CN108047086B - Compositions containing unnatural amino acids and polypeptides, methods involving unnatural amino acids and polypeptides, and uses thereof - Google Patents

Compositions containing unnatural amino acids and polypeptides, methods involving unnatural amino acids and polypeptides, and uses thereof Download PDF

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CN108047086B
CN108047086B CN201810031829.6A CN201810031829A CN108047086B CN 108047086 B CN108047086 B CN 108047086B CN 201810031829 A CN201810031829 A CN 201810031829A CN 108047086 B CN108047086 B CN 108047086B
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amino acid
unnatural amino
alkylene
polypeptide
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CN108047086A (en
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苗振伟
刘俊杰
赛·诺曼
罗素·德瑞弗
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Ambrx Inc
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Abstract

Disclosed herein are unnatural amino acids and polypeptides comprising at least one unnatural amino acid, as well as methods for making these unnatural amino acids and polypeptides. Unnatural amino acids, either by themselves or as part of a polypeptide, can comprise a wide variety of possible functional groups, but typically have at least one oxime, carbonyl, dicarbonyl, and/or hydroxylamine group. Also disclosed herein are non-natural amino acid polypeptides that are further modified post-translationally, methods for effecting such modifications, and methods for purifying such polypeptides. Typically, the modified unnatural amino acid polypeptide comprises at least one oxime, carbonyl, dicarbonyl, and/or hydroxylamine group. Further disclosed are methods of using these natural amino acid polypeptides and modified natural amino acid polypeptides, including therapeutic, diagnostic, and other biotechnological uses.

Description

Compositions containing unnatural amino acids and polypeptides, methods involving unnatural amino acids and polypeptides, and uses thereof
Description of the division
The present application is a divisional application of patent application having a filing date of 12/21 2005, a filing number of 201510093838.4, a title of "composition containing unnatural amino acid and polypeptide, a method involving unnatural amino acid and polypeptide, and uses thereof", a patent application having a filing number of 201510093838.4 is a divisional application of patent application having a filing date of 12/21 2005, a filing number of 200580044328.2 (PCT/US 2005/046618), a title of "composition containing unnatural amino acid and polypeptide, a method involving unnatural amino acid and polypeptide, and uses of unnatural amino acid and polypeptide".
Cross reference to related applications
The present application claims the benefits of U.S. provisional application No. 60/638,418 to 22 nd month 2004, U.S. provisional application No. 60/638,527 to 22 nd month 2004, U.S. provisional application No. 60/639,195 to 22 nd month 2004, U.S. provisional application No. 60/696,210 to 1 st month 7 2005, U.S. provisional application No. 60/696,302 to 1 st month 7 2005 and U.S. provisional application No. 60/696,068 to 1 st month 7 2005, the disclosures of which are incorporated herein by reference in their entirety.
Background
The ability to incorporate non-genetically encoded amino acids (i.e., an "unnatural amino acid") into proteins allows for the introduction of chemical functional groups that can provide naturally occurring functional groups (such as epsilon-NH of lysine) 2 thiol-SH of cysteine, imino group of histidine, etc.). Certain chemical functional groups are known to be inert to the functional groups present in the 20 common genetically encoded amino acids, but are bound toFunctional groups on unnatural amino acids react cleanly and efficiently to form stable linkages.
Methods are available today to selectively introduce chemical functional groups that are not present in proteins, which are chemically inert to all functional groups present in the 20 common genetically encoded amino acids and which can be used to effectively and selectively react with reagents comprising certain functional groups to form stable covalent bonds.
Disclosure of Invention
Described herein are methods, compositions, techniques, and strategies for making, purifying, characterizing, and using unnatural amino acids, unnatural amino acid polypeptides, and modified unnatural amino acid polypeptides. One aspect is methods, compositions, techniques and strategies for derivatizing unnatural amino acids and/or unnatural amino acid polypeptides. In one embodiment, the methods, compositions, techniques and strategies involve chemical derivatization, in other embodiments biological derivatization, in other embodiments physical derivatization, and in other embodiments combinations of derivatizations. In other or additional embodiments, these derivatives are regioselective. In other or additional embodiments, these derivatives are regiospecific. In other or additional embodiments, these derivations are rapid at ambient temperature. In other or additional embodiments, these derivatizations occur in aqueous solutions. In other or additional embodiments, these derivatizations occur at a pH between about 4 and about 10. In other or additional embodiments, these derivatizations are stoichiometric, near stoichiometric or quasi-stoichiometric in the reagents containing the unnatural amino acid and derivatizing reagents by addition of an accelerator. In other or additional embodiments, methods are provided that allow stoichiometric, near stoichiometric or near stoichiometric binding of a desired group to an unnatural amino acid polypeptide by adding a promoter. Strategies, reaction mixtures, synthesis conditions that allow stoichiometric, near stoichiometric or near stoichiometric binding of the desired groups to the unnatural amino acid polypeptide by addition of promoters are provided in other or additional embodiments.
One aspect is an unnatural amino acid for oxime bond-based chemical derivatization of peptides and proteins. In other or additional embodiments, the unnatural amino acid is incorporated into a polypeptide, i.e., these embodiments are unnatural amino acid polypeptides. In other or additional embodiments, the unnatural amino acid is functionalized on its side chain such that it reacts with the derivative molecule to create an oxime bond. In other or additional embodiments, an unnatural amino acid polypeptide that can be reacted with a derivative molecule to produce an oxime-containing unnatural amino acid polypeptide. In other or additional embodiments, the unnatural amino acid is selected from amino acids with carbonyl, dicarbonyl, acetal, hydroxylamine, or oxime side chains. In other or additional embodiments, the unnatural amino acid is selected from amino acids with protected or masked carbonyl, dicarbonyl, hydroxylamine, or oxime side chains. In other or additional embodiments, the unnatural amino acid includes an oxime-masked side chain. In other or additional embodiments, the unnatural amino acid comprises a carbonyl or dicarbonyl side chain, where the carbonyl or dicarbonyl is selected from the group consisting of a ketone or an aldehyde. In another embodiment is an unnatural amino acid containing a functional group capable of forming an oxime upon treatment with a suitably functionalized co-reactant. In another or additional embodiment, the unnatural amino acid is similar in structure to the natural amino acid, but contains one of the aforementioned functional groups. In another or other embodiment, the unnatural amino acid is similar to phenylalanine or tyrosine (aromatic amino acid); in yet another embodiment, the unnatural amino acid is similar to alanine and leucine (hydrophobic amino acids). In one embodiment, the unnatural amino acid has properties that differ from those of the natural amino acid. In one embodiment, these different properties are chemical reactivity of the side chains, which in another embodiment allows the side chains of the unnatural amino acid to undergo a reaction, while as units of the polypeptide, the side chains of even naturally occurring amino acid units in the same polypeptide do not undergo the aforementioned reaction. In another embodiment, the side chain of the unnatural amino acid has a chemistry orthogonal to the side chain of the naturally occurring amino acid. In another embodiment, the side chain of the unnatural amino acid includes a moiety that comprises an electrophile; in another embodiment, the electrophilic moiety on the side chain of the unnatural amino acid can undergo nucleophilic attack to produce an oxime-derived protein. In any of the foregoing embodiments in this paragraph, the unnatural amino acid can exist as a separate molecule or can be incorporated into a polypeptide of any length; if the latter, the polypeptides may further incorporate naturally occurring or unnatural amino acids.
Another aspect is a hydroxylamine substituted molecule for use in the preparation of oxime bond based derivatized unnatural amino acid polypeptides. In another embodiment is a hydroxylamine substituted molecule for derivatizing a carbonyl or dicarbonyl containing unnatural amino acid polypeptide by forming an oxime bond between the derivatizing molecule and the carbonyl or dicarbonyl containing unnatural amino acid polypeptide. In other embodiments, the aforementioned carbonyl-or dicarbonyl-containing unnatural amino acid polypeptide is a keto-containing unnatural amino acid polypeptide. In other or additional embodiments, the non-natural amino acid containing a carbonyl or dicarbonyl group includes a side chain selected from a ketone or an aldehyde. In other or additional embodiments, the hydroxylamine-substituted molecule comprises a member selected from the group consisting of: marking; a dye; a polymer; a water-soluble polymer; derivatives of polyethylene glycol; a photocrosslinking agent; a cytotoxic compound; a drug; affinity labeling; a photoaffinity label; a reactive compound; a resin; a second protein or polypeptide analog; an antibody or antibody fragment; a metal chelator; a cofactor; a fatty acid; a carbohydrate; a polynucleotide; DNA; RNA; an antisense polynucleotide; saccharides, water-soluble dendrimers, cyclodextrins, biomaterials; a nanoparticle; spin labeling; a fluorophore-containing moiety; a radioactive portion; novel functional groups; groups that interact covalently or non-covalently with other molecules; a light-shielding portion; an actinic radiation excitable moiety, a ligand, a photoisomerisable moiety; biotin; biotin analogues; a moiety incorporating a heavy atom; a chemically cleavable group; a photocleavable group; an extended side chain; a carbon-bonded sugar; a redox active agent; an aminothiopropionic acid; a toxic moiety; isotopically labeled moieties; a biophysical probe; phosphorescent groups; a chemiluminescent group; an electron dense group; a magnetic group; an insertion group; a chromophore; an energy transfer agent; a bioactive agent; a detectable label; a small molecule; inhibitory ribonucleic acid, radionucleotide; a neutron capture agent; derivatives of biotin; a quantum dot; nano transitorin; a radiotransferrin; abzymes, activating complex activators, viruses, adjuvants, aglycones, allergens, angiostatin, anti-hormones, antioxidants, aptamers, guide RNAs, saponins, shuttle vectors, macromolecules, pseudoepitopes, receptors, reverse micelles, and any combination thereof. In other or additional embodiments, the hydroxylamine-substituted molecule is a hydroxylamine-substituted polyethylene glycol (PEG) molecule. In another embodiment, the side chains of the unnatural amino acid have a chemistry orthogonal to those of the naturally occurring amino acid that allows for selective reaction of the unnatural amino acid with the hydroxylamine-substituted molecule. In another embodiment, the side chain of the unnatural amino acid includes an electrophile-containing moiety that selectively reacts with a hydroxylamine-containing molecule; in another embodiment, the electrophilic moiety on the side chain of the unnatural amino acid can undergo nucleophilic attack to produce an oxime-derived protein. Another aspect related to the embodiments described in this paragraph is the modified unnatural amino acid polypeptide resulting from the reaction of the derivative molecule with the unnatural amino acid polypeptide. Other embodiments include any further modifications to the modified unnatural amino acid polypeptide.
