among Figure 24 with containing diamines difunctionality PEG connection base.
Example 18
This example in detail makes the hGH Pegylation as provide among Figure 25 with Heterobifunctional connection base.
Example 19
This example in detail makes two hGH polypeptide dimerizations as provide among Figure 26 with containing azanol trifunctional connection base, makes hGH dimer Pegylation subsequently.
Example 20
Clone and the expression of the modified polypeptide of this example in detail in Escherichia coli.Use the translation system of introducing that comprises quadrature tRNA (O-tRNA) and quadrature aminoacyl tRNA synthetase (O-RS) to express the polypeptide that contains alpha-non-natural amino acid.O-RS preferentially utilizes alpha-non-natural amino acid to make the O-tRNA aminoacylization.Then, the selection codon of translation system response coding inserts alpha-non-natural amino acid in the polypeptide.Can be used for incorporating into O-tRNA and the amino acid of O-RS and the U.S. patent application case the 10/126th that polynucleotide sequence is described in " In Vivo Incorporation of Unnatural AminoAcids " by name of alpha-non-natural amino acid, the United States Patent (USP) application case the 10/126th of No. 927 and by name " Methods and Compositions for theProduction of Orthogonal tRNA-Aminoacyl tRNA Synthetase Pairs ", in No. 931, described patent is incorporated herein by reference.Also can use following O-RS and O-tRNA sequence:
Figure A200680049995D02311
Figure A200680049995D02321
With containing modified gene and quadrature aminoacyl tRNA synthetase/tRNA plasmid transformation escherichia coli, thereby alpha-non-natural amino acid locus specificity ground is incorporated in the polypeptide (to required alpha-non-natural amino acid tool specificity).That grows in the medium that contains the specific alpha-non-natural amino acid between about 0.01 to about 100mM under 37 ℃ expresses modified polypeptide through transformed into escherichia coli with high fidelity and efficient.E. coli host cell produces the polypeptide that contains alpha-non-natural amino acid of His mark with the form of inclusion body or aggregation.Aggregation is being dissolved and affinity purification in the 6M guanidine hydrochloride under the sex change condition.By under about 4 ℃ in about 50mM TRIS-HCl (about pH8.0) and about 40 μ M CuSO 4And dialysis is carried out folding whole night again in about 2% (w/v) sodium lauroyl sarcosine (Sarkosyl).Subsequently to about 20mMTRIS-HCl (about pH8.0) and about 100mM NaCl and about 2mM CaCl 2The dialysis material is removed the His label subsequently.Referring to people such as Boissel, (1993) 268:15983-93.The method of purified polypeptide is confirmed in affiliated field as everyone knows and by SDS-PAGE, Western engram analysis or electron spray ionisation ion trap mass spectrometry etc.
Following case description is measured and the activity in vitro and in vivo of more modified therapeutic activity non-natural amino acid polypeptides and the active in vitro and in vivo method of therapeutic activity natural amino acid polypeptide.
Example 21: cell is in conjunction with calibrating
Under 0 ℃, there is not or exists various concentration (volume: 10 μ l) under the situation of GH, the hGH of un-marked or GM-CSF and in existence 125Under the situation of I-GH (about 100,000cpm or 1ng), in duplicate with cell (3 * 10 6Individual) cultivate (cumulative volume: 120 μ l) that in PBS/1% BSA (100 μ l), reaches 90 minutes.The ice-cold FCS of 200 μ l that subsequently cell is suspended again and be tiled in the 350 μ l plastic centrifuge tubes goes up and centrifugal (1000g; 1 minute).Collect centrifugal and in gamma counter (Packard), centrifugal and supernatant are counted separately by clipping the pipe end.
Determine that with the combination (cpm) (non-specific binding) that deducts in the total binding under the situation that does not have competitor (bipartite mean value) under the situation of the GH that has 100 times of excessive un-marked specificity is in conjunction with (cpm).Measure the non-specific binding of employed each cell type.Use is with a kind of 125The I-GH preparation different date carry out experiment and its should manifest in uniformity. 125I-GH represents with the GH acceptor is celliferous and combines.The natural GH or the hGH of un-marked suppress combination in the dose dependent mode, and GM-CSF or other negative control are quite different.Similar with natural GH, the hGH competition is with natural 125The ability of the combination of I-GH shows two kinds of forms of the same identification of described acceptor.
Example 22: make the in vivo research of hGH Pegylation via heterocyclic bond
To mouse or rat throwing and PEG-hGH, not modified hGH and buffering solution.The result will show, compare with not modified hGH, and Pegylation hGH of the present invention has the higher activity and the half life period of prolongation, and this is indicated by the body weight of remarkable increase.
Example 23: the hGH of binding and non-binding and the measurement of the in vivo half life period of its variant
All zooperies all are to carry out in the facility that AAALAC authorizes and according to the scheme that the management of laboratory animal and the use committee (Institutional Animal Care and Use Committee) of St.Louis University ratified.With rat be housed in individually have 12 hours the daytime/cage in the room of night circulation in.Certified Purina rodent food 5001 is provided and lets alone free drinking water to animal.For going the hypophysis rat, contain 5% glucose in the drinking water in addition.
Example 24: pharmacokinetic study
Before entering zoopery, assess the quality of each Pegylation mutant hGH by three calibratings.(Invitrogen, Carlsbad CA) run 4-12% acrylamide NuPAGE Bis-Tris gel about the purity that glue is checked PEG-hGH (via the heterocyclic bond Pegylation) by run the glue buffer solution with MES SDS under non-reduced condition.With Coomassie blue (Coomassie blue) with gel-colored.According to density measurement scanning, the bands of a spectrum of PEG-hGH are pure greater than 95%.By using from Charles River Laboratories (Wilmington, KTA MA) 2The dynamics LAL of kit examines and determine the endotoxin content of testing among each PEG-hGH, and every dose of described content is less than 5EU.Utilize the biologically active of IM-9 pSTAT5 biological assay evaluation PEG-hGH, and through confirming EC 50Value is less than 15nM.
Compared to each other and compare at the pharmacokinetic properties of the growth hormone compound of from the male Sprague-Dawley rat (261-425g) that Charles River Laboratories obtains, PEG being modified with somatotropin without Pegylation.Underwent operative is installed in the arteria carotis conduit for blood collecting.After successfully conduit being installed, before administration, animal is assigned in the treatment group (three to six every group).To the subcutaneous 1mg/kg compound that gives the 0.41-0.55ml/kg dose volume of animal via.When each time point, collect blood sample and put it in the microcentrifugal tube that is coated with EDTA via built-in conduit.Centrifugal back is collected blood plasma and is stored up to analysis down at-80 ℃.Use from BioSource International (Camarillo, CA) or Diagnostic Systems Laboratories (Webster, TX) antibody sandwich somatotropin ELISA kit is measured compound concentration.Use the reference material calculating concentration corresponding with the analog that is given.Use modeling program WinNonlin (Pharsight, 4.1 versions) estimation pharmacokinetic parameter.Use utilizes the non-compartment model analysis of linear-up/log-down trapezoidal integration and to the even weighting of concentration data.
After to rat single subcutaneous administration, under regular intervals, obtain plasma concentration.Give the single dose of rat (every group of n=3-6 only) 1mg/kg protein.Comprise with the hGH polypeptide (his-hGH) of hGH wild-type protein (WHO hGH), His mark or at each place of 6 diverse locations with the covalently bound alpha-non-natural amino acid of 30kDa PEG to acetyl phenyl alanine through the hGH of His mark polypeptide with WHO hGH and (his)-hGH compares.Obtain plasma sample under the time interval and evaluate the compound of being injected as described in rule.Following table shows that single dose is thrown and the pharmacokinetic parameter value of various hGH polypeptide.By non-compartment model analysis (Pharsight, 4.1 versions) assessment concentration time curve.The value of being showed is mean value (+/-standard deviation).Cmax: Cmax; Last eventually T1/2: t1/2; AUC 0-＞inf: be extrapolated to infinitely-great area under the concentration-time curve; MRT: mean residence time; Cl/f: apparent total plasma clearance; And Vz/f: the apparent volume of distribution during latter stage.According to observations, hGH compares with contrast, and 30KPEG-pAF92 (his) hGH makes circulation obviously prolong, increase serum half-life and increases biological usability.
Table: to the pharmacokinetic parameter value of subcutaneous throwing of normal male Sprague-Dawley rat single and single dose 1mg/kg
Figure A200680049995D02341
Figure A200680049995D02351
Example 25: drug efficacy study
Obtain to go the hypophysis male Sprague-Dawley rat from Charles River Laboratories.Exenterate hypophysis when age in 3-4 week.Animal is shaked down time in three weeks, monitor body weight during this period.Be included in before the treatment beginning in 7 day time the body weight increment and be the animal of 0-8g and it is assigned in the treatment group at random.To rat through subcutaneous throwing and single dose or every day dosage.In whole research process, every day and continuously rat being weighed, anesthesia, blood drawing and administration (working as where applicable).Use the heparinize capillary to collect blood and put it into the microcentrifugal tube that is coated with EDTA from orbital sinus.Store up to analysis down by centrifugal separation plasma and at-80 ℃.Draw average (+/-S.D.) plasma concentration is to the figure in the time interval.
Peptide IGF-1 is the member of somatomedin or insulin-like growth factor family.Many growth-promoting effects of IGF-1 mediating growth hormone.Use competitive desmoenzyme immunoassays kit to measure IGF-1 concentration at the rat/mouse IGF-1 reference material that is provided (Diagnosic Systems Laboratories).Make rat remove hypophysis.Through subcutaneous give rat (every group n=5-7 only) single dose or every day dosage.Weigh to rat every day continuously, anesthesia, blood drawing and administration (working as where applicable).Obtain the hGH ((his) hGH) of placebo treatment, wild type hGH (hGH), His mark and comprise the body weight result to the hGH polypeptide of acetyl phenyl alanine covalently bound with 30kDa PEG at the 35th and the 92nd.The weight increase that 30KPEG-pAF35 (his) hGH compound causes the 9th day the time is different from 30KPEG-pAF92 (his) hGH compound (p＜0.0005) statistically according to observations, because observe higher weight increase.Use two tails distribute, not in pairs, equate variance, check to be determined to throw by t to comprise the influence that behind the hGH polypeptide of the non-naturally encoded amino acids of Pegylation, has the circulating plasma IGF-1 content of significant difference with single dose.