Another aspect is a carbonyl or dicarbonyl substituted molecule for use in the preparation of oxime bond based derivatized unnatural amino acid polypeptides. In another embodiment is a carbonyl or dicarbonyl substituted molecule for derivatizing an oxime-containing unnatural amino acid polypeptide by an oxime exchange reaction between the derivatizing molecule and an oxime-containing peptide or protein. In another embodiment is a carbonyl or dicarbonyl substituted molecule that can undergo oxime exchange with an oxime-containing unnatural amino acid polypeptide at a pH ranging between about 4 and about 8. In another embodiment is a carbonyl or dicarbonyl substituted molecule for derivatizing an oxime-containing or hydroxylamine-containing unnatural amino acid polypeptide by forming an oxime bond between the derivatized molecule and the oxime-containing (thus forming a new oxime bond by an exchange-type reaction) or hydroxylamine-containing unnatural amino acid polypeptide. In another embodiment, the carbonyl-or dicarbonyl-substituted molecule is an aldehyde-substituted molecule. In other embodiments, the carbonyl-or dicarbonyl-substituted molecules comprise a moiety selected from the group consisting of: marking; a dye; a polymer; a water-soluble polymer; derivatives of polyethylene glycol; a photocrosslinking agent; a cytotoxic compound; a drug; affinity labeling; a photoaffinity label; a reactive compound; a resin; a second protein or polypeptide analog; an antibody or antibody fragment; a metal chelator; a cofactor; a fatty acid; a carbohydrate; a polynucleotide; DNA; RNA; an antisense polynucleotide; saccharides, water-soluble dendrimers, cyclodextrins, biomaterials; a nanoparticle; spin labeling; a fluorophore-containing moiety; a radioactive portion; novel functional groups; groups that interact covalently or non-covalently with other molecules; a light-shielding portion; an actinic radiation excitable moiety, a ligand, a photoisomerisable moiety; biotin; biotin analogues; a moiety incorporating a heavy atom; a chemically cleavable group; a photocleavable group; an extended side chain; a carbon-bonded sugar; a redox active agent; an aminothiopropionic acid; a toxic moiety; isotopically labeled moieties; a biophysical probe; phosphorescent groups; a chemiluminescent group; an electron dense group; a magnetic group; an insertion group; a chromophore; an energy transfer agent; a bioactive agent; a detectable label; a small molecule; inhibitory ribonucleic acid, radionucleotide; a neutron capture agent; derivatives of biotin; a quantum dot; nano transitorin; a radiotransferrin; abzymes, activating complex activators, viruses, adjuvants, aglycones, allergens, angiostatin, anti-hormones, antioxidants, aptamers, guide RNAs, saponins, shuttle vectors, macromolecules, pseudoepitopes, receptors, reverse micelles, and any combination thereof. In other or additional embodiments, the aldehyde-substituted molecule is an aldehyde-substituted polyethylene glycol (PEG) molecule. In another embodiment, the side chains of the unnatural amino acid have a chemistry orthogonal to those of the naturally occurring amino acid that allows the unnatural amino acid to selectively react with molecules substituted with carbonyl or dicarbonyl groups. In another embodiment, the side chain of the unnatural amino acid includes a moiety that selectively reacts with a carbonyl-or dicarbonyl-containing molecule, e.g., an oxime group or a hydroxylamine group; in another embodiment, the nucleophilic moiety on the side chain of the unnatural amino acid can undergo electrophilic attack to produce an oxime-derivatized protein. Another aspect associated with the embodiments described in this paragraph is a modified unnatural amino acid polypeptide that results from the reaction of a derivative molecule with a unnatural amino acid polypeptide. Other embodiments include any further modifications to the modified unnatural amino acid polypeptide.
Another aspect is a mono-, di-and multi-functional linker for producing oxime-based derivatized unnatural amino acid polypeptides. In one embodiment are molecular linkers (bi-functional as well as multi-functional) that can be used to link carbonyl-or dicarbonyl-containing unnatural amino acid polypeptides to other molecules. In another embodiment are molecular linkers (bi-functional as well as multi-functional) useful for linking an oxime-or hydroxylamine-containing unnatural amino acid polypeptide to other molecules. In another embodiment, the carbonyl-or dicarbonyl-containing unnatural amino acid polypeptide comprises a ketone and/or aldehyde side chain. In embodiments utilizing an oxime-or hydroxylamine-containing unnatural amino acid polypeptide, the molecular linker contains a carbonyl or dicarbonyl group at one end thereof; in other embodiments, the carbonyl or dicarbonyl is selected from aldehyde or ketone groups. In other or additional embodiments, the hydroxylamine-substituted linker molecule is a hydroxylamine-substituted polyethylene glycol (PEG) linker molecule. In other or additional embodiments, the carbonyl-or dicarbonyl-substituted linker molecule is a carbonyl-or dicarbonyl-substituted polyethylene glycol (PEG) linker molecule. In other embodiments, the phrase "other molecules" includes, by way of example only, proteins, other polymers, and small molecules. In other or additional embodiments, the hydroxylamine-containing linker molecule comprises the same or equivalent groups on all ends, such that upon reaction with a carbonyl-or dicarbonyl-containing non-natural amino acid polypeptide, the resulting product is a homomultimer of the carbonyl-or dicarbonyl-containing non-natural amino acid polypeptide. In other embodiments, the homodimerization is a homodimerization. In other or additional embodiments, the carbonyl-or dicarbonyl-containing molecular linker comprises the same or equivalent groups at all ends, such that upon reaction with an oxime-or hydroxylamine-containing non-natural amino acid polypeptide, the resulting product is a homo-multimer of the oxime-or hydroxylamine-containing non-natural amino acid polypeptide. In other embodiments, the homodimerization is a homodimerization. In another embodiment, the side chains of the unnatural amino acid have a chemistry orthogonal to those of the naturally occurring amino acid that allows the unnatural amino acid to selectively react with the hydroxylamine-substituted linker molecule. In another embodiment, the side chains of the unnatural amino acid have a chemistry orthogonal to those of the naturally occurring amino acid that allows the unnatural amino acid to selectively react with carbonyl or dicarbonyl substituted linker molecules. In another embodiment, the side chain of the unnatural amino acid includes an electrophile-containing moiety that selectively reacts with a hydroxylamine-containing linker molecule; in another embodiment, the electrophilic moiety on the side chain of the unnatural amino acid can undergo nucleophilic attack by a linker molecule comprising hydroxylamine to produce an oxime-derivatized protein. Another aspect associated with the embodiments described in this paragraph is the linked "modified or unmodified" unnatural amino acid polypeptide resulting from the reaction of the linker molecule with the unnatural amino acid polypeptide. Other embodiments include any further modifications to the linked "modified or unmodified" unnatural amino acid polypeptides.
One aspect is a method of derivatizing proteins by condensation of carbonyl or dicarbonyl groups with hydroxylamine reactants to produce oximido products. Included within this aspect are methods for producing oxime-derived protein adduct-derived proteins based on the condensation of a carbonyl-or dicarbonyl-containing reactant with a hydroxylamine-containing reactant. In additional or other embodiments is a method of derivatizing a ketone-containing protein with a hydroxylamine-functionalized polyethylene glycol (PEG) molecule. In additional or other aspects are methods of derivatizing oxime-containing proteins by an oxime exchange reaction between a carbonyl-or dicarbonyl-containing derivatizing molecule and an oxime-containing peptide or protein. In additional or other aspects, hydroxylamine-substituted molecules may include proteins, other polymers, and small molecules.
Another aspect is a method of chemically synthesizing hydroxylamine-substituted molecules for derivatizing ketone-substituted proteins. Another aspect is a method of chemically synthesizing hydroxylamine-substituted molecules for derivatizing aldehyde-substituted proteins. In one embodiment, hydroxylamine substituted molecules may include peptides, other polymers (unbranched and branched), and small molecules. In one embodiment is a method of preparing a hydroxylamine-substituted molecule suitable for derivatizing carbonyl-or dicarbonyl-containing unnatural amino acid polypeptides, including, by way of example only, ketone-containing unnatural amino acid polypeptides. In another or additional embodiment, the unnatural amino acid is site-specifically incorporated during in vivo translation of the protein. In another or additional embodiment, nucleophilic attack of hydroxylamine-substituted molecules by carbonyl or dicarbonyl groups allows site-specific derivatization of such carbonyl-or dicarbonyl-containing unnatural amino acids to form oxime-derivatized polypeptides in a site-specific manner. In another or additional embodiment, the method of making a hydroxylamine-substituted molecule provides a method of obtaining a variety of site-specific derivative polypeptides. In another or additional embodiment is a method of synthesizing a hydroxylamine-functionalized polyethylene glycol (PEG) molecule.