Example 26: comprise the safety of non-naturally encoded amino acid whose Pegylation hGH (via the heterocyclic bond Pegylation) and/or the human clinical trial of effect
The more subcutaneous throwing of purpose and the safety that comprises non-naturally encoded amino acid whose Pegylation recombined human hGH and pharmacokinetics and one or more commercially available hGH products (include 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)) safety and pharmacokinetics.
In this research of patient registration 18 ages in 20-40 year scope and body weight between the healthy volunteer of 60-90kg.Individual will not have clinically significantly unusual hematology or serum chemistry laboratory evaluation, and have negative urine toxicology screening, HIV screening and hepatitis B surface antibody.They should not have in the following sign any one: hypertension; Any primary blood disease medical history; The medical history of significant liver, kidney, cardiovascular, stomach and intestine, apparatus urogenitalis, metabolism, sacred disease; The medical history of anaemia or epilepsy; Known susceptibility to product, PEG or the human serum albumin in bacterium or mammal source; Custom and frequent consumption contain the beverage of caffeine (caffeine); Participate in any other clinical testing or in 30 days of research beginning, failed blood or offered blood; In 3 months of research beginning, be exposed to hGH; Sick in 7 days of research beginning; And before research, have during the clinical labororatory assessment during physical examination or in 14 days of research beginning significantly unusual.Can be at all individualities of safety evaluation, and collect the whole blood gleanings as scheduled for pharmacokinetic analysis.All researchs all are to carry out under the situation of Ethics Committee (institutional ethics committee) approval and patient's agreement.
Research and design this will be for the Phase I in the healthy male volunteers, single center, open-label, at random, the research of rotating of two cycles.18 individualities are assigned in two treatments one of sequence set (every group of 9 individualities) at random.Use the comprise non-naturally encoded amino acid whose Pegylation hGH and the selected commercially available prod of equal dose to throw and GH by single subcutaneous injection through two individually dosed phases on thigh top.The dispensing dosage of commercially available prod and frequency are followed the indication in the packaging label.Can will use other administration, administration frequency or other parameter of commercially available prod to be added in the research by comprising other group of individuals when needing.14 days phase buffer of each administration period interval (washout period).At least 12 hours and 72 hours afterwards rather than between the administration phase before the administration in interim each of two administrations are confined to the research center with individuality.If other administration, frequency or other parameter of the Pegylation of desire test simultaneously hGH can be added other group of individuals so.Approval can be used in this research for the human multiple GH composite that uses.Humatrope TM(EliLilly ﹠amp; Co.), Nutropin TM(Genentech), Norditropin TM(Novo-Nordisk), Genotropin TM(Pfizer) and Saizen/Serostim TM(Serono) be the commercially available GH product of approval for human use.The experiment deployment thing of hGH is the Pegylation hGH that comprises non-naturally encoded amino acids.
Blood sampling is before throwing and hGH and extract a series of blood by direct venipuncture afterwards.When administration precontract 30 minutes, 20 minutes and 10 minutes, obtain venous blood sample (5mL) to be used to measure Serum GH concentration during roughly following time after (3 baseline samples) and the administration: 30 minutes and 1,2,5,8,12,15,18,24,30,36,48,60 and 72 hour.Each serum sample is divided into two aliquots.All serum samples all are to store down at-20 ℃.Serum sample transports on dry ice.Before the 1st day initial dose immediately, before the 4th day morning, the 16th day administration immediately and carry out fasting clinical laboratory tests (hematology, serum chemistry and urinalysis) the 19th day morning.
Bioanalytical method use ELISA kit program (Diagnostic Systems Laboratory[DSL], Webster TX) mensuration Serum GH concentration.
Immediate record vital signs when safety is determined at after the preceding and each administration of each administration (the 1st day and the 16th day) 6,24,48 and 72 hours.It is on the basis of the incidence that the is based upon adverse events change of comparing baseline with type and clinical laboratory tests that safety is measured.In addition, the preceding change of research is compared in assessment vital signs measurements (comprising blood pressure) with the physical examination result.
Data analysis deducts the average baselining GH concentration of averaging and measuring via the GH content of three samples collecting when coming before the self administration of medication 30,20 and 10 minutes by value after each administration, comes at serum-concentration value after the baseline GH concentration correction administration before the administration.If Serum GH concentration is lower than the quantitative level of calibrating before the administration, Serum GH concentration is not included in the calculating of mean value before the so described administration.By determining pharmacokinetic parameter at the serum-concentration data of baseline GH concentration correction.On Digital Equipment Corporation VAX 8600 computer systems, use the BIOAVL software of latest edition to calculate pharmacokinetic parameter by the model independent solution.Determine following pharmacokinetic parameter: peak serum concentration (C Max); Reach the time (t of peak serum concentration Max); Use that linear trapezoid method then calculates from time zero blood sampling time (AUC to the end 0-72) area under the concentration-time curve (AUC); And from end elimination at the end of elimination factor constant calculations half life period (t 1/2).The elimination factor constant be end last by linear concentration logarithm-time plot in the range of linearity linear regression of neighboring data point estimate.Calculate mean value, standard deviation (SD) and the coefficient of variation (CV) of pharmacokinetic parameter at treatment each time.The ratio of calculating parameter mean value (composite of the composite of preservation/non-preservation).
The incidence of safety results adverse events is equal distribution between the treatment group.Compare not exist clinically with clinical laboratory tests or blood pressure before baseline or the research and change significantly, and compare before physical examination result and vital signs measured value and the research and do not have obvious change.It is similar that the security features of two treatment groups should seem.
Pharmacokinetics result will (include but not limited to Humatrope at one or more commercially available hGH products that receive single dose when each measured time point TM(Eli Lilly ﹠amp; Co.), Nutropin TM(Genentech), Norditropin TM(Novo-Nordisk), Genotropin TM(Pfizer) and Saizen/Serostim TM(Serono)) the average serum GH concentration time curve of all 18 individualities (not proofreading and correct at baseline GH content) is compared with the Pegylation hGH that comprises non-naturally encoded amino acids afterwards.Baseline GH concentration should be in the normal physiological scope before all individual administrations.By determining pharmacokinetic parameter at the serum data of average baselining GH concentration correction before the administration, and definite C MaxAnd t MaxSelected clinical control reagent (Humatrope TM(Eli Lilly ﹠amp; Co.), Nutropin TM(Genentech), Norditropin TM(Novo-Nordisk), Genotropin TM(Pfizer), Saizen/Serostim TM(Serono)) average t MaxRemarkable t less than the Pegylation hGH that comprises non-naturally encoded amino acids MaxCompare with the t1/2 of the Pegylation hGH that comprises non-naturally encoded amino acids, the t1/2 value of the commercially available hGH product of being tested is significantly less.
Although this research is to carry out in the healthy male individuality, similarly absorb feature and security features but be expected at will exist in other patient colonies, described colony such as: suffer from cancer or chronic renal failure sex patient, paediatrics patients with renal failure, be in the patient in body stored Procedure (autologous predeposit program) or the patient of predetermined optional operation.
In a word, the Pegylation hGH that comprises non-naturally encoded amino acids of subcutaneous throwing and single dose will be safe and be that the healthy male individuality is well tolerable.Based on suitable adverse events incidence, clinical labororatory's value, vital signs and physical examination result, the hGH of commercial form will equate with the security features that comprises the Pegylation hGH of non-naturally encoded amino acids.The Pegylation hGH that comprises non-naturally encoded amino acids provides great clinical efficacy for patient and health care supplier potentially.
Example 27: the water miscible comparison of Pegylation hGH and non-Pegylation hGH
Be dissolvable in water the hGH polypeptide (his-hGH) of hGH wild-type protein (WHO hGH) in the 100 μ L water, His mark or comprise with the covalently bound alpha-non-natural amino acid of 30kDa PEG by mensuration the amount of the hGH polypeptide of the His mark of acetyl phenyl alanine is obtained the water-soluble of indivedual polypeptide at the 92nd.The amount of Pegylation hGH is greater than the amount of WHO hGH and hGH, and this Pegylation that shows non-natural amino acid polypeptides can increase water-soluble.
Example 28: the in vivo research of modified therapeutic activity non-natural amino acid polypeptides
The prostate cancer tumor xenogeneic graft is implanted in the mouse body, it is divided into two groups subsequently.With therapeutic activity natural amino acid polypeptide treated every day with modified therapeutic activity non-natural amino acid polypeptides treatment and another group one group of every day.Measure tumor size every day, and as indicated in reducing as tumor size with the group of modified therapeutic activity non-natural amino acid polypeptides treatment, compare with therapeutic activity natural amino acid polypeptide, modified therapeutic activity non-natural amino acid polypeptides has improved therapeutic efficiency.
Example 29: the in vivo research of modified therapeutic activity non-natural amino acid polypeptides
The prostate cancer tumor xenogeneic graft is implanted in the mouse body, it is divided into two groups subsequently.With therapeutic activity natural amino acid polypeptide treated every day with modified therapeutic activity non-natural amino acid polypeptides treatment and another group one group of every day.Measure tumor size every day, and as indicated in reducing as tumor size with the group of modified therapeutic activity non-natural amino acid polypeptides treatment, compare with therapeutic activity natural amino acid polypeptide, modified therapeutic activity non-natural amino acid polypeptides has improved therapeutic efficiency.
Following case description is measured and the activity in vitro and in vivo of more modified therapeutic activity non-natural amino acid polypeptides and the active in vitro and in vivo method of therapeutic activity natural amino acid polypeptide.
Example 30: the active and affine force measurement of non-natural amino acid polypeptides
This example in detail non-natural amino acid polypeptides activity and non-natural amino acid polypeptides are to the affine force measurement of its acceptor, combination collocation thing or part.
Express and separate according to the known method of one of ordinary skill in the art at the non-natural amino acid polypeptides acceptor, in conjunction with the protein of collocation thing or part.Use Biocore TMThe network analysis non-natural amino acid polypeptides combines with its acceptor.Similarly, can will be used for this calibrating in conjunction with collocation thing or part.
Recommend as manufacturer, use the soluble recepter that standard amine coupling program will about 600-800 RU to be fixed in Biacore TMOn the CM5 chip.Inject HBS-EP buffer solution (Biacore with the flow rate of 40 μ l/min from the teeth outwards TM, Pharmacia) wild type of the various concentration in or modified or not modified non-natural amino acid polypeptides reach 4-5 minute, and monitoring is dissociated and reached 15 minutes after injection.By 4.5M MgCl 215 pulse per second (PPS)s make surface regeneration.After at least 100 regeneration cycle, observe binding affinity and only lose seldom (1-5%).The reference cell that uses no sessile receptor is to deduct any buffer solution volume effect and non-specific binding.
Utilize BiaEvaluation 4.1 software (BIACORE TM) handle from the dynamics binding data of modified or not modified non-natural amino acid polypeptides titration experiments acquisition.Ratio (k with indivedual speed constants Off/ k On) calculated equilibrium dissociation constant (Kd).
Set up the stable cell lines of acceptor, combination collocation thing or the part of expressing non-natural amino acid polypeptides.Utilization contains acceptor, constructs the body electroporation of cells in conjunction with collocation thing or Ligand cDNA.Before the clone, transfectional cell was recovered 48 hours.By use at the antibody padding of acceptor differentiate expressed receptor, in conjunction with the transfection body of collocation thing or part, and at the FACS array (BD Biosciences, San Diego are analyzed on CA).Behind the repetition subclone of further counting the required transfection body of wheel, set up the cell clone of stable transfection.Described cell is used for cell in conjunction with calibrating.
Under 0 ℃, there is not or exists various concentration (volume: 10 μ l) under the situation of the natural amino acid polypeptide of un-marked or negative control polypeptide and in existence 125Under the situation of I-(modified) non-natural amino acid polypeptides (about 100,000cpm or 1ng), in duplicate with cell (3 * 10 6Individual) cultivate (cumulative volume: 120 μ l) that in PBS/1% BSA (100 μ l), reaches 90 minutes.The ice-cold FCS of 200 μ l that subsequently cell is suspended again and be tiled in the 350 μ l plastic centrifuge tubes goes up and centrifugal (1000g; 1 minute).Collect centrifugal and in gamma counter (Packard), centrifugal and supernatant are counted separately by clipping the pipe end.
Deduct non-specific binding with the total binding under the situation that does not have competitor (bipartite mean value) and determine that specificity is in conjunction with (cpm).Measure the non-specific binding of employed each cell type.Use is with a kind of 125I-(modified) non-natural amino acid polypeptides preparation different date carry out experiment and its should manifest in uniformity. 125I-(modified) non-natural amino acid polypeptides represents with acceptor, combines albumen or the celliferous combination of part.The natural amino acid polypeptide of un-marked suppresses combination in the dose dependent mode, and the negative control polypeptide is quite different.
Example 31: the in vivo research of modified therapeutic activity non-natural amino acid polypeptides
Mouse or rat are thrown and modified therapeutic activity non-natural amino acid polypeptides, therapeutic activity natural amino acid polypeptide and buffering solution.The result will show, compare with therapeutic activity natural amino acid polypeptide, and modified therapeutic activity non-natural amino acid polypeptides has the half life period of higher active and prolongation.
Example 32: the modified therapeutic activity non-natural amino acid polypeptides of binding and non-binding and the measurement of the in vivo half life period of its variant
All zooperies all are to carry out in the facility that AAALAC authorizes and according to the scheme that the management of laboratory animal and the use committee (Institutional Animal Care and Use Committee) of St.Louis University ratified.With rat be housed in individually have 12 hours the daytime/cage in the room of night circulation in.Certified Purina rodent food 5001 is provided and lets alone free drinking water to animal.
Example 33: pharmacokinetic study
Before entering zoopery, by the quality of three each modified therapeutic activity non-natural amino acid polypeptides of calibrating assessment.(Invitrogen, Carlsbad CA) run 4-12% acrylamide NuPAGE Bis-Tris gel about the purity that glue is checked modified therapeutic activity non-natural amino acid polypeptides by run the glue buffer solution with MES SDS under non-reduced condition.With Coomassie blue with gel-colored.According to density measurement scanning, the bands of a spectrum of modified therapeutic activity non-natural amino acid polypeptides are pure greater than 95%.By using from Charles River Laboratories (Wilmington, KTA MA) 2The dynamics LAL of kit examines and determine the endotoxin content of testing in each modified therapeutic activity non-natural amino acid polypeptides, and every dose of described content is less than 5EU.Utilize the bioactive cell that characterizes modified therapeutic activity non-natural amino acid polypeptides to examine and determine the biologically active of evaluating described polypeptide.
From the male Sprague-Dawley rat (261-425g) that Charles River Laboratories obtains that the pharmacokinetic properties of modified therapeutic activity non-natural amino acid polypeptides compound is compared to each other and compare with therapeutic activity natural amino acid polypeptide.Underwent operative is installed in the arteria carotis conduit for blood collecting.After successfully conduit being installed, before administration, animal is assigned in the treatment group (three to six every group).To subcutaneous about 0.41 the about 1mg/kg compound that gives of animal via to about 0.55ml/kg dose volume.When each time point, collect blood sample and put it in the microcentrifugal tube that is coated with EDTA via built-in conduit.Store up to analysis down at centrifugal back collection blood plasma and at-80 ℃.Use from BioSource International (Camarillo, CA) or Diagnostic Systems Laboratories (Webster, TX) antibody sandwich ELISA kit is measured compound concentration.Use the reference material calculating concentration corresponding with the analog that is given.Use modeling program WinNonlin (Pharsight, 4.1 versions) assessment pharmacokinetic parameter.Use utilizes the non-compartment model analysis of linear-up/log-down trapezoidal integration and to the even weighting of concentration data.Drawing data figure is to obtain Cmax subsequently: Cmax; Last eventually T1/2: t1/2; AUC 0-＞inf: be extrapolated to infinitely-great area under the concentration-time curve; MRT: mean residence time; Cl/f: apparent total plasma clearance; And Vz/f: the apparent volume of distribution during latter stage.
Example 34: drug efficacy study
Obtain male Sprague-Dawley rat from Charles River Laboratories.Animal is shaked down time in three weeks, during this period the monitoring biological characteristic relevant with the natural amino acid polypeptide.The animal that these biological characteristics is had the change of acceptable level is assigned in the treatment group at random.To subcutaneous rat throwing and single dose or every day dosage modified non-natural amino acid polypeptides.In whole research, with rat every day and anesthesia successively, blood drawing and administration (working as where applicable) and measurement associated biomolecule feature.Use the heparinize capillary to collect blood and put it into the microcentrifugal tube that is coated with EDTA from orbital sinus.Store up to analysis down by centrifugal separation plasma and at-80 ℃.Plasma concentration behind the acquisition single subcutaneous administration in the rat body.
Example 35: the safety of modified therapeutic activity non-natural amino acid polypeptides and/or the human clinical trial of effect
The more subcutaneous throwing of purpose and the safety of modified therapeutic activity non-natural amino acid polypeptides and the safety and the pharmacokinetics of pharmacokinetics and therapeutic activity natural amino acid polypeptide.
In this research of patient registration 18 ages in 20-40 year scope and body weight between the healthy volunteer of 60-90kg.Individual will not have clinically significantly unusual hematology or serum chemistry laboratory evaluation, and have negative urine toxicology screening, HIV screening and hepatitis B surface antibody.They should not have in the following sign any one: hypertension; Any primary blood disease medical history; The medical history of significant liver, kidney, cardiovascular, stomach and intestine, apparatus urogenitalis, metabolism, sacred disease; The medical history of anaemia or epilepsy; Known susceptibility to product, PEG or the human serum albumin in bacterium or mammal source; Custom and frequent consumption contain the beverage of caffeine (caffeine); Participate in any other clinical testing or in 30 days of research beginning, failed blood or offered blood; In 3 months of research beginning, be exposed to therapeutic activity natural amino acid polypeptide; Sick in 7 days of research beginning; And before research, have during the clinical labororatory assessment during physical examination or in 14 days of research beginning significantly unusual.Can be at all individualities of safety evaluation, and collect the whole blood gleanings as scheduled for pharmacokinetic analysis.All researchs all are to carry out under the situation of Ethics Committee (institutional ethicscommittee) approval and patient's agreement.
Research and design this will be for the Phase I in the healthy male volunteers, single center, open-label, at random, the research of rotating of two cycles.18 individualities are assigned in two treatments one of sequence set (every group of 9 individualities) at random.Use the modified therapeutic activity non-natural amino acid polypeptides of equal dose to throw and therapeutic activity natural amino acid polypeptide by single subcutaneous injection through two individually dosed phases on thigh top.Can other administration, administration frequency or other parameter be added in the research by comprising other group of individuals when needing.14 days phase buffer of each administration period interval.At least 12 hours and 72 hours afterwards rather than between the administration phase before the administration in interim each of two administrations are confined to the research center with individuality.If desire is tested other administration, frequency or other parameter of modified therapeutic activity non-natural amino acid polypeptides simultaneously, can add other group of individuals so.
Blood sampling is before throwing and modified therapeutic activity non-natural amino acid polypeptides or therapeutic activity natural amino acid polypeptide and extract a series of blood by direct venipuncture afterwards.When administration precontract 30 minutes, 20 minutes and 10 minutes, obtain venous blood sample (5mL) to be used to measure the serum-concentration of modified therapeutic activity non-natural amino acid polypeptides or therapeutic activity natural amino acid polypeptide during roughly following time after (3 baseline samples) and the administration: 30 minutes and 1,2,5,8,12,15,18,24,30,36,48,60 and 72 hour.Each serum sample is divided into two aliquots.All serum samples all are to store down at-20 ℃.Serum sample transports on dry ice.Before the 1st day initial dose immediately, before the 4th day morning, the 16th day administration immediately and carry out fasting clinical laboratory tests (hematology, serum chemistry and urinalysis) the 19th day morning.
Bioanalytical method use ELISA kit program (Diagnostic Systems Laboratory[DSL], Webster TX) the mensuration serum-concentration.
Immediate record vital signs when safety is determined at after the preceding and each administration of each administration (the 1st day and the 16th day) 6,24,48 and 72 hours.It is on the basis of the incidence that the is based upon adverse events change of comparing baseline with type and clinical laboratory tests that safety is measured.In addition, the preceding change of research is compared in assessment vital signs measurements (comprising blood pressure) with the physical examination result.