Another aspect is a method of chemically synthesizing a carbonyl or dicarbonyl substituted molecule for derivatizing an oxime substituted unnatural amino acid polypeptide. In one embodiment, the carbonyl-or dicarbonyl-substituted molecule is a ketone-substituted molecule. In one embodiment, the carbonyl-or dicarbonyl-substituted molecule is an aldehyde-substituted molecule. In another embodiment, the carbonyl-or dicarbonyl-substituted molecules comprise proteins, polymers (unbranched and branched), and small molecules. In another or additional embodiment, these methods complement techniques capable of site-specific incorporation of unnatural amino acids during in vivo translation of a protein. In another or additional embodiment is a general method of preparing a carbonyl-or dicarbonyl-substituted molecule suitable for reaction with an oxime-containing unnatural amino acid polypeptide to provide a site-specifically derivatized unnatural amino acid polypeptide. In another or additional embodiment is a method of synthesizing a carbonyl or dicarbonyl substituted polyethylene glycol (PEG) molecule.
Another aspect is a method of chemically derivatizing carbonyl-or dicarbonyl-substituted unnatural amino acid polypeptides using a bifunctional linker comprising hydroxylamine. In one embodiment is a method of linking a hydroxylamine-substituted linker to a carbonyl or dicarbonyl substituted protein by a condensation reaction to produce an oxime bond. In other or additional embodiments, the carbonyl-or dicarbonyl-substituted unnatural amino acid is a ketone-substituted unnatural amino acid. In other or additional embodiments, the unnatural amino acid polypeptide is site-specifically derivatized and/or the three-dimensional structure is derivatized with precise control using a bifunctional linker comprising hydroxylamine. In one embodiment, these methods are used to attach molecular linkers (including, but not limited to, monofunctional, difunctional, and multifunctional linkers) to carbonyl-containing or dicarbonyl-containing (including, but not limited to, ketone-containing and aldehyde-containing) unnatural amino acid polypeptides, where at least one of the linker termini contains a hydroxylamine group that can be linked to the carbonyl-containing or dicarbonyl-containing unnatural amino acid polypeptide by an oxime linkage. In another or additional embodiment, these linkers are used to link carbonyl-or dicarbonyl-containing unnatural amino acid polypeptides with other molecules, including, for example, proteins, other polymers (branched and unbranched), and small molecules.
In some embodiments, the unnatural amino acid polypeptide is linked to a water-soluble polymer. In some embodiments, the water-soluble polymer includes a polyethylene glycol moiety. In some embodiments, the polyethylene glycol molecule is a difunctional polymer. In some embodiments, the bifunctional polymer is linked to the second polypeptide. In some embodiments, the second polypeptide is the same as the first polypeptide, and in other embodiments, the second polypeptide is a different polypeptide. In some embodiments, the unnatural amino acid polypeptide comprises at least two amino acids that are linked to a water-soluble polymer (including polyethylene glycol moieties).
In some embodiments, the non-natural amino acid polypeptide includes substitutions, additions, or deletions that increase the affinity of the non-natural amino acid polypeptide for the receptor. In some embodiments, the non-natural amino acid polypeptide comprises a substitution, addition, or deletion that increases the stability of the non-natural amino acid polypeptide. In some embodiments, the non-natural amino acid polypeptide includes substitutions, additions, or deletions that increase the water solubility of the non-natural amino acid polypeptide. In some embodiments, the unnatural amino acid polypeptide includes a substitution, addition, or deletion that increases the solubility of the produced unnatural amino acid polypeptide in the host cell. In some embodiments, the unnatural amino acid polypeptide includes a substitution, addition, or deletion that modulates protease resistance, serum half-life, immunogenicity, and/or expression relative to an amino acid polypeptide that does not have a substitution, addition, or deletion.
In some embodiments, the unnatural amino acid polypeptide is an agonist, a partial agonist, an antagonist, a partial antagonist, or a reverse agonist. In some embodiments, the agonist, partial agonist, antagonist, partial antagonist, or reverse agonist comprises an unnatural amino acid that is bonded to a water-soluble polymer. In some embodiments, the water-soluble polymer includes a polyethylene glycol moiety. In some embodiments, polypeptides comprising unnatural amino acids bonded to water-soluble polymers, for example, can prevent dimerization of the corresponding receptors. In some embodiments, a polypeptide comprising an unnatural amino acid bonded to a water-soluble polymer modulates the binding of the polypeptide to a binding partner, ligand, or receptor. In some embodiments, a polypeptide comprising an unnatural amino acid bonded to a water-soluble polymer modulates one or more property or activity of the polypeptide.
In some embodiments, the selector codon is selected from the group consisting of an amber codon (amber codon), an ochre codon (ochre codon), an opal codon (opal codon), a unique codon (unique codon), a rare codon (rare codon), an unnatural codon (unique codon), a five-base codon (five-base codon), and a four-base codon (four-base codon).
Also described herein are methods of making non-natural amino acid polypeptides linked to water-soluble polymers. In some embodiments, the method comprises contacting an isolated polypeptide comprising an unnatural amino acid with a water-soluble polymer that comprises a moiety that reacts with the unnatural amino acid. In some embodiments, the incorporated unnatural amino acid is reactive with a water-soluble polymer that is unreactive with any of the 20 common amino acids. In some embodiments, the water-based polymer includes polyethylene glycol moieties. The molecular weight of the polymer may be in a wide range including, but not limited to, a range between about 100Da and about 100,000Da or higher. The molecular weight of the polymer may be between about 100Da and about 100,000Da, including, but not limited to, 100,000Da, 95,000Da, 90,000Da, 85,000Da, 80,000Da, 75,000Da, 70,000Da, 65,000Da, 60,000Da, 55,000Da, 50,000Da, 45,000Da, 40,000Da, 35,000Da, 30,000Da, 25,000Da, 20,000Da, 15,000Da, 10,000Da, 9,000Da, 8,000Da, 7,000Da, 6,000Da, 5,000Da, 4,000Da, 3,000Da, 2,000Da, 1,000Da, 900Da, 800Da, 700Da, 600Da, 500Da, 400Da, 300Da, 200Da, and 100Da. In some embodiments, the molecular weight of the polymer is between about 100Da and 50,000 Da. In some embodiments, the molecular weight of the polymer is between about 100Da and 40,000 Da. In some embodiments, the molecular weight of the polymer is between about 1,000da and 40,000 da. In some embodiments, the molecular weight of the polymer is between about 5,000da and 40,000 da. In some embodiments, the molecular weight of the polymer is between about 10,000da and 40,000 da. In some embodiments, the polyethylene glycol molecule is a branched polymer. The molecular weight of the branched PEG may be between about 1,000da and about 100,000da, including, but not limited to, 100,000da, 95,000da, 90,000da, 85,000da, 80,000da, 75,000da, 70,000da, 65,000da, 60,000da, 55,000da, 50,000da, 45,000da, 40,000da, 35,000da, 30,000da, 25,000da, 20,000da, 15,000da, 10,000da, 9,000da, 8,000da, 7,000da, 6,000da, 5,000da, 4,000da, 3,000da, 2,000da and 1,000da. In some embodiments, the branched PEG has a molecular weight between about 1,000da and 50,000 da. In some embodiments, the branched PEG has a molecular weight between about 1,000da and 40,000 da. In some embodiments, the branched PEG has a molecular weight between about 5,000da and 40,000 da. In some embodiments, the branched PEG has a molecular weight between about 5,000da and 20,000 da.
Also described herein are compositions comprising at least one unnatural amino acid described herein and a pharmaceutically acceptable carrier. In some embodiments, the unnatural amino acid is bonded to the water-soluble polymer. Also described herein are pharmaceutical compositions comprising a pharmaceutically acceptable carrier and a polypeptide, wherein at least one amino acid is substituted with an unnatural amino acid. In some embodiments, the unnatural amino acid comprises a sugar moiety. In some embodiments, the water-soluble polymer is linked to the polypeptide through a sugar moiety. Also described herein are unnatural amino acids, unnatural amino acid polypeptides, and prodrugs of modified unnatural amino acid polypeptides; compositions comprising these prodrugs and a pharmaceutically acceptable carrier are further described herein. Also described herein are unnatural amino acids, unnatural amino acid polypeptides, and metabolites of modified unnatural amino acid polypeptides; these metabolites may have desirable activities that complement or synergistically enhance the activities of the unnatural amino acid, unnatural amino acid polypeptide, and modified unnatural amino acid polypeptides. Also described herein are the use of the unnatural amino acids, unnatural amino acid polypeptides, and modified unnatural amino acid polypeptides described herein to provide a desired metabolite to an organism (comprising a patient in need of such a metabolite).
Also described herein are cells comprising a polynucleotide encoding a polypeptide and comprising a selector codon. In some embodiments, the cell includes an orthogonal RNA synthetase and/or an orthogonal tRNA for substituting an unnatural amino acid into a polypeptide. In some embodiments, the cells are in cell culture, while in other embodiments are cells that are part of multicellular organisms (including amphibians, reptiles, birds, and mammals). In any of the cell embodiments, other embodiments comprise expression of a polynucleotide that produces the unnatural amino acid polypeptide. In other embodiments are organisms that can utilize the unnatural amino acids described herein to produce unnatural amino acid polypeptides (including modified unnatural amino acid polypeptides). In other embodiments are organisms containing the unnatural amino acids, unnatural amino acid polypeptides, and/or modified unnatural amino acid polypeptides described herein. These organisms include unicellular organisms and multicellular organisms, which include amphibians, reptiles, birds and mammals. In some embodiments, the unnatural amino acid polypeptide is produced in vitro. In some embodiments, the unnatural amino acid polypeptide is produced in a cell lysate. In some embodiments, the unnatural amino acid polypeptide is produced by ribosome translation.
Also described herein are methods of making polypeptides comprising unnatural amino acids. In some embodiments, the method comprises culturing a cell comprising a polynucleotide encoding a polypeptide, an orthogonal RNA synthetase, and/or an orthogonal tRNA under conditions that allow for expression of the polypeptide; and purifying the polypeptide from the cells and/or the culture medium.