Data analysis deducts the average baselining concentration of averaging and measuring via the level of three samples collecting when coming before the self administration of medication 30,20 and 10 minutes by value after each administration, comes to proofread and correct serum-concentration after the administration at baseline concentrations before the administration.If serum-concentration is lower than the quantitative level of calibrating before the administration, serum-concentration is not included in the calculating of mean value before the so described administration.Determine pharmacokinetic parameter by the serum-concentration data of proofreading and correct at baseline concentrations.On DigitalEquipment Corporation VAX 8600 computer systems, use the BIOAVL software of latest edition to calculate pharmacokinetic parameter by the model independent solution.Determine following pharmacokinetic parameter: peak serum concentration (C Max); Reach the time (t of peak serum concentration Max); Use that linear trapezoid method then calculates from time zero blood sampling time (AUC to the end 0-72) area under the concentration-time curve (AUC); And from end elimination at the end of elimination factor constant calculations half life period (t 1/2).The elimination factor constant be end last by linear concentration logarithm-time plot in the range of linearity linear regression of neighboring data point estimate.Calculate mean value, standard deviation (SD) and the coefficient of variation (CV) of pharmacokinetic parameter at treatment each time.The ratio of calculating parameter mean value (composite of the composite of preservation/non-preservation).
The incidence of safety results adverse events is equal distribution between the treatment group.Compare not exist clinically with clinical laboratory tests or blood pressure before baseline or the research and change significantly, and compare before physical examination result and vital signs measured value and the research and do not have obvious change.It is similar that the security features of two treatment groups should seem.
Pharmacokinetics result compares the modified therapeutic activity non-natural amino acid polypeptides of all 18 individualities after modified therapeutic activity non-natural amino acid polypeptides that receives single dose or therapeutic activity natural amino acid polypeptide or the average serum concentration-time graph of therapeutic activity natural amino acid polypeptide (proofreading and correct at baseline values) when each measured time point.Baseline concentrations should be in the normal physiological scope before all individual administrations.By determining pharmacokinetic parameter at the serum data of average baselining concentration correction before the administration, and definite C MaxAnd t MaxThe average t of therapeutic activity natural amino acid polypeptide MaxObvious t than modified therapeutic activity non-natural amino acid polypeptides MaxShort.Compare with the t1/2 of modified therapeutic activity non-natural amino acid polypeptides, the t1/2 value of therapeutic activity natural amino acid polypeptide obviously will be lacked.
Although this research is to carry out in the healthy male individuality, similarly absorb feature and security features but be expected at will exist in other patient colonies, described colony such as: suffer from cancer or chronic renal failure sex patient, paediatrics patients with renal failure, be in the patient in the body stored Procedure or the patient of predetermined optional operation.
In a word, the modified therapeutic activity non-natural amino acid polypeptides of subcutaneous throwing and single dose will be safe and be that the healthy male individuality is well tolerable.Based on suitable adverse events incidence, clinical labororatory's value, vital signs and physical examination result, the security features of modified therapeutic activity non-natural amino acid polypeptides and therapeutic activity natural amino acid polypeptide will equate.Modified therapeutic activity non-natural amino acid polypeptides provides great clinical efficacy potentially to patient and health care supplier.
Should be appreciated that, example as herein described and embodiment are only for illustration purposes, and will be to one of ordinary skill in the art give chapter and verse various modifications or the change of described example and embodiment, and described modification and changing all will be included in the scope of the spirit and scope of the application's case and the claims of enclosing.The mode that the open case of all that this paper quoted, patent and patent application case are all quoted in full is incorporated herein and is used for all purposes.
Sequence table
SEQ? ID#? Sequence Note Protein, nucleic acid, tRNA or RS
1 CCGGCGGTAGTTCAGCAGGGCAGAACGGCGGAC TCTAAATCCGCATGGCGCTGGTTCAAATCCGGCC CGCCGGACCA Methanococcus jannaschii mtRNA Tyr CUA tRNA
2 CCCAGGGTAGCCAAGCTCGGCCAACGGCGACGG ACTCTAAATCCGTTCTCGTAGGAGTTCGAGGGTT CGAATCCCTTCCCTGGGACCA HLAD03; Optimize amber and suppress tRNA tRNA
3 GCGAGGGTAGCCAAGCTCGGCCAACGGCGACGG ACTTCCTAATCCGTTCTCGTAGGAGTTCGAGGGTT CGAATCCCTCCCCTCGCACCA HL325A; Optimize the AGGA frameshift suppressor tRNA tRNA
4 MDEFEMIKRNTSEIISEEELREVLKKDEKSAGIGFEP SGKIHL GHYLQIKKMIDLQNAGFDIIILLADLHAYLNQKGEL DEIRKIG DYNKKVFEAMGLKAKYVYGSTFQLDKDYTLNVY RLALKT TLKRARRSMELIAREDENPKVAEVIYPIMQVNTYY YLGVDV AVGGMEQRKIHMLARELLPKKVVCIHNPVLTGLDG EGKMS SSKGNFIAVDDSPEEIRAKIKKAYCPAGVVEGNPIM EIAKYFL EYPLTIKRPEKFGGDLTVNSYEELESLFKNKELHPM DLKNA VAEELIKILEPIRKRL Be used to incorporate into aminoacyl tRNA synthetase p-Az-PheRS (6) to azido-L-phenyl alanine RS
5 MDEFEMIKRNTSEIISEEELREVLKKDEKSAGIGFEP SGKIHL GHYLQKKMIDLQNAGFDIIILLADLHAYLNQKGEL DEIRKIG The aminoacyl tRNA that is used to incorporate into benzoyl-L-phenyl alanine closes RS
DYNKKVFEAMGLKAKYVYGSSFQLDKDYTLNVY RLALKT TLKRARRSMELIAREDENPKVAEVIYPIMQVNTSH YLGVDV AVGGMEQRKIHMLARELLPKKVVCIHNPVLTGLDG EGKMS SSKGNFIAVDDSPEEIRAKIKKAYCPAGVVEGNPIM EIAKYFL EYPLTIKRPEKFGGDLTVNSYEELESLFKNKELHPM DLKNA VAEELIKILEPIRKRL Become enzyme p-BpaRS (1)
6? MDEFEMIKRNTSEIISEEELREVLKKDEKAAIGFEPS GKIHLG HYLQIKKMIDL QNAGFDIIILLADLHAYLNQKGELDEIRKIGDYNKK VFEAMG LKAKYVYGSPFQLDKDYTLNVYRLALKTTLKRAR RSMELIA REDENPKVAEVIYPIMQVNAIYLAVDVAVGGMEQR KIHML ARELLPKKVVCIHNPVLTGLDGEGKMSSSKGNFIA VDDSPEE IRAKIKKAYCPAGVVEGNPIMEIAKYFLEYPLTIKRP EKFGG DLTVNSYEELESLFKNKELHPMDLKNAVAEELIKIL EPIRKR L Be used to incorporate into the aminoacyl tRNA synthetase propargyl-PheRS of propargyl-phenyl alanine 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?YPLTI KRPEK?FGGDL?TVNSY EELES LFKNK?ELHPM?DLKNA?VAEEL?IKILE?PIRKRL Be used to incorporate into the aminoacyl tRNA synthetase propargyl-PheRS of propargyl-phenyl alanine 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 KKVVC?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-phenyl alanine RS
SEQ? ID#? Sequence Note Protein, nucleic acid, tRNA or
RS
9 MDEFEMIKRNTSEIISEEELREVLKKDEKSATIGFEP SGKIHL GHYLQIKKMIDLQNAGFDIIILLADLHAYLNQKGEL DEIRKIG DYNKKVFEAMGLKAKYVYGSNFQLDKDYTLNVY RLALKT TLKRARRSMELIAREDENPKVAEVIYPIMQVNPLH YQGVDV AVGGMEQRKIHMLARELLPKKVVCIHNPVLTGLD GEGKMS SSKGNFIAVDDSPEEIRAKIKKAYCPAGVVEGNPIM EIAKYFL EYPLTIKRPEKFGGDLTVNSYEELESLFKNKELHPM DLKNA VAEELIKILEPIRKRL Be used to incorporate into aminoacyl tRNA synthetase p-Az-PheRS (1) to azido-phenyl alanine RS
10 MDEFEMIKRNTSEIISEEELREVLKKDEKSATIGFEP SGKIHL GHYLQDIKKMIDLQNAGFDIIILLADLHAYLNQKG ELDEIRKIG DYNKKVFEAMGLKAKYVYGSSFQLDKDYTLNVY RLALKT TLKRARRSMELIAREDENPKVAEVIYPIMQVNPLH YQGVDV AVGGMEQRKIHMLARELLPKKVVCIHNPVLTGLD GEGKMS SSKGNFIAVDDSPEEIRAKIKKAYCPAGVVEGNPIM ELAKYFL EYPLTIKRPEKFGGDLTVNSYEELESLFKNKELHPM DLKNA VAEELIKILEPIRKRL Be used to incorporate into aminoacyl tRNA synthetase p-Az-PheRS (3) to azido-phenyl alanine RS
11 MDEFEMIKRNTSEIISEEELREVLKKDEKSALIGFEP SGKIHL GHYLQIKKMIDLQNAGFDIIILLADLHAYLNQKGEL DEIRKIG DYNKKVFEAMGLKAKYVYGSTFQLDKDYTLNVY RLALKT TLKRARRSMELIAREDENPKVAEVIYPIMQVNPVH YQGVDV AVGGMEQRKIHMLARELLPKKVVCIHNPVLTGLD GEGKMS SSKGNFIAVDDSPEEIRAKIKKAYCPAGVVEGNPIM EIAKYFL EYPLTIKRPEKFGGDLTVNSYEELESLFKNKELHPM DLKNA VAEELIKILEPIRKRL Be used to incorporate into aminoacyl tRNA synthetase p-Az-PheRS (4) to azido-phenyl alanine RS
12 MDEFEMIKRNTSEIISEEELREVLKKDEKSATIGFEP SGKIHL GHYLQIKKMIDLQNAGFDIIILLADLHAYLNQKGEL DEIRKIG DYNKKVFEAMGLKAKYVYGSSFQLDKDYTLNVY Be used to incorporate into aminoacyl tRNA synthetase p-Az-PheRS (2) to azido-phenyl alanine RS
RLALKT TLKRARRSMELIAREDENPKVAEVIYPIMQVNPSH YQGVDV AVGGMEQRKIHMLARELLPKKVVCIHNPVLTGLD GEGKMS SSKGNFLAVDDSPEEIRAKIKKAYCPAGVVEGNPIM EIAKYFL EYPLTIKRPEKFGGDLTVNSYEELESLFKNKELHPM DLKNA VAEELIKILEPIRKRL