Also described herein are libraries of unnatural amino acids described herein or libraries of unnatural amino acid polypeptides described herein, or libraries of modified unnatural amino acid polypeptides described herein, or a combination thereof. Also described herein are arrays containing at least one unnatural amino acid, at least one unnatural amino acid polypeptide, and/or at least one modified unnatural amino acid. Also described herein are arrays comprising at least one polynucleotide encoding a polypeptide and comprising a selector codon. The arrays described herein can be used to screen for the production of unnatural amino acid polypeptides in an organism (either by detecting transcription of a polynucleotide encoding the polypeptide or by detecting translation of the polypeptide).
Also described herein are methods of screening for a desired activity of a library described herein, or a library of other compounds and/or polypeptides and/or polynucleotides described herein using an array described herein. The use of this activity data from library screening for the development and discovery of new therapeutic agents, as well as the therapeutic agents themselves, is also described herein.
Also described herein are methods of increasing the therapeutic half-life, serum half-life, or circulation time of a polypeptide. In some embodiments, the method comprises replacing any one or more amino acids in the naturally occurring polypeptide with at least one unnatural amino acid and/or coupling the polypeptide to a water-soluble polymer.
Also described herein are methods of treating a patient in need of such treatment with an effective amount of a pharmaceutical composition, wherein the pharmaceutical composition comprises a polypeptide comprising an unnatural amino acid and a pharmaceutically acceptable carrier. In some embodiments, the unnatural amino acid is coupled to a water-soluble polymer.
In other or alternative embodiments is a method of treating a disorder, condition, or disease, the method comprising administering a therapeutically effective amount of an unnatural amino acid polypeptide comprising at least one unnatural amino acid, where the unnatural amino acid is selected from the group consisting of an oxime-containing unnatural amino acid, a carbonyl-containing unnatural amino acid, a dicarbonyl-containing unnatural amino acid, and a hydroxylamine-containing unnatural amino acid. In other embodiments, these unnatural amino acids are incorporated into polypeptides described herein by biosynthesis. In other or alternative embodiments, the unnatural amino acid polypeptides include at least one unnatural amino acid selected from the group consisting of amino acids of formulas I-XVIII, formulas XXX-XXXIV (A and B), and formulas XXXX-XXXXIII.
In other or alternative embodiments is a method of treating a disorder, condition, or disease, the method comprising administering a therapeutically effective amount of a non-natural amino acid polypeptide comprising at least one oxime-containing non-natural amino acid, and the resulting biosynthetic oxime-containing non-natural amino acid polypeptide enhances the bioavailability of the polypeptide relative to a homologous naturally-occurring amino acid polypeptide.
In other or alternative embodiments are methods of treating a disorder, condition, or disease, the method comprising administering a therapeutically effective amount of a non-natural amino acid polypeptide comprising at least one oxime-containing non-natural amino acid, and the resulting biosynthetic oxime-containing non-natural amino acid polypeptide increases the safety profile of the polypeptide relative to a homologous naturally-occurring amino acid polypeptide.
In other or alternative embodiments is a method of treating a disorder, condition, or disease, the method comprising administering a therapeutically effective amount of a non-natural amino acid polypeptide comprising at least one oxime-containing non-natural amino acid, and the resulting biosynthetic oxime-containing non-natural amino acid polypeptide increases the water solubility of the polypeptide relative to the homologous naturally-occurring amino acid polypeptide.
In other or alternative embodiments is a method of treating a disorder, condition, or disease, the method comprising administering a therapeutically effective amount of a non-natural amino acid polypeptide comprising at least one oxime-containing non-natural amino acid, and the resulting biosynthetic oxime-containing non-natural amino acid polypeptide increases the therapeutic half-life of the polypeptide relative to the homologous naturally-occurring amino acid polypeptide.
In other or alternative embodiments is a method of treating a disorder, condition, or disease, the method comprising administering a therapeutically effective amount of a non-natural amino acid polypeptide comprising at least one oxime-containing non-natural amino acid, and the resulting biosynthetic oxime-containing non-natural amino acid polypeptide increases the serum half-life of the polypeptide relative to the homologous naturally-occurring amino acid polypeptide.
In other or alternative embodiments is a method of treating a disorder, condition, or disease, the method comprising administering a therapeutically effective amount of a non-natural amino acid polypeptide comprising at least one oxime-containing non-natural amino acid, and the resulting biosynthetic oxime-containing non-natural amino acid polypeptide extends the circulation time of the polypeptide relative to a homologous naturally-occurring amino acid polypeptide.
In other or alternative embodiments is a method of treating a disorder, condition, or disease, the method comprising administering a therapeutically effective amount of a non-natural amino acid polypeptide comprising at least one oxime-containing non-natural amino acid, and the resulting biosynthesized oxime-containing non-natural amino acid polypeptide modulates the biological activity of the polypeptide relative to a homologous naturally-occurring amino acid polypeptide.
In other or alternative embodiments is a method of treating a disorder, condition, or disease, the method comprising administering a therapeutically effective amount of a non-natural amino acid polypeptide comprising at least one oxime-containing non-natural amino acid, and the resulting biosynthetic oxime-containing non-natural amino acid polypeptide modulates the immunogenicity of the polypeptide relative to a homologous naturally-occurring amino acid polypeptide.
It is to be understood that the methods and compositions described herein are not limited to the particular methods, protocols, cell lines, constructs, and reagents described herein and may vary as well. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the methods and compositions described herein which will be limited only by the appended claims.
As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs as described herein. Although any methods, devices, and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention described herein, the preferred methods, devices, and materials are now described.
All publications and patents mentioned herein are incorporated herein by reference in their entirety for the purpose of describing and disclosing, for example, the constructs and methods described in the publications, which might be used in connection with the invention described herein. The disclosure discussed herein provides disclosure of this application only prior to the filing date of this application. Nothing herein is to be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason.
The term "affinity tag" as used herein refers to a tag that binds reversibly or irreversibly to another molecule to modify it, destroy it, or form a compound therewith. For example, affinity tags include enzymes and their matrices, or antibodies and their antigens.
The terms "alkoxy", "alkylamino" and "alkylthio" are used in their conventional sense and refer to an alkyl group bonded to the molecule through an oxygen atom, amino group, sulfur atom, respectively.
Unless otherwise indicated, the term "alkyl" (by itself or as part of another molecule) means a straight or branched chain, or cyclic hydrocarbon group, or a combination thereof, which may be fully saturated, monounsaturated, or polyunsaturated and may contain both divalent and multivalent groups having the indicated number of carbon atoms (i.e., C1-C10 means 1 to 10 carbons). Examples of saturated hydrocarbon groups include, but are not limited to, homologs and isomers of groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl, (cyclohexyl) methyl, cyclopropylmethyl, (e.g., n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. Unsaturated alkyl is alkyl having one or more double or triple bonds. Examples of unsaturated alkyl groups include, but are not limited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2- (butadienyl), 2, 4-pentadienyl, 3- (1, 4-pentadienyl), ethynyl, 1-propynyl and 3-propynyl, 3-butynyl and higher homologs and isomers. Unless otherwise indicated, the term "alkyl" also means those derivatives that include alkyl groups as defined in more detail herein, such as "heteroalkyl", "haloalkyl" and "homoalkyl".
The term "alkylene" (by itself or as part of another molecule) means a divalent group derived from an alkane, such as from (-CH) 2 (-) n, wherein n may be from 1 to about 24. By way of example only, these groups include, but are not limited to, groups having 10 or fewer carbon atoms, such as structure-CH 2 CH 2 -and-CH 2 CH 2 CH 2 CH 2 -. "lower alkyl" or "lower alkylene" is a shorter chain alkyl or alkylene group typically having 8 or fewer carbon atoms. Unless otherwise indicated, the term "alkylene" is also meant to include those groups described herein as "heteroalkylene".
The term "amino acid" refers to naturally occurring amino acids and non-natural amino acids, as well as amino acid analogs and amino acid mimics that function in a manner similar to naturally occurring amino acids. Naturally encoded amino acids are 20 common amino acids (alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine), pyrolysine and selenocysteine. Amino acid analogs refer to compounds that have the same basic chemical structure as a naturally occurring amino acid (e.g., only the alpha carbon to which hydrogen, carboxyl, amino, and R groups are bound). These analogs can have modified R groups (e.g., norleucine) or can have modified peptide backbones while retaining the same basic chemical structure as a naturally occurring amino acid. Non-limiting examples of amino acid analogs include homoserine, norleucine, methionine oxysulfide, methionine methyl sulfonium.
Amino acids may be referred to herein by their name, by their commonly known three letter symbols, or by the one-letter symbols recommended by the IUPAC-IUB biochemical nomenclature committee (IUPAC-IUB Biochemical Nomenclature Commission). In addition, nucleotides may be referred to by their commonly accepted single-letter codes.
"amino terminal modifying group" refers to any molecule that can be attached to a terminal amino group. For example, these terminal amino groups may be at the ends of polymeric molecules, wherein these polymeric molecules include, but are not limited to, polypeptides, polynucleotides, and polysaccharides. The terminal modifying groups include, but are not limited to, various water-soluble polymers, peptides or proteins. By way of example only, the terminal modifying group comprises polyethylene glycol or serum albumin. The terminal modifying group can be used to alter the therapeutic characteristics of the polymeric molecule, including, but not limited to, increasing the serum half-life of the peptide.
By "antibody fragment" is meant any form of antibody other than the full length form. Antibody fragments herein include antibodies that are smaller components present within the whole length antibody, as well as engineered antibodies. Antibody fragments include, but are not limited to, fv, fc, fab, and (Fab') 2, single chain Fv (scFv), diabody, triabody, tetrabody, bifunctional hybrid antibodies, CDR1, CDR2, CDR3, combinations of CDRs, variable regions, framework regions, constant regions, heavy, light and variable regions, and alternative framework non-antibody molecules, bispecific antibodies and analogs thereof (Maynard and Georgiou,2000, annu. Rev. Biomed. 2:339-76; hudson,1998, curr. Opin. Biotechnol. 9:395-402). Another functional substructure is a single chain Fv (scFv) comprising the variable regions of the immunoglobulin heavy and light chains covalently linked by a peptide linker (S-z Hu et al, 1996,Cancer Research,56,3055-3061). These small (Mr 25,000) proteins generally maintain specificity and affinity for antigens in a single polypeptide and can provide the appropriate building blocks for larger antigen-specific molecules. Unless specifically indicated otherwise, statements and claims using the term "antibody" expressly encompass "antibody fragments.
As used herein, the term "aromatic" or "aryl" refers to a closed ring structure having at least one ring with a conjugated pi electron system and comprising carbocyclic and heterocyclic aryl groups (or "heteroaryl" or "heteroaromatic"). Carbocyclic or heterocyclic aromatic groups may contain 5 to 20 ring atoms. The term encompasses covalently bonded monocyclic or fused polycyclic (i.e., rings sharing adjacent pairs of carbon atoms) groups. The aromatic groups may be unsubstituted or substituted. Non-limiting examples of "aromatic" or "aryl" groups include phenyl, 1-naphthyl, 2-naphthyl, 4-biphenyl, anthracenyl, and phenanthrenyl. The substituents of each of the above aryl and heteroaryl ring systems are selected from the group of acceptable substituents described herein.
For simplicity, the term "aromatic" or "aryl" when used in combination with other terms, including but not limited to aryloxy, arylthioxy, aralkyl, includes both aryl and heteroaryl rings as defined above. Thus, the term "aralkyl" or "alkaryl" is meant to include those groups in which an aryl group is attached to an alkyl group (including, but not limited to, benzyl, phenethyl, pyridylmethyl, and the like), wherein the alkyl group includes those alkyl groups in which a carbon atom (including, but not limited to, methylene) has been replaced by a heteroatom (by way of example only, an oxygen atom). Examples of such aryl groups include, but are not limited to, phenoxymethyl, 2-pyridyloxymethyl, 3- (1-naphthyloxy) propyl, and the like.
As used herein, the term "arylene" refers to a divalent aryl group. Non-limiting examples of "arylene" include phenylene, pyridylene, pyrimidinylene, and thiophenylene. The substituents of the arylene groups are selected from the group of acceptable substituents described herein.
"bifunctional polymer", also referred to as "bifunctional linker", refers to a polymer comprising two functional groups capable of specifically reacting with other moieties to form covalent or non-covalent bonds. These moieties may include, but are not limited to, pendant groups on natural or unnatural amino acids or peptides containing such natural or unnatural amino acids. By way of example only, the bifunctional linker may have a functional group that is reactive with a group on the first peptide and another functional group that is reactive with a group on the second peptide, thereby forming a conjugate comprising the first peptide, the bifunctional linker, and the second peptide. Numerous procedures and linker molecules for linking a variety of compounds to peptides are known. See, for example, european patent application No. 188,256; U.S. Pat. nos. 4,671,958, 4,659,839, 4,414,148, 4,699,784; 4,680,338; and 4,569,789, which are incorporated by reference herein in their entirety. "multifunctional polymer" is also referred to as "multifunctional linker" and refers to a polymer that includes two or more functional groups capable of reacting with other moieties. These moieties may comprise, but are not limited to, natural or unnatural amino acids or pendant groups (including, but not limited to, pendant amino acid groups) on peptides containing these natural or unnatural amino acids to form covalent or noncovalent bonds. The difunctional or polyfunctional polymer may be of any desired length or molecular weight and may be selected to provide a particular desired spacing or conformation between one or more molecules bonded to the compound and the molecule or compound to which it is bound.
As used herein, the term "bioavailability" refers to the rate and extent at which a substance or active portion thereof is delivered from a pharmaceutical dosage form and becomes available at the site of action or in the general circulation. Increased bioavailability refers to increasing the rate and extent at which a substance or active portion thereof is delivered from a pharmaceutical dosage form and becomes available at the site of action or in the general circulation. For example, an increase in bioavailability may be indicated as an increase in the concentration of a substance or active portion thereof in blood when compared to other substances or active portions. Non-limiting examples of methods of assessing increased bioavailability are given in examples 88-92. This method can be used to assess the bioavailability of any polypeptide.
As used herein, the term "bioactive molecule," "bioactive moiety" or "bioactive agent" means any substance that can affect the biological system, pathway, any physical or biochemical property of a molecule, or interaction associated with an organism, including, but not limited to, viruses, bacteria, phages, transductors, prions, insects, fungi, plants, animals, and humans. In particular, as used herein, bioactive molecules include, but are not limited to, any substance intended for diagnosing, curing, alleviating, treating or preventing a disease in a human or other animal, or otherwise enhancing the physical or mental health of a human or animal. Examples of bioactive molecules include, but are not limited to, peptides, proteins, enzymes, small molecule drugs, hard drugs, soft drugs, carbohydrates, inorganic atoms or molecules, dyes, lipids, nucleosides, radionuclides, oligonucleotides, toxins, cells, viruses, liposomes, microparticles, and micelles. Classes of bioactive agents suitable for use in the methods and compositions described herein include, but are not limited to, drugs, prodrugs, radionuclides, imaging agents, polymers, antibiotics, fungicides, antivirals, anti-inflammatory agents, antitumor agents, cardiovascular agents, anxiolytic agents, hormones, growth factors, steroids, toxins of microbial origin, and the like.
By "modulating biological activity" is meant increasing or decreasing the reactivity of a polypeptide, altering the selectivity of a polypeptide, increasing or decreasing the substrate selectivity of a polypeptide. Analysis of the altered biological activity can be performed by comparing the biological activity of the non-native polypeptide to the biological activity of the native polypeptide.
As used herein, the term "biological material" refers to materials of biological origin, including, but not limited to, materials obtained from bioreactors and/or by recombinant methods and techniques.
As used herein, the term "biophysical probe" refers to a probe that can detect or monitor a structural change in a molecule. These molecules include, but are not limited to, proteins and "biophysical probes" may be used to detect or monitor interactions of proteins with other macromolecules. Examples of biophysical probes include, but are not limited to, spin labels, fluorophores, and photoactivatable groups.
As used herein, the term "biosynthesis" refers to any method that utilizes a translation system (cellular or non-cellular) that includes the use of at least one of the following components: a polynucleotide, a codon, a tRNA, and a ribosome. For example, the unnatural amino acid can be "biosynthetically incorporated" into a unnatural amino acid polypeptide using part VIII "in vivo production of a polypeptide comprising the unnatural amino acid," as well as the methods and techniques described in non-limiting example 14. In addition, methods for selecting suitable unnatural amino acids that can be "biosynthetically incorporated" into unnatural amino acid polypeptides are described in non-limiting examples 15-16.
As used herein, the term "biotin analog" (or also referred to as "biotin mimetic") is any molecule that binds with high affinity to avidin and/or streptavidin in addition to biotin.
The term "carbonyl" as used herein means a moiety containing a moiety selected from the group consisting of-C (O) -, -S (O) 2 -and-C (S) -groups including, but not limited to, groups containing at least one ketone group and/or at least one aldehyde group and/or at least one ester group and/or at least one carboxylic acid group and/or at least one thioester group. These carbonyl groups include ketones, aldehydes, carboxylic acids, esters, and thioesters. In addition, these groups may be part of a linear, branched or cyclic molecule.
The term "carboxy-terminal modifying group" refers to any molecule that can be attached to a terminal carboxy group. For example, these terminal carboxyl groups may be at the ends of polymeric molecules, wherein these polymeric molecules include, but are not limited to, polypeptides, polynucleotides, and polysaccharides. The terminal modifying groups include, but are not limited to, various water-soluble polymers, peptides or proteins. By way of example only, the terminal modifying group comprises polyethylene glycol or serum albumin. The terminal modifying group can be used to alter the therapeutic characteristics of the polymeric molecule, including, but not limited to, increasing the serum half-life of the peptide.
As used herein, the term "chemically cleavable group" (also referred to as "chemically labile") refers to a group that decomposes or cleaves upon exposure to an acid, base, oxidizing agent, reducing agent, chemical initiator, or free radical initiator.
As used herein, the term "chemiluminescent group" refers to a group that emits light due to a chemical reaction without additional heating. By way of example only, luminol (5-amino-2, 3-dihydro-1, 4-phthalazinedione) is reacted with an oxidizing agent (e.g., hydrogen peroxide (H) 2 O 2 ) Reaction to give the excited state product (3-aminophthalate, 3-APA).
As used herein, the term "chromophore" refers to a molecule that absorbs light at the visible, UV, or IR wavelengths.
As used herein, the term "cofactor" refers to an atom or molecule necessary for the action of a macromolecule. Cofactors include, but are not limited to, inorganic ions, coenzymes, proteins, or some other factor necessary for the activity of the enzyme. Examples include heme in hemoglobin, magnesium in chlorophyll, and metal ions of proteins.
As used herein, "co-folding" refers to a refolding process, reaction, or method that uses at least two molecules that interact with each other and that causes an unfolded or improperly folded molecule to be converted into a properly folded molecule. Merely by way of example, a "co-fold" uses at least two polypeptides that interact with each other and cause an unfolded or improperly folded polypeptide to be converted into a native, properly folded polypeptide. These polypeptides may contain natural amino acids and/or at least one unnatural amino acid.