13 MDEFEMIKRNTSEIISEEELREVLKKDEKSALIGFEP SGKIHL GHYLQIKKMIDLQNAGFDIIILLADLHAYLNQKGEL DEIRKIG DYNKKVFEAMGLKAKYVYGSEFQLDKDYTLNVY RLALKT TLKRARRSMELIAREDENPKVAEVIYPIMQVNGCH YRGVDV AVGGMEQRKIHMLARELLPKKVVCIHNPVLTGLD GEGKMS SSKGNFIAVDDSPEEIRAKIKKAYCPAGVVEGNPIM EIAKYFL EYPLTIKRPEKFGGDLTVNSYEELESLFKNKELHPM DLKNA VAEELIKILEPIRKRL Be used to incorporate into aminoacyl tRNA synthetase (LW1) to azido-phenyl alanine RS
14 MDEFEMIKRNTSEIISEEELREVLKKDEKSALIGFEP SGKIHL GHYLQIKKMIDLQNAGFDIIILLADLHAYLNQKGEL DEIRKIG DYNKKVFEAMGLKAKYVYGSEFQLDKDYTLNVY RLALKT TLKRARRSMELIAREDENPKVAEVIYPIMQVNGTH YRGVDV AVGGMEQRKIHMLARELLPKKVVCIHNPVLTGLD GEGKMS SSKGNFIAVDDSPEEIRAKIKKAYCPAGVVEGNPIM EIAKYFL EYPLTIKRPEKFGGDLTVNSYEELESLFKNKELHPM DLKNA VAEELIKILEPIRKRL Be used to incorporate into aminoacyl tRNA synthetase (LW5) to azido-phenyl alanine RS
15 MDEFEMIKRNTSEIISEEELREVLKKDEKSAAIGFEP SGKIHL GHYLQIKKMIDLQNAGFDIIILLADLHAYLNQKGEL DEIRKIG DYNKKVFEAMGLKAKYVYGSEFQLDKDYTLNVY RLALKT TLKRARRSMELIAREDENPKVAEVIYPIMQVNGGH YLGVDV IVGGMEQRKIHMLARELLPKKVVCIHNPVLTGLDG EGKMSS SKGNFIAVDDSPEEIRAKIKKAYCPAGVVEGNPIMEI AKYFL Be used to incorporate into aminoacyl tRNA synthetase (LW6) to azido-phenyl alanine RS
EYPLTIKRPEKFGGDLTVNSYEELESLFKNKELHPM DLKNA VAEELIKILEPIRKRL
16 MDEFEMIKRNTSEIISEEELREVLKKDEKSAAIGFEP SGKIHL GHYLQKKMIDLQNAGFDIIILLADLHAYLNQKGEL DEIRKIG DYNKKVFEAMGLKAKYVYGSRFQLDKDYTLNVY RLALKT TLKRARRSMELIAREDENPKVAEVIYPIMQVNVfflY DGVDV AVGGMEQRKIHMLARELLPKKVVCIHNPVLTGLDG EGKMS SSKGNFIAVDDSPEEIRAKIKKAYCPAGVVEGNPIM EIAKYFL EYPLTIKRPEKFGGDLTVNSYEELESLFKNKELHPM DLKNA VAEELKILEPIRKRL Be used to incorporate into aminoacyl tRNA synthetase (AzPheRS-5) to azido-phenyl alanine RS
17 MDEFEMIKRNTSEIISEEELREVLKKDEKSAGIGFEP SGKIHL GHYLQKKMIDLQNAGFDIIILLADLHAYLNQKGEL DEIRKIG DYNKKVFEAMGLKAKYVYGSTFQLDKDYTLNVY RLALKT TLKRARRSMELIAREDENPKVAEVIYPIMQVNTYY YLGVDV AVGGMEQREIHMLARELLPKKVVCIHNPVLTGLDG EGKMS SSKGNFIAVDDSPEEIRAKIKKAYCPAGVVEGNPIM EIAKYFL EYPLTIKRPEKFGGDLTVNSYEELESLFKNKELHPM DLKNA VAEELIKILEPIRKRL Be used to incorporate into aminoacyl tRNA synthetase (AzPheRS-6) to azido-phenyl alanine RS

Claims (41)

1. compound, it comprises structure 1 or 2:
Figure A200680049995C00021
Wherein:
A is optional, and when existing, it is the low-carbon (LC) alkylidene, be substituted low-carbon (LC) alkylidene, low-carbon (LC) cycloalkylidene, be substituted the low-carbon (LC) cycloalkylidene, the low-carbon (LC) alkenylene, be substituted low-carbon (LC) alkenylene, alkynylene, the inferior assorted alkyl of low-carbon (LC), be substituted inferior assorted alkyl, the inferior Heterocyclylalkyl of low-carbon (LC), be substituted low-carbon (LC) inferior Heterocyclylalkyl, arlydene, be substituted arlydene, inferior heteroaryl, be substituted inferior heteroaryl, alkarylene, be substituted alkarylene, inferior aralkyl or be substituted inferior aralkyl;
B is optional, and when existing, its at one end with contain the connection base that two amine moieties are connected, described connection base is selected from the group that is made up of following each group: the low-carbon (LC) alkylidene, be substituted low-carbon (LC) alkylidene, low-carbon (LC) alkenylene, be substituted the low-carbon (LC) alkenylene, the inferior assorted alkyl of low-carbon (LC), be substituted the low-carbon (LC) Asia mix alkyl ,-O-(alkylidene or be substituted alkylidene)-,-S-(alkylidene or be substituted alkylidene)-,-C (O) R ＂-,-S (O) k(alkylidene or be substituted alkylidene)-(wherein k is 1,2 or 3) ,-C (O)-(alkylidene or be substituted alkylidene)-,-C (S)-(alkylidene or be substituted alkylidene)-,-NR ＂-(alkylidene or be substituted alkylidene)-,-CON (R ＂)-(alkylidene or be substituted alkylidene)-,-CSN (R ＂)-(alkylidene or be substituted alkylidene)-and-N (R ＂) CO-(alkylidene or be substituted alkylidene)-, wherein each R ＂ is independently for H, alkyl or be substituted alkyl;
T 1Be bond or CH 2And T 2Be CH;
Wherein each optional substituting group is independently selected from low-carbon alkyl, is substituted low-carbon alkyl, low-carbon naphthenic, is substituted low-carbon naphthenic, low-carbon (LC) thiazolinyl, is substituted the low-carbon (LC) thiazolinyl, alkynyl, the assorted alkyl of low-carbon (LC), is substituted assorted alkyl, low-carbon (LC) Heterocyclylalkyl, is substituted the low-carbon (LC) Heterocyclylalkyl, aryl, is substituted aryl, heteroaryl, is substituted heteroaryl, alkaryl, is substituted alkaryl, aralkyl or is substituted aralkyl;
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 3And R 4Be H, halogen, low-carbon alkyl or be substituted low-carbon alkyl, perhaps R independently of one another 3And R 4Or two R 3Group forms cycloalkyl or Heterocyclylalkyl according to circumstances;
Or-A-B-contain two amine moieties form together comprise at least one two amido, through protecting two amidos or bicyclic cycloalkyl through covering two amidos or Heterocyclylalkyl;
Or-B-contain two amine moiety groups form together comprise at least one two amido, through protecting two amidos or dicyclo or tricyclic naphthenes base or cyclophane base or Heterocyclylalkyl through covering two amidos;
Wherein-A-B-contains on two amine moieties at least one amido according to circumstances for through protection amine;
Or its active metabolite, salt or pharmaceutically acceptable prodrug or solvate.
2. compound according to claim 1, wherein A is the low-carbon (LC) alkylidene that is substituted or is unsubstituted, or the arlydene that is selected from the group that is made up of phenylene, inferior pyridine radicals, inferior pyrimidine radicals or inferior thienyl that is unsubstituted or is substituted.
3. compound according to claim 1, wherein B be the low-carbon (LC) alkylidene, be substituted the low-carbon (LC) alkylidene ,-O-(alkylidene or be substituted alkylidene)-,-C (O)-(alkylidene or be substituted alkylidene)-,-CON (R ＂)-(alkylidene or be substituted alkylidene)-,-S (alkylidene or be substituted alkylidene)-,-S (O) (alkylidene or be substituted alkylidene)-or-S (O) 2(alkylidene or be substituted alkylidene)-.
4. compound according to claim 3, wherein B is-O (CH 2)-,-NHCH 2-,-C (O)-(CH 2)-,-CONH-(CH 2)-,-SCH 2-,-S (=O) CH 2-or-S (O) 2CH 2-.
5. compound according to claim 1, wherein R 1Be H, tertbutyloxycarbonyl, 9-fluorenyl methoxy carbonyl, N-acetyl group, tetrafluoro acetyl group or benzene methoxycarbonyl group.
6. compound according to claim 1, wherein R 1Be resin, amino acid, polypeptide or polynucleotide.
7. compound according to claim 1, wherein R 2Be OH, O-methyl, O-ethyl or the O-tert-butyl group.
8. compound according to claim 1, wherein R 2Be resin, amino acid, polypeptide or polynucleotide.
9. compound according to claim 8, wherein R 2Be polynucleotide.
10. compound according to claim 9, wherein R 2Be ribonucleic acid.
11. compound according to claim 10, wherein R 2Be tRNA.
12. compound according to claim 11, wherein said tRNA specific recognition is selected codon.
13. compound according to claim 12, wherein said selection codon is selected from the group that is made up of following each thing: amber codon, ochre codon, opal codon, unique codon, rare codon, non-natural codon, five base codons and four base codons.
14. compound according to claim 13, wherein R 2For suppressing tRNA.
15. compound according to claim 1, it is corresponding with structure 3 or 4:
Each R wherein aBe independently selected from the group that forms by following each group: H, halogen, alkyl, be substituted alkyl ,-N (R ＂) 2,-C (O) N (R ＂) 2,-OR ＂ and-S (O) kR ＂, wherein k is 1,2 or 3, wherein each R ＂ is H, alkyl independently or is substituted alkyl.
16. compound according to claim 15, it is selected from the group that is made up of following each thing:
Figure A200680049995C00042
Figure A200680049995C00043
Or its salt; Or incorporate the polypeptide of any described compound in any position.
17. compound according to claim 16, wherein said-B-contain on two amine moieties at least one amido through protection.
18. compound according to claim 17, wherein said amine protecting group are selected from the group that is made up of following each group:
Figure A200680049995C00045
With
Figure A200680049995C00046
19. compound according to claim 18, it is selected from the group that is made up of following each thing:
Figure A200680049995C00051
With
Figure A200680049995C00052
Or its salt; Or incorporate the polypeptide of any described compound in any position.
20. a peptide species, it also has at least a compound according to claim 1.