As used herein, a "comparison window" refers to a segment of any one of the contiguous positions of a sequence and a reference sequence that is used to compare the same number of contiguous positions after the two sequences are optimally aligned. These contiguous locations include, but are not limited to, a group of about 20 to about 600 consecutive units, including about 50 to about 200 consecutive units, and about 100 to about 150 consecutive units. By way of example only, these sequences include polypeptides and polypeptides containing unnatural amino acids, where the contiguous units include, but are not limited to, natural amino acids and unnatural amino acids. In addition, by way of example only, these sequences comprise polynucleotides in which the nucleotides are the corresponding contiguous units. Sequence alignment methods for comparison are well known in the art. Optimal sequence alignments for comparison can be made by methods including, but not limited to, the local homology algorithm of Smith and Waterman (1970) adv. Appl. Math.2:482c, the homology alignment algorithm of Needleman and Wunsch (1970) J.mol. Biol.48:443, the similarity search method of Pearson and Lipman (1988) Proc. Nat' l. Acad. Sci. USA 85:2444, computerized implementation of these algorithms (GAP, BESTFIT, FASTA and TFASTA in Wisconsin genetics software package (Wisconsin Genetics Software Package) (Genetics Computer Group,575Science Dr., madison, wis.), or manual alignment (see, e.g., ausubel et al, current Protocols in Molecular Biology (1995) supplement).
For example, algorithms that can be used to determine percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al (1997) Nuc. Acids Res.25:3389-3402, and Altschul et al (1990) J.mol. Biol.215:403-410, respectively. The software for performing BLAST analysis is publicly available through the national center for biotechnology information (National Center for Biotechnology Information). The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as default a word length (W) of 11, an expected value (E) of 10, m=5, n= -4, and a comparison of the two strands. For amino acid sequences, the BLASTP program uses a word length of 3, an expected value (E) of 10 and a BLOSUM62 scoring matrix (see Henikoff and Henikoff (1992) proc. Natl. Acad. Sci. Usa 89:10915) for alignment (B) of 50, an expected value (E) of 10, m=5, n= -4 and a comparison of the two chains as default values. The BLAST algorithm is typically performed with the "low complexity" filter off.
The BLAST algorithm also performs statistical analysis of the similarity between two sequences (see, e.g., karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5787). One measure of similarity provided by the BLAST algorithm is the minimum sum probability (P (N)), which provides an indication of the probability that a match between two nucleotide or amino acid sequences will occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, or less than about 0.01, or less than about 0.001.
The term "conservatively modified variants" applies to both natural and unnatural amino acid sequences, as well as to both natural and unnatural nucleic acid sequences, and combinations thereof. With respect to a particular nucleic acid sequence, "conservatively modified variants" refers to those natural and unnatural amino acid sequences that encode identical or essentially identical, or where the natural and unnatural nucleic acids do not encode natural and unnatural amino acid sequences (for essentially identical sequences). For example, due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For example, codons GCA, GCC, GCG and GCU both encode the amino acid alanine. Thus, at each position where alanine is specified by a codon, the codon can be changed to any corresponding codon without changing the encoded polypeptide. These nucleic acid variations are "silent variations," which are one of the conservatively modified variations. Thus, for example, each native or non-native nucleic acid sequence herein encoding a native or non-native polypeptide also describes each possible silent variation of the native or non-native nucleic acid. It will be appreciated by those skilled in the art that each codon in a natural or non-natural nucleic acid (except AUG, which is typically only a codon for methionine, and TGG, which is typically only a codon for tryptophan) can be modified to produce a functionally identical molecule. Thus, each silent variation of a natural or unnatural nucleic acid which encodes a natural or unnatural polypeptide is implied in each such sequence.
With respect to amino acid sequences, individual substitutions, deletions or additions to nucleic acid, peptide, polypeptide or protein sequences that alter, add or delete a single natural and unnatural amino acid or a small percentage of natural and unnatural amino acids in the coding sequence are "conservatively modified variants" where the alteration results in the deletion of an amino acid, the addition of an amino acid, or the substitution of a natural and unnatural amino acid with a chemically similar amino acid. Conservative substitutions providing functionally similar natural amino acids are well known in the art. These conservatively modified variants differ from the polymorphic variants, interspecies homologs, and alleles of the methods and compositions described herein and do not exclude such. The following eight groups each contain amino acids that are conservative substitutions for one another:
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) Phenylalanine (F), tyrosine (Y), tryptophan (W);
7) Serine (S), threonine (T); and
8) Cysteine (C), methionine (M)
See, e.g., cright on, proteins: structures and Molecular Properties (WH Freeman & Co.; version 2 (month 12 1993)).
Unless otherwise indicated, the terms "cycloalkyl" and "heterocycloalkyl" (by itself or in combination with other terms) refer to the cyclic forms of "alkyl" and "heteroalkyl," respectively. Thus, cycloalkyl or heterocycloalkyl groups include saturated, partially unsaturated, and fully unsaturated ring linkages. In addition, for heterocycloalkyl, the heteroatom may occupy the position where the heterocycle is attached to the remainder of the molecule. Heteroatoms may include, but are not limited to, oxygen, nitrogen, or sulfur. Examples of cycloalkyl groups include, but are not limited to, cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Examples of heterocycloalkyl groups include, but are not limited to, 1- (1, 2,5, 6-tetrahydropyridinyl), 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothiophen-2-yl, tetrahydrothiophen-3-yl, 1-piperazinyl, 2-piperazinyl, and the like. In addition, the term encompasses polycyclic structures including, but not limited to, double and triple cyclic ring structures. Similarly, the term "heterocycloalkylene" (by itself or as part of another molecule) means a divalent group derived from a heterocycloalkyl group, and the term "cycloalkylene" (by itself or as part of another molecule) means a divalent group derived from a cycloalkyl group.
As used herein, the term "cyclodextrin" refers to a cyclic carbohydrate consisting of at least 6 to 8 glucose molecules in a ring composition. The outside of the ring contains a water-soluble group; at the center of the ring is a relatively nonpolar cavity capable of accommodating small molecules.
As used herein, the term "cytotoxic" refers to a compound that damages cells.
Such as the bookAs used herein, "denaturant" refers to any compound or substance that causes reversible stretching of a polymer. By way of example only, a "denaturant" may cause reversible stretching of a protein. The strength of the denaturant will be determined by the nature and concentration of the particular denaturant. For example, denaturants include, but are not limited to, chaotropes, detergents, water miscible organic solvents, phospholipids, or combinations thereof. Non-limiting examples of chaotropic agents include, but are not limited to, urea, guanidine, and sodium thiocyanate. Non-limiting examples of detergents may include (but are not limited to): strong detergents such as sodium dodecyl sulfate or polyoxyethylene ether (e.g., tween or Triton detergents), sodium dodecyl sarcosinate (Sarkosyl); mild nonionic detergents (e.g., digitonin); mild cationic detergents, e.g. N- >2,3- (dioleyloxy) -propyl-N, N-trimethylammonium; mild ionic detergents (e.g., sodium cholate or sodium deoxycholate); or zwitterionic detergents, which include, but are not limited to, sulfobetaines (zwifergent), 3- (3-cholesteryl-propyl) dimethylamino-1-propane sulfate (CHAPS), and 3- (3-cholesteryl-propyl) dimethylamino-2-hydroxy-1-propane sulfonate (CHAPSO). Non-limiting examples of water miscible organic solvents include, but are not limited to, acetonitrile, lower alkanols (especially C 2 -C 4 Alkanols, such as ethanol or isopropanol), or lower alkanediols (C 2 -C 4 Alkylene glycols, such as ethylene glycol) may be used as denaturants. Non-limiting examples of phospholipids include, but are not limited to, naturally occurring phospholipids such as phosphatidylethanolamine, lecithin, phosphatidylserine, and phosphatidylinositol; or synthetic phospholipid derivatives or variants, such as dihexanoyl lecithin or diheptanoyl lecithin.
As used herein, the term "detectable label" refers to a label that can be observed using analytical techniques including, but not limited to, fluorescence, chemiluminescence, electron spin resonance, ultraviolet/visible absorption spectroscopy, mass spectrometry, nuclear magnetic resonance, and electrochemical methods.
The term "dicarbonyl" as used herein means a compound containing at least two moieties selected from the group consisting of-C (O) -, S (O) -, -S (O) 2 -and-C (S) -part of the group consisting ofIncluding, but not limited to, 1, 2-dicarbonyl, 1, 3-dicarbonyl, and 1, 4-dicarbonyl, and groups containing at least one ketone group and/or at least one aldehyde group and/or at least one ester group and/or at least one carboxylic acid group and/or at least one thioester group. These dicarbonyl groups include diketones, ketoaldehydes, ketoacids, ketoesters and ketothioesters. In addition, these groups may be part of a linear, branched or cyclic molecule. The two moieties in the dicarbonyl may be the same or different, and may include substituents that would produce, for example only, an ester, ketone, aldehyde, thioester, or amide at either of the two moieties.
As used herein, the term "drug" refers to any substance used in the prevention, diagnosis, alleviation, treatment or cure of a disease or condition.
As used herein, the term "dye" refers to a soluble dye material that contains a chromophore.
As used herein, the term "effective amount" refers to a sufficient amount of an agent or compound administered that will alleviate to some extent the symptoms of one or more diseases or conditions being treated. The result may be a reduction and/or alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. For example, the agent or compound administered includes, but is not limited to, a natural amino acid polypeptide, a non-natural amino acid polypeptide, a modified natural amino acid polypeptide, or a modified non-amino acid polypeptide. Compositions containing these natural amino acid polypeptides, unnatural amino acid polypeptides, modified natural amino acid polypeptides, or modified unnatural amino acid polypeptides can be administered for prophylactic, enhancing, and/or therapeutic treatment. The appropriate "effective" amount in any individual case can be determined using techniques such as dose escalation studies.
As used herein, the term "electron dense group" refers to a group that scatters electrons when irradiated with an electron beam. These groups include, but are not limited to, ammonium molybdate, bismuth subnitrate, cadmium iodide, 99%, carbohydrazide, ferric chloride hexahydrate, hexamethylenetetramine, 98.5%, anhydrous indium trichloride, lanthanum nitrate, lead acetate trihydrate, lead citrate trihydrate, lead nitrate, periodic acid, phosphomolybdic acid, phosphotungstic acid, potassium ferricyanide, potassium ferrocyanide, ruthenium red, silver nitrate, silver protein (Ag assay: 8.0-8.5%) "strong", tetraphenylporphin silver (S-TPPS), sodium chloroaurate, sodium tungstate, thallium nitrate, thiosemicarbazide (TSC), uranyl acetate, uranyl nitrate, and vanadyl sulfate.
As used herein, the term "energy transfer agent" refers to a molecule that can contribute or accept energy from another molecule. By way of example only, fluorescence Resonance Energy Transfer (FRET) is a dipole-dipole coupling process by which excited state energy of a fluorescent donor molecule is non-radiatively transferred to an unexcited acceptor molecule, which then fluoresces at a longer wavelength to emit donated energy.
The term "enhancing" means increasing or extending the effectiveness or duration of a desired effect. For example, an effect of a "enhancing" therapeutic agent refers to the ability to increase or extend the efficacy or duration, effect, of the therapeutic agent during treatment of a disease, disorder, or condition. As used herein, "an effective enhancing amount" refers to an amount sufficient to enhance the effect of a therapeutic agent during treatment of a disease, disorder, or condition. When used in a patient, the amount effective for this use will depend on the severity and course of the disease, disorder or condition, previous treatments, the health status of the patient and the response to the drug, and the discretion of the attendant physician.
As used herein, the term "eukaryotic organism" refers to an organism belonging to the phylogenetic eukaryotic domain (domain Eucarya), which includes, but is not limited to, animals (including, but not limited to, mammals, insects, reptiles, birds, etc.), ciliates, plants (including, but not limited to, monocots, dicots, and algae), fungi, yeasts, flagellates, microsporidians, and protozoa.
As used herein, the term "fatty acid" refers to a carboxylic acid having a hydrocarbon side chain of about C6 or longer.
As used herein, the term "fluorophore" refers to a molecule that upon excitation emits a photon and thereby fluoresces.
As used herein, the terms "functional group", "active moiety", "activating group", "leaving group", "reactive site", "chemically reactive group" and "chemically reactive moiety" refer to a portion or unit of a molecule that undergoes a chemical reaction. These terms are somewhat synonymous in the chemical arts and are used herein to refer to the portion of a molecule that performs some function or activity and is reactive with other molecules.
The term "halogen" includes fluorine, chlorine, iodine and bromine.
As used herein, the term "haloacyl" refers to an acyl group containing a halogen moiety, which includes, but is not limited to, -C (O) CH 3 、-C(O)CF 3 、-C(O)CH 2 OCH 3 And the like.
As used herein, the term "haloalkyl" refers to an alkyl group containing a halogen moiety, including (but not limited to) -CF 3 and-CH 2 CF 3 And the like.
As used herein, the term "heteroalkyl" refers to a straight or branched chain or cyclic hydrocarbon group, or a combination thereof, consisting of an alkyl group and at least one heteroatom selected from the group consisting of O, N, si and S, and wherein the nitrogen and sulfur atoms may optionally be oxidized and the nitrogen heteroatom may optionally be quaternized. Heteroatoms O, N and S and Si may be located at any internal position of the heteroalkyl group or at a position where the alkyl group is attached to the remainder of the molecule. Examples include, but are not limited to, -CH 2 -CH 2 -O-CH 3 、-CH 2 -CH 2 -NH-CH 3 、-CH 2 -CH 2 -N(CH 3 )-CH 3 、-CH 2 -S-CH 2 -CH 3 、-CH 2 -CH 2 ,-S(O)-CH 3 、-CH 2 -CH 2 -S(O) 2 -CH 3 、-CH＝CH-O-CH 3 、-Si(CH 3 ) 3 、-CH 2 -CH＝N-OCH 3 -ch=ch-N (CH 3 )-CH 3 . In addition, up to two heteroatoms may be contiguous, such as-CH 2 -NH-OCH 3 and-CH 2 -O-Si(CH 3 ) 3 。
As used hereinAs used herein, the term "heteroalkylene" refers to a divalent group derived from a heteroalkyl group, e.g., from-CH 2 -CH 2 -S-CH 2 CH 2 -and-CH 2 -S-CH 2 -CH 2 -NH-CH 2 Illustrated (but not limiting). For heteroalkylene groups, the same or different heteroatoms may also occupy one or both ends of the chain (including, but not limited to, alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, aminooxyalkylene, and the like). Further, with respect to the alkylene and heteroalkylene linking groups, the direction of the linking group is not implied by the direction in which the formula of the linking group is written. For example, formula-C (O) 2 R' -represents-C (O) 2 R '-and-R' C (O) 2 -both.
As used herein, the term "heteroaryl" or "heteroaromatic" refers to an aryl group containing at least one heteroatom selected from N, O and S; wherein the nitrogen and sulfur atoms may optionally be oxidized and the nitrogen atoms may optionally be quaternized. Heteroaryl groups may be substituted or unsubstituted. Heteroaryl groups may be attached to the remainder of the molecule through heteroatoms. Non-limiting examples of heteroaryl groups include 1-pyrrolyl, 2-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidinyl, 4-pyrimidinyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl, 1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl, 3-quinolinyl and 6-quinolinyl.
The term "higher alkyl" as used herein refers to an alkyl group that is a hydrocarbyl group.
As used herein, the term "identical" refers to the same two or more sequences or subsequences. In addition, as used herein, the term "substantially identical" refers to a sequence of two or more having a percentage of the same consecutive units when compared and aligned for maximum correspondence through a comparison window or designated area as measured using a comparison algorithm or by manual alignment and visual inspection. For example only, two or more sequences may be "substantially identical" if the consecutive units are about 60% identical, about 65% identical, about 70% identical, about 75% identical, about 80% identical, about 85% identical, about 90% identical, or about 95% identical over a specified region. These percentages describe the "percent identity" of two or more sequences. The identity of the sequences may be present over a region of at least about 75-100 consecutive units in length, over a region of about 50 consecutive units in length, or (if not specified) over the entire sequence. This definition also relates to the complement of the test sequence. By way of example only, two or more polypeptide sequences are identical when the amino acid residues are identical, while two or more polypeptide sequences are "substantially identical" if the amino acid residues are about 60% identical, about 65% identical, about 70% identical, about 75% identical, about 80% identical, about 85% identical, about 90% identical, or about 95% identical over a designated region. The identity may be over a region of at least about 75-100 amino acids in length, over a region of about 50 amino acids in length, or (if not specified) over the entire sequence of the polypeptide sequence. In addition, by way of example only, two or more polynucleotide sequences are identical when the nucleic acid residues are identical, and two or more polynucleotide sequences are "substantially identical" if the nucleic acid residues are about 60% identical, about 65% identical, about 70% identical, about 75% identical, about 80% identical, about 85% identical, about 90% identical, or about 95% identical over a designated region. The identity may be present over a region of at least about 75-100 nucleic acids in length, over a region of about 50 nucleic acids in length, or (if not specified) over the entire sequence of the polynucleotide sequence.
Regarding sequence comparison, typically one sequence serves as a reference sequence against which a test sequence is compared. When using the sequence comparison algorithm, the test sequence and the reference sequence are entered into the computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters may be used, or alternative parameters may be specified. The sequence comparison algorithm then calculates the percent sequence identity of the test sequence relative to the reference sequence based on the program parameters.
As used herein, the term "immunogenic" refers to an antibody that is responsive to administration of a therapeutic agent. Immunogenicity against therapeutic unnatural amino acid polypeptides can be obtained using quantitative and qualitative assays for detecting antibodies to unnatural amino acid polypeptides in biological fluids. Such assays include, but are not limited to, radioimmunoassays (RIA), enzyme-linked immunosorbent assays (ELISA), luminescent Immunoassays (LIA), and Fluorescent Immunoassays (FIA). Analysis of immunogenicity against a therapeutic unnatural amino acid polypeptide includes comparing the antibody response after administration of the therapeutic unnatural amino acid polypeptide to the antibody response after administration of the therapeutic natural amino acid polypeptide.
As used herein, the term "intercalating agent" (also referred to as an "intercalating group") refers to a chemical that can intercalate into the intramolecular space of a molecule or the intermolecular space between molecules. By way of example only, an intercalator or intercalating group may be a molecule that intercalates into the stacked bases of a DNA duplex.
As used herein, the term "separating" refers to separating and removing a component of interest from a component of no interest. The isolated material may be in a dry or semi-dry state, or in solution, including but not limited to an aqueous solution. The isolated component may be in a homogeneous state or the isolated component may be part of a pharmaceutical composition comprising additional pharmaceutically acceptable carriers and/or excipients. Purity and homogeneity can be determined using analytical chemistry techniques including, but not limited to, polyacrylamide gel electrophoresis or high performance liquid chromatography. In addition, when a component of interest is isolated and is the predominant species present in a formulation, the component is described herein as being substantially purified. As used herein, the term "purified" refers to a component of interest that is at least 85% pure, at least 90% pure, at least 95% pure, at least 99% or more pure. By way of example only, a nucleic acid or protein is "isolated" when it does not contain at least some cellular components associated therewith in its natural state, or the nucleic acid or protein has been concentrated to a level higher than that at which it is produced in vivo or in vitro. Also, for example, a gene is isolated when separated from an open reading frame that is outside of the gene and encodes a protein other than the gene of interest.