21. polypeptide according to claim 20, wherein said polypeptide for the protein of the therapeutic protein homology that is selected from the group that forms by required polypeptide.
22. compound according to claim 1, wherein said compound can and contain dicarbapentaborane reagent under temperate condition and react in the aqueous solution.
23. compound according to claim 22, wherein said compound and the described reaction that contains dicarbapentaborane reagent have at least a in the following feature: (i) take place in about 4 to about 10 pH value scope; (ii) be created in heterocyclic bond stable under the biotic factor; (iii) has locus specificity; (iv) can reversibly destroy the tertiary structure of polypeptide; (v) at room temperature take place rapidly; (vi) be easy under aqueous conditions, take place; (vii) when described compound and the described ratio that contains dicarbapentaborane reagent be that about being easy to 1 to 1 time taken place; Or (viii) have regioselectivity and/or regiospecificity.
24. compound according to claim 22, wherein said compound and the described reaction that contains dicarbapentaborane reagent have at least four kinds in the following feature: (i) take place in about 4 to about 10 pH value scope; (ii) be created in heterocyclic bond stable under the biotic factor; (iii) has locus specificity; (iv) can reversibly destroy the tertiary structure of polypeptide; (v) at room temperature take place rapidly; (vi) be easy under aqueous conditions, take place; (vii) when described compound and the described ratio that contains dicarbapentaborane reagent be that about being easy to 1 to 1 time taken place; Or (viii) have regioselectivity and/or regiospecificity.
25. compound according to claim 22, wherein said temperate condition are 2 to 10 pH value.
26. compound according to claim 22, wherein said temperate condition are 4 to 9 pH value.
27. compound according to claim 1, wherein said compound can be stablized 1 month in the aqueous solution at least.
28. compound according to claim 1, wherein said compound can be stablized under 2 to 10 pH value.
29. compound according to claim 28, wherein said compound can be stablized under 4 to 9 pH value.
30. compound according to claim 27, wherein said compound can stablize at least 2 weeks.
31. compound according to claim 30, wherein said compound can be stablized 5 days at least.
32. a compound, it has formula (XXXVIII) or structure (XXXIX):
Figure A200680049995C00061
Wherein:
A is optional, and when existing, it is the low-carbon (LC) alkylidene, be substituted low-carbon (LC) alkylidene, low-carbon (LC) cycloalkylidene, be substituted the low-carbon (LC) cycloalkylidene, the low-carbon (LC) alkenylene, be substituted low-carbon (LC) alkenylene, alkynylene, the inferior assorted alkyl of low-carbon (LC), be substituted inferior assorted alkyl, the inferior Heterocyclylalkyl of low-carbon (LC), be substituted low-carbon (LC) inferior Heterocyclylalkyl, arlydene, be substituted arlydene, inferior heteroaryl, be substituted inferior heteroaryl, alkarylene, be substituted alkarylene, inferior aralkyl or be substituted inferior aralkyl;
B is optional, and when existing, its at one end with contain the connection base that two amine moieties are connected, described connection base is selected from the group that is made up of following each group: the low-carbon (LC) alkylidene, be substituted low-carbon (LC) alkylidene, low-carbon (LC) alkenylene, be substituted the low-carbon (LC) alkenylene, the inferior assorted alkyl of low-carbon (LC), be substituted the low-carbon (LC) Asia mix alkyl ,-O-(alkylidene or be substituted alkylidene)-,-S-(alkylidene or be substituted alkylidene)-,-C (O) R ＂-,-S (O) k(alkylidene or be substituted alkylidene)-(wherein k is 1,2 or 3) ,-C (O)-(alkylidene or be substituted alkylidene)-,-C (S)-(alkylidene or be substituted alkylidene)-,-NR ＂-(alkylidene or be substituted alkylidene)-,-CON (R ＂)-(alkylidene or be substituted alkylidene)-,-CSN (R ＂)-(alkylidene or be substituted alkylidene)-and-N (R ＂) CO-(alkylidene or be substituted alkylidene)-, wherein each R ＂ is independently for H, alkyl or be 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 3And R 4Be H, halogen, low-carbon alkyl or be substituted low-carbon alkyl, perhaps R independently of one another 3And R 4Or two R 3Group forms cycloalkyl or Heterocyclylalkyl according to circumstances;
Z 1Be bond, CR 7R 7, O, S, NR ＇, CR 7R 7-CR 7R 7, CR 7R 7-O, O-CR 7R 7, CR 7R 7-S, S-CR 7R 7, CR 7R 7-NR ＇, NR ＇-CR 7R 7
R ＇ is H, alkyl or is substituted alkyl;
Z 2Be selected from the group that forms by following each group: bond ,-C (O)-,-C (S)-, the C that is substituted according to circumstances 1-C 3Alkylidene, the C that is substituted according to circumstances 1-C 3Alkenylene and the assorted alkyl that is substituted according to circumstances;
Each R 6And R 7Be independently selected from the group that forms by following each group: H, alkyl, be substituted alkyl, thiazolinyl, be substituted thiazolinyl, alkynyl, be substituted alkynyl, alkoxyl, be substituted alkoxyl, alkyl alkoxy, be substituted alkyl alkoxy, polyalkylene oxide, be substituted polyalkylene oxide, aryl, be substituted aryl, heteroaryl, be substituted heteroaryl, alkaryl, be substituted alkaryl, aralkyl, be substituted aralkyl ,-(alkylidene or be substituted alkylidene)-ON (R ＂) 2,-(alkylidene or be substituted alkylidene)-C (O) SR ＂ ,-(alkylidene or be substituted alkylidene)-S-S-(aryl or be substituted aryl) ,-C (O) R ＂ ,-C (O) 2R ＂ or-C (O) N (R ＂) 2, wherein each R ＂ independently for hydrogen, alkyl, be substituted alkyl, thiazolinyl, be substituted thiazolinyl, alkoxyl, be substituted alkoxyl, aryl, be substituted aryl, heteroaryl, alkaryl, be substituted alkaryl, aralkyl or be substituted aralkyl;
Or any two adjacent R 7Group forms 5 yuan to 8 yuan heterocycles, cycloalkyl or aromatic rings that are substituted according to circumstances together; Wherein said optional substituting group is selected from halogen, OH, C 1-6Alkyl, C 1-6Alkoxyl, halogen-C 1-6Alkyl, halogen-C 1-6Alkoxyl, aryl, halogen aryl or heteroaryl;
Condition is Z 1Add Z 2Provide and be no more than 3 annular atomses;
R 5For H, alkyl, be substituted alkyl, thiazolinyl, be substituted thiazolinyl, alkynyl, be substituted alkynyl, alkoxyl, be substituted alkoxyl, alkyl alkoxy, be substituted alkyl alkoxy, polyalkylene oxide, be substituted polyalkylene oxide, aryl, be substituted aryl, heteroaryl, be substituted heteroaryl, alkaryl, be substituted alkaryl, aralkyl, be substituted aralkyl ,-(alkylidene or be substituted alkylidene)-ON (R ＂) 2,-(alkylidene or be substituted alkylidene)-C (O) SR ＂ ,-(alkylidene or be substituted alkylidene)-S-S-(aryl or be substituted aryl) ,-C (O) R ＂ ,-C (O) 2R ＂ or-C (O) N (R ＂) 2, wherein each R ＂ independently for hydrogen, alkyl, be substituted alkyl, thiazolinyl, be substituted thiazolinyl, alkoxyl, be substituted alkoxyl, aryl, be substituted aryl, heteroaryl, alkaryl, be substituted alkaryl, aralkyl, be substituted aralkyl;
Or R 5Be L-X, wherein X is selected from the group that is made up of required functional group; And L is optional, and when existing, it is the connection base that is selected from the group that is made up of following each group: alkylidene, be substituted alkylidene, alkenylene, be substituted alkenylene ,-O-,-O-(alkylidene or be substituted alkylidene)-,-S-,-S-(alkylidene or be substituted alkylidene)-,-S (O) k-(wherein k is 1,2 or 3) ,-S (O) k(alkylidene or be substituted alkylidene)-,-C (O)-,-C (O)-(alkylidene or be substituted alkylidene)-,-C (S)-,-C (S)-(alkylidene or be substituted alkylidene)-,-N (R ＇)-,-NR ＇-(alkylidene or be substituted alkylidene)-,-C (O) N (R ＇)-,-CON (R ＇)-(alkylidene or be substituted alkylidene)-,-CSN (R ＇)-,-CSN (R ＇)-(alkylidene or be substituted alkylidene)-,-N (R ＇) CO-(alkylidene or be substituted alkylidene)-,-N (R ＇) C (O) O-,-(alkylidene or be substituted alkylidene)-O-N=CR ＇-,-(alkylidene or be substituted alkylidene)-C (O) NR ＇-(alkylidene or be substituted alkylidene)-,-(alkylidene or be substituted alkylidene)-S (O) k-(alkylidene or be substituted alkylidene)-S-,-(alkylidene or be substituted alkylidene)-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 each R ＇ is H, alkyl independently or is substituted alkyl;
Or its active metabolite, salt or pharmaceutically acceptable prodrug or solvate.
33. compound according to claim 32, it has formula (XLI) or structure (XLII):
Figure A200680049995C00081
R wherein aBe independently selected from the group that forms by following each group: H, halogen, alkyl, be substituted alkyl ,-N (R ＇) 2,-C (O) R ＇-,-C (O) N (R ＇) 2,-OR ＇ and-S (O) kR ＇, wherein k is 1,2 or 3.
34. compound according to claim 33, it has following structure:
Or
35. a compound, it has the structure that is selected from the group that is made up of following each structure:
Figure A200680049995C00084
With
Figure A200680049995C00085
Wherein:
A is optional, and when existing, it is the low-carbon (LC) alkylidene, be substituted low-carbon (LC) alkylidene, low-carbon (LC) cycloalkylidene, be substituted the low-carbon (LC) cycloalkylidene, the low-carbon (LC) alkenylene, be substituted low-carbon (LC) alkenylene, alkynylene, the inferior assorted alkyl of low-carbon (LC), be substituted inferior assorted alkyl, the inferior Heterocyclylalkyl of low-carbon (LC), be substituted low-carbon (LC) inferior Heterocyclylalkyl, arlydene, be substituted arlydene, inferior heteroaryl, be substituted inferior heteroaryl, alkarylene, be substituted alkarylene, inferior aralkyl or be substituted inferior aralkyl;
B is optional, and when existing, its at one end with contain the connection base that two amine moieties are connected, described connection base is selected from the group that is made up of following each group: the low-carbon (LC) alkylidene, be substituted low-carbon (LC) alkylidene, low-carbon (LC) alkenylene, be substituted the low-carbon (LC) alkenylene, the inferior assorted alkyl of low-carbon (LC), be substituted the low-carbon (LC) Asia mix alkyl ,-O-(alkylidene or be substituted alkylidene)-,-S-(alkylidene or be substituted alkylidene)-,-C (O) R ＂-,-S (O) k(alkylidene or be substituted alkylidene)-(wherein k is 1,2 or 3) ,-C (O)-(alkylidene or be substituted alkylidene)-,-C (S)-(alkylidene or be substituted alkylidene)-,-NR ＂-(alkylidene or be substituted alkylidene)-,-CON (R ＂)-(alkylidene or be substituted alkylidene)-,-CSN (R ＂)-(alkylidene or be substituted alkylidene)-and-N (R ＂) CO-(alkylidene or be substituted alkylidene)-, wherein each R ＂ is independently for H, alkyl or be 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 3And R 4Be H, halogen, low-carbon alkyl or be substituted low-carbon alkyl, perhaps R independently of one another 3And R 4Or two R 3Group forms cycloalkyl or Heterocyclylalkyl according to circumstances;
Z 1Be bond, CR 5R 5, CR 5R 5-CR 5R 5, CR 5R 5-O, O-CR 5R 5, S-CR 5R 5, NR 5-CR 5R 5, CR 5R 5-S, CR 5R 5-NR 5
Z 2Be selected from the group that forms by following each group: the C that is substituted according to circumstances 1-C 3Alkylidene, the C that is substituted according to circumstances 1-C 3Alkenylene, the assorted alkyl and the N that are substituted according to circumstances;
Z 3Be independently selected from the group that forms by following each group: bond, the C that is substituted according to circumstances 1-C 4Alkylidene, the C that is substituted according to circumstances 1-C 4Alkenylene, the assorted alkyl that is substituted according to circumstances ,-O-,-S-,-C (O)-,-C (S)-and-N (R ＇)-; Condition is at least one Z 3It is not bond;
T 3For bond, C (R) (R), O or S, and R is H, halogen, alkyl, is substituted alkyl, cycloalkyl or is substituted cycloalkyl; Condition is to work as T 3During for O or S, R not can be halogen;
R 6Independently for H, alkyl, be substituted alkyl, thiazolinyl, be substituted thiazolinyl, alkynyl, be substituted alkynyl, alkoxyl, be substituted alkoxyl, alkyl alkoxy, be substituted alkyl alkoxy, polyalkylene oxide, be substituted polyalkylene oxide, aryl, be substituted aryl, heteroaryl, be substituted heteroaryl, alkaryl, be substituted alkaryl, aralkyl, be substituted aralkyl ,-(alkylidene or be substituted alkylidene)-ON (R ＂) 2,-(alkylidene or be substituted alkylidene)-C (O) SR ＂ ,-(alkylidene or be substituted alkylidene)-S-S-(aryl or be substituted aryl) ,-C (O) R ＂ ,-C (O) 2R ＂ or-C (O) N (R ＂) 2, wherein each R ＂ independently for hydrogen, alkyl, be substituted alkyl, thiazolinyl, be substituted thiazolinyl, alkoxyl, be substituted alkoxyl, aryl, be substituted aryl, heteroaryl, alkaryl, be substituted alkaryl, aralkyl or be substituted aralkyl;
Condition is Z 1Add Z 2Provide and be no more than 3 annular atomses;
R 5For H, alkyl, be substituted alkyl, thiazolinyl, be substituted thiazolinyl, alkynyl, be substituted alkynyl, alkoxyl, be substituted alkoxyl, alkyl alkoxy, be substituted alkyl alkoxy, polyalkylene oxide, be substituted polyalkylene oxide, aryl, be substituted aryl, heteroaryl, be substituted heteroaryl, alkaryl, be substituted alkaryl, aralkyl, be substituted aralkyl ,-(alkylidene or be substituted alkylidene)-ON (R ＂) 2,-(alkylidene or be substituted alkylidene)-C (O) SR ＂ ,-(alkylidene or be substituted alkylidene)-S-S-(aryl or be substituted aryl) ,-C (O) R ＂ ,-C (O) 2R ＂ or-C (O) N (R ＂) 2, wherein each R ＂ independently for hydrogen, alkyl, be substituted alkyl, thiazolinyl, be substituted thiazolinyl, alkoxyl, be substituted alkoxyl, aryl, be substituted aryl, heteroaryl, alkaryl, be substituted alkaryl, aralkyl, be substituted aralkyl;
Or R 5Be L-X, wherein X is selected from the group that is made up of required functional group; And L is optional, and when existing, it is the connection base that is selected from the group that is made up of following each group: alkylidene, be substituted alkylidene, alkenylene, be substituted alkenylene ,-O-,-O-(alkylidene or be substituted alkylidene)-,-S-,-S-(alkylidene or be substituted alkylidene)-,-S (O) k-(wherein k is 1,2 or 3) ,-S (O) k(alkylidene or be substituted alkylidene)-,-C (O)-,-C (O)-(alkylidene or be substituted alkylidene)-,-C (S)-,-C (S)-(alkylidene or be substituted alkylidene)-,-N (R ＇)-,-NR ＇-(alkylidene or be substituted alkylidene)-,-C (O) N (R ＇)-,-CON (R ＇)-(alkylidene or be substituted alkylidene)-,-CSN (R ＇)-,-CSN (R ＇)-(alkylidene or be substituted alkylidene)-,-N (R ＇) CO-(alkylidene or be substituted alkylidene)-,-N (R ＇) C (O) O-,-(alkylidene or be substituted alkylidene)-O-N=CR ＇-,-(alkylidene or be substituted alkylidene)-C (O) NR ＇-(alkylidene or be substituted alkylidene)-,-(alkylidene or be substituted alkylidene)-S (O) k-(alkylidene or be substituted alkylidene)-S-,-(alkylidene or be substituted alkylidene)-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 each R ＇ is H, alkyl independently or is substituted alkyl;
Or its active metabolite, salt or pharmaceutically acceptable prodrug or solvate.
36. compound according to claim 35, it is selected from the group that is made up of following each thing:
Figure A200680049995C00111
R wherein aBe independently selected from the group that forms by following each group: H, halogen, alkyl, be substituted alkyl ,-N (R ＇) 2,-C (O) N (R ＇) 2,-OR ＇ and-S (O) kR ＇, wherein k is 1,2 or 3.
37. compound according to claim 36, it is selected from the group that is made up of following each thing:
Figure A200680049995C00112
With
Figure A200680049995C00113
38. a compound, it is selected from the group that is made up of following each thing:
With
Wherein:
A is optional, and when existing, it is the low-carbon (LC) alkylidene, be substituted low-carbon (LC) alkylidene, low-carbon (LC) cycloalkylidene, be substituted the low-carbon (LC) cycloalkylidene, the low-carbon (LC) alkenylene, be substituted low-carbon (LC) alkenylene, alkynylene, the inferior assorted alkyl of low-carbon (LC), be substituted inferior assorted alkyl, the inferior Heterocyclylalkyl of low-carbon (LC), be substituted low-carbon (LC) inferior Heterocyclylalkyl, arlydene, be substituted arlydene, inferior heteroaryl, be substituted inferior heteroaryl, alkarylene, be substituted alkarylene, inferior aralkyl or be substituted inferior aralkyl;
B is optional, and when existing, its at one end with contain the connection base that two amine moieties are connected, described connection base is selected from the group that is made up of following each group: the low-carbon (LC) alkylidene, be substituted low-carbon (LC) alkylidene, low-carbon (LC) alkenylene, be substituted the low-carbon (LC) alkenylene, the inferior assorted alkyl of low-carbon (LC), be substituted the low-carbon (LC) Asia mix alkyl ,-O-(alkylidene or be substituted alkylidene)-,-S-(alkylidene or be substituted alkylidene)-,-C (O) R ＂-,-S (O) k(alkylidene or be substituted alkylidene)-(wherein k is 1,2 or 3) ,-C (O)-(alkylidene or be substituted alkylidene)-,-C (S)-(alkylidene or be substituted alkylidene)-,-NR ＂-(alkylidene or be substituted alkylidene)-,-CON (R ＂)-(alkylidene or be substituted alkylidene)-,-CSN (R ＂)-(alkylidene or be substituted alkylidene)-and-N (R ＂) CO-(alkylidene or be substituted alkylidene)-, wherein each R ＂ is independently for H, alkyl or be 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 3And R 4Be H, halogen, low-carbon alkyl or be substituted low-carbon alkyl, perhaps R independently of one another 3And R 4Or two R 3Group forms cycloalkyl or Heterocyclylalkyl according to circumstances;
Z 1Be bond, CR 5R 5, CR 5R 5-CR 5R 5, CR 5R 5-O, O-CR 5R 5, S-CR 5R 5, NR 5-CR 5R 5, CR 5R 5-S, CR 5R 5-NR 5
Z 3Be independently selected from the group that forms by following each group: bond, the C that is substituted according to circumstances 1-C 4Alkylidene, the C that is substituted according to circumstances 1-C 4Alkenylene, the assorted alkyl that is substituted according to circumstances ,-O-,-S-,-C (O)-,-C (S)-and-N (R ＇)-;
M 2For
Figure A200680049995C00131
Figure A200680049995C00132
Or Wherein (a) expression and the bond of described B group, and (b) bond of the indivedual positions in expression and the described heterocyclic radical;
Figure A200680049995C00134
Or
Figure A200680049995C00135
Wherein (a) expression and the bond of described B group, and (b) bond of indivedual positions in expression and the described heterocyclic radical;
M 4For
Figure A200680049995C00136
Or
Figure A200680049995C00137
Wherein (a) expression and the bond of described B group, and (b) bond of indivedual positions in expression and the described heterocyclic radical;
T 3For bond, C (R) (R), O or S, and R is H, halogen, alkyl, is substituted alkyl, cycloalkyl or is substituted cycloalkyl;
R 6Independently for H, alkyl, be substituted alkyl, thiazolinyl, be substituted thiazolinyl, alkynyl, be substituted alkynyl, alkoxyl, be substituted alkoxyl, alkyl alkoxy, be substituted alkyl alkoxy, polyalkylene oxide, be substituted polyalkylene oxide, aryl, be substituted aryl, heteroaryl, be substituted heteroaryl, alkaryl, be substituted alkaryl, aralkyl, be substituted aralkyl ,-(alkylidene or be substituted alkylidene)-ON (R ＂) 2,-(alkylidene or be substituted alkylidene)-C (O) SR ＂ ,-(alkylidene or be substituted alkylidene)-S-S-(aryl or be substituted aryl) ,-C (O) R ＂ ,-C (O) 2R ＂ or-C (O) N (R ＂) 2, wherein each R ＂ independently for hydrogen, alkyl, be substituted alkyl, thiazolinyl, be substituted thiazolinyl, alkoxyl, be substituted alkoxyl, aryl, be substituted aryl, heteroaryl, alkaryl, be substituted alkaryl, aralkyl or be substituted aralkyl;
R 5For H, alkyl, be substituted alkyl, thiazolinyl, be substituted thiazolinyl, alkynyl, be substituted alkynyl, alkoxyl, be substituted alkoxyl, alkyl alkoxy, be substituted alkyl alkoxy, polyalkylene oxide, be substituted polyalkylene oxide, aryl, be substituted aryl, heteroaryl, be substituted heteroaryl, alkaryl, be substituted alkaryl, aralkyl, be substituted aralkyl ,-(alkylidene or be substituted alkylidene)-ON (R ＂) 2,-(alkylidene or be substituted alkylidene)-C (O) SR ＂ ,-(alkylidene or be substituted alkylidene)-S-S-(aryl or be substituted aryl) ,-C (O) R ＂ ,-C (O) 2R ＂ or-C (O) N (R ＂) 2, wherein each R ＂ independently for hydrogen, alkyl, be substituted alkyl, thiazolinyl, be substituted thiazolinyl, alkoxyl, be substituted alkoxyl, aryl, be substituted aryl, heteroaryl, alkaryl, be substituted alkaryl, aralkyl, be substituted aralkyl;
Or R 5Be L-X, wherein X is selected from the group that is made up of required functional group; And L is optional, and when existing, it is the connection base that is selected from the group that is made up of following each group: alkylidene, be substituted alkylidene, alkenylene, be substituted alkenylene ,-O-,-O-(alkylidene or be substituted alkylidene)-,-S-,-S-(alkylidene or be substituted alkylidene)-,-S (O) k-(wherein k is 1,2 or 3) ,-S (O) k(alkylidene or be substituted alkylidene)-,-C (O)-,-C (O)-(alkylidene or be substituted alkylidene)-,-C (S)-,-C (S)-(alkylidene or be substituted alkylidene)-,-N (R ＇)-,-NR ＇-(alkylidene or be substituted alkylidene)-,-C (O) N (R ＇)-,-CON (R ＇)-(alkylidene or be substituted alkylidene)-,-CSN (R ＇)-,-CSN (R ＇)-(alkylidene or be substituted alkylidene)-,-N (R ＇) CO-(alkylidene or be substituted alkylidene)-,-N (R ＇) C (O) O-,-(alkylidene or be substituted alkylidene)-O-N=CR ＇-,-(alkylidene or be substituted alkylidene)-C (O) NR ＇-(alkylidene or be substituted alkylidene)-,-(alkylidene or be substituted alkylidene)-S (O) k-(alkylidene or be substituted alkylidene)-S-,-(alkylidene or be substituted alkylidene)-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 each R ＇ is H, alkyl independently or is substituted alkyl;
Or its active metabolite, salt or pharmaceutically acceptable prodrug or solvate.
39. according to the described compound of claim 38, it is selected from the group that is made up of following each thing:
Figure A200680049995C00141
With
Figure A200680049995C00142
R wherein aBe independently selected from the group that forms by following each group: H, halogen, alkyl, be substituted alkyl ,-N (R ＇) 2,-C (O) R ＇-,-C (O) N (R ＇) 2,-OR ＇ and-S (O) kR ＇, wherein k is 1,2 or 3.
40. a compound, it is selected from the group that is made up of following each thing:
Figure A200680049995C00151
With
Figure A200680049995C00152
Wherein:
A is optional, and when existing, it is the low-carbon (LC) alkylidene, be substituted low-carbon (LC) alkylidene, low-carbon (LC) cycloalkylidene, be substituted the low-carbon (LC) cycloalkylidene, the low-carbon (LC) alkenylene, be substituted low-carbon (LC) alkenylene, alkynylene, the inferior assorted alkyl of low-carbon (LC), be substituted inferior assorted alkyl, the inferior Heterocyclylalkyl of low-carbon (LC), be substituted low-carbon (LC) inferior Heterocyclylalkyl, arlydene, be substituted arlydene, inferior heteroaryl, be substituted inferior heteroaryl, alkarylene, be substituted alkarylene, inferior aralkyl or be substituted inferior aralkyl;
B is optional, and when existing, its at one end with contain the connection base that two amine moieties are connected, described connection base is selected from the group that is made up of following each group: the low-carbon (LC) alkylidene, be substituted low-carbon (LC) alkylidene, low-carbon (LC) alkenylene, be substituted the low-carbon (LC) alkenylene, the inferior assorted alkyl of low-carbon (LC), be substituted the low-carbon (LC) Asia mix alkyl ,-O-(alkylidene or be substituted alkylidene)-,-S-(alkylidene or be substituted alkylidene)-,-C (O) R ＂-,-S (O) k(alkylidene or be substituted alkylidene)-(wherein k is 1,2 or 3) ,-C (O)-(alkylidene or be substituted alkylidene)-,-C (S)-(alkylidene or be substituted alkylidene)-,-NR ＂-(alkylidene or be substituted alkylidene)-,-CON (R ＂)-(alkylidene or be substituted alkylidene)-,-CSN (R ＂)-(alkylidene or be substituted alkylidene)-and-N (R ＂) CO-(alkylidene or be substituted alkylidene)-, wherein each R ＂ is independently for H, alkyl or be 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 3And R 4Be H, halogen, low-carbon alkyl or be substituted low-carbon alkyl, perhaps R independently of one another 3And R 4Or two R 3Group forms cycloalkyl or Heterocyclylalkyl according to circumstances;
R 6Independently for H, alkyl, be substituted alkyl, thiazolinyl, be substituted thiazolinyl, alkynyl, be substituted alkynyl, alkoxyl, be substituted alkoxyl, alkyl alkoxy, be substituted alkyl alkoxy, polyalkylene oxide, be substituted polyalkylene oxide, aryl, be substituted aryl, heteroaryl, be substituted heteroaryl, alkaryl, be substituted alkaryl, aralkyl, be substituted aralkyl ,-(alkylidene or be substituted alkylidene)-ON (R ＂) 2,-(alkylidene or be substituted alkylidene)-C (O) SR ＂ ,-(alkylidene or be substituted alkylidene)-S-S-(aryl or be substituted aryl) ,-C (O) R ＂ ,-C (O) 2R ＂ or-C (O) N (R ＂) 2, wherein each R ＂ independently for hydrogen, alkyl, be substituted alkyl, thiazolinyl, be substituted thiazolinyl, alkoxyl, be substituted alkoxyl, aryl, be substituted aryl, heteroaryl, alkaryl, be substituted alkaryl, aralkyl or be substituted aralkyl;
R 5For H, alkyl, be substituted alkyl, thiazolinyl, be substituted thiazolinyl, alkynyl, be substituted alkynyl, alkoxyl, be substituted alkoxyl, alkyl alkoxy, be substituted alkyl alkoxy, polyalkylene oxide, be substituted polyalkylene oxide, aryl, be substituted aryl, heteroaryl, be substituted heteroaryl, alkaryl, be substituted alkaryl, aralkyl, be substituted aralkyl ,-(alkylidene or be substituted alkylidene)-ON (R ＂) 2,-(alkylidene or be substituted alkylidene)-C (O) SR ＂ ,-(alkylidene or be substituted alkylidene)-S-S-(aryl or be substituted aryl) ,-C (O) R ＂ ,-C (O) 2R ＂ or-C (O) N (R ＂) 2, wherein each R ＂ independently for hydrogen, alkyl, be substituted alkyl, thiazolinyl, be substituted thiazolinyl, alkoxyl, be substituted alkoxyl, aryl, be substituted aryl, heteroaryl, alkaryl, be substituted alkaryl, aralkyl, be substituted aralkyl;
Or R 5Be L-X, wherein X is selected from the group that is made up of required functional group; And L is optional, and when existing, it is the connection base that is selected from the group that is made up of following each group: alkylidene, be substituted alkylidene, alkenylene, be substituted alkenylene ,-O-,-O-(alkylidene or be substituted alkylidene)-,-S-,-S-(alkylidene or be substituted alkylidene)-,-S (O) k-(wherein k is 1,2 or 3) ,-S (O) k(alkylidene or be substituted alkylidene)-,-C (O)-,-C (O)-(alkylidene or be substituted alkylidene)-,-C (S)-,-C (S)-(alkylidene or be substituted alkylidene)-,-N (R ＇)-,-NR ＇-(alkylidene or be substituted alkylidene)-,-C (O) N (R ＇)-,-CON (R ＇)-(alkylidene or be substituted alkylidene)-,-CSN (R ＇)-,-CSN (R ＇)-(alkylidene or be substituted alkylidene)-,-N (R ＇) CO-(alkylidene or be substituted alkylidene)-,-N (R ＇) C (O) O-,-(alkylidene or be substituted alkylidene)-O-N=CR ＇-,-(alkylidene or be substituted alkylidene)-C (O) NR ＇-(alkylidene or be substituted alkylidene)-,-(alkylidene or be substituted alkylidene)-S (O) k-(alkylidene or be substituted alkylidene)-S-,-(alkylidene or be substituted alkylidene)-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 each R ＇ is H, alkyl independently or is substituted alkyl;
Or its active metabolite, salt or pharmaceutically acceptable prodrug or solvate.
41. according to the described compound of claim 40, it is selected from the group that is made up of following each thing:
Figure A200680049995C00171
With
Figure A200680049995C00172
R wherein aBe independently selected from the group that forms by following each group: H, halogen, alkyl, be substituted alkyl ,-N (R ＇) 2,-C (O) R ＇-,-C (O) N (R ＇) 2,-OR ＇ and-S (O) kR ＇, wherein k is 1,2 or 3.
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Publication number Priority date Publication date Assignee Title
CN110551203A (en) * 2019-09-25 2019-12-10 成都奥达生物科技有限公司 Exenatide analogue

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110551203A (en) * 2019-09-25 2019-12-10 成都奥达生物科技有限公司 Exenatide analogue

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