As used herein, the term "label" refers to a substance that is incorporated into a compound and is readily detectable, whereby its physical distribution can be detected and/or monitored.
As used herein, the term "linkage" refers to a bond or chemical moiety formed by a chemical reaction between a functional group of a linker and another molecule. These linkages may include, but are not limited to, covalent and non-covalent linkages, and these chemical moieties may include, but are not limited to, ester, carbonate, imine, phosphate, hydrazone, acetal, orthoester, peptide linkages, and oligonucleotide linkages. Hydrolytically stable linkage means that the linkage is substantially stable in water and does not react with water for long periods of time (perhaps even indefinitely) at a suitable pH, including but not limited to under physiological conditions. Hydrolytically unstable or degradable linkages means that the linkages can degrade in water or aqueous solutions (including, for example, blood). Enzymatically labile or degradable linkages means that the linkages can be degraded by one or more enzymes. By way of example only, PEG and related polymers may include degradable linkages in the polymer backbone or in linker groups between the polymer backbone and one or more terminal functional groups of the polymer molecule. These degradable linkages include, but are not limited to, ester linkages formed from the reaction of PEG carboxylic acid or activated PEG carboxylic acid with alcohol groups on the bioactive agent, where these ester groups are typically hydrolyzed under physiological conditions to release the bioactive agent. Other hydrolytically degradable linkages include, but are not limited to, carbonate linkages; imine bonds formed by the reaction of an amine with an aldehyde; a phosphate bond formed by reacting an alcohol with a phosphate group; hydrazone bonds as the reaction product of hydrazides with aldehydes; an acetal bond as a reaction product of an aldehyde and an alcohol; orthoester linkages as the reaction product of formate and alcohol; peptide bonds formed by amino groups (including, but not limited to, on the end of a polymer such as PEG) with carboxyl groups of a peptide; and oligonucleotide linkages formed from phosphoramidite groups, including but not limited to at the ends of the polymer, with the 5' hydroxyl groups of the oligonucleotide.
As used herein, the term "medium" refers to any medium used to grow and collect cells and/or products expressed and/or secreted by such cells. These "media" include, but are not limited to, solutions, solids, semisolids, or rigid vectors that can support or contain any host cells including, for example, bacterial host cells, yeast host cells, insect host cells, plant host cells, eukaryotic host cells, mammalian host cells, CHO cells, prokaryotic host cells, e.coli (e.coli) or Pseudomonas host cells, and cell contents. These "media" include, but are not limited to, media in which the host cell has been grown in which the polypeptide has been secreted, including media before or after the proliferation step. These "media" also include, but are not limited to, buffers or reagents containing host cell lysates, such as polypeptides produced intracellularly and host cells lyse or divide to release the polypeptides.
As used herein, the term "metabolite" refers to a derivative of a compound (e.g., a natural amino acid polypeptide, an unnatural amino acid polypeptide, a modified natural amino acid polypeptide, or a modified unnatural amino acid polypeptide) that is formed upon metabolism of the compound (e.g., a natural amino acid polypeptide, an unnatural amino acid polypeptide, a modified natural amino acid polypeptide, or a modified unnatural amino acid polypeptide). The term "pharmaceutically active metabolite" or "active metabolite" refers to a biologically active derivative of a compound (e.g., a natural amino acid polypeptide, an unnatural amino acid polypeptide, a modified natural amino acid polypeptide, or a modified unnatural amino acid polypeptide) that is formed upon metabolism of the compound (e.g., a natural amino acid polypeptide, an unnatural amino acid polypeptide, a modified natural amino acid polypeptide, or a modified unnatural amino acid polypeptide).
As used herein, the term "metabolism" refers to the sum of the processes by which a particular substance is altered by an organism. These processes include, but are not limited to, hydrolysis reactions and reactions catalyzed by enzymes. Additional information on metabolism can be obtained from The Pharmacological Basis of Therapeutics, 9 th edition, mcGraw-Hill (1996). By way of example only, a natural amino acid polypeptide, a non-natural amino acid polypeptide, a modified natural amino acid polypeptide, or a metabolite of a modified non-natural amino acid polypeptide may be identified by administering the natural amino acid polypeptide, the non-natural amino acid polypeptide, the modified natural amino acid polypeptide, or the modified non-natural amino acid polypeptide to a host and analyzing a tissue sample from the host, or by culturing the natural amino acid polypeptide, the non-natural amino acid polypeptide, the modified natural amino acid polypeptide, or the modified non-natural amino acid polypeptide with hepatocytes in vitro and analyzing the resulting compound.
As used herein, the term "metal chelator" refers to a molecule that forms a metal complex with a metal ion. For example, these molecules may form two or more coordination bonds with the central metal ion and may form a ring structure.
As used herein, the term "metal-containing moiety" refers to a group containing a metal ion, atom or particle. These moieties include, but are not limited to, cisplatin (cispratin), chelated metal ions (such as nickel, iron, and platinum), and metal nanoparticles (such as nickel, iron, and platinum).
As used herein, the term "moiety bound to a heavy atom" refers to a group bound to an ion that is typically a heavier atom than carbon. Such ions or atoms include, but are not limited to, silicon, tungsten, gold, lead, and uranium.
As used herein, the term "modified" refers to the presence of alterations to a natural amino acid, a non-natural amino acid, a natural amino acid polypeptide, or a non-natural amino acid polypeptide. These alterations or modifications may be obtained by post-synthesis modification of natural amino acids, unnatural amino acids, natural amino acid polypeptides or unnatural amino acid polypeptides, or by co-translational or post-translational modification of natural amino acids, unnatural amino acids, natural amino acid polypeptides or unnatural amino acid polypeptides. The form "modified or unmodified" means that the natural amino acid, unnatural amino acid, natural amino acid polypeptide or unnatural amino acid polypeptide in question is optionally modified, i.e. the natural amino acid, unnatural amino acid, natural amino acid polypeptide or unnatural amino acid polypeptide in question can be modified or unmodified.
As used herein, the term "modulated serum half-life" refers to a positive or negative change in the circulatory half-life of a modified biologically active molecule relative to its non-unmodified form. For example, modified bioactive molecules include, but are not limited to, natural amino acids, unnatural amino acids, natural amino acid polypeptides, or unnatural amino acid polypeptides. For example, serum half-life is measured by taking blood samples at various time points after administration of a biologically active molecule or modified biologically active molecule and determining the concentration of that molecule in each sample. The correlation of serum concentration with time allows calculation of serum half-life. For example, the modulated serum half-life may be an increase in serum half-life, which may enable improved dosing regimens or avoid toxic effects. This serum increase may be at least about two times, at least about three times, at least about five times, or at least about ten times. Non-limiting examples of methods for assessing serum half-life increase are given in examples 88-92. This method can be used to assess the serum half-life of any polypeptide.
As used herein, the term "modulated therapeutic half-life" refers to a positive or negative change in the half-life of a therapeutically effective amount of a modified bioactive molecule relative to its unmodified form. For example, modified bioactive molecules include, but are not limited to, natural amino acids, unnatural amino acids, natural amino acid polypeptides, or unnatural amino acid polypeptides. For example, the therapeutic half-life is measured by measuring the pharmacokinetic and/or pharmacodynamic properties of the molecule at various time points after administration. The increased therapeutic half-life may enable a particular beneficial dosing regimen, a particular beneficial total dose, or avoid undue effects. For example, increased therapeutic half-life may result from increased potency, increased or decreased binding of a modified molecule to its target, increased or decreased another parameter or mechanism of action of an unmodified molecule, or increased or decreased degradation of a molecule by an enzyme (such as, for example only, a protease). Non-limiting examples of methods of assessing increased therapeutic half-life are given in examples 88-92. This method can be used to assess the therapeutic half-life of any polypeptide.
As used herein, the term "nanoparticle" refers to particles having a particle size between about 500nm and about 1 nm.
As used herein, the term "near stoichiometric" refers to a molar ratio of compounds that participate in a chemical reaction of about 0.75 to about 1.5.
As used herein, the term "non-eukaryotic" refers to a non-eukaryotic organism. For example, the non-eukaryotic organism may belong to Eubacteria (Eubacteria), including but not limited to Escherichia coli, thermophilic thermus (Thermus thermophilus) or stearothermophilus (Bacillus stearothermophilus), pseudomonas fluorescens (Pseudomonas fluorescens), pseudomonas aeruginosa (Pseudomonas aeruginosa), pseudomonas putida (Pseudomonas putida), phylogenetic domains, or Archaea (archea), including but not limited to methanococcus jannaschii (Methanococcus jannaschii), methanobacterium thermoautotrophicum (Methanobacterium thermoautotrophicum), archaebacterium scintillans (Archaeoglobus fulgidus), rhodococcus furiosus (Pyrococcus furiosus), rhodococcus garvieae (Pyrococcus horikoshii), aerothermus (Aeuropyrum pernix), or halophilus (halobacteria), such as haloxyfocalia volvuli (Haloferax volcanii) and halophila species NRC-1, or phylogenetic domains.
"unnatural amino acid" refers to an amino acid that is not one of the 20 common amino acids or pyrolysine or selenocysteine. Other terms that may be used synonymously with the term "unnatural amino acid" are "unnatural encoded amino acid", "unnatural amino acid", "non-naturally occurring amino acid", as well as various hyphenated and non-hyphenated forms thereof. The term "unnatural amino acid" includes, but is not limited to, an amino acid that naturally occurs by modification of a naturally encoded amino acid (including, but not limited to, 20 common amino acids or pyrolysines and selenocysteine) but is not incorporated into a growing polypeptide chain by the translation complex itself. Examples of naturally occurring amino acids that are not naturally encoded include, but are not limited to, N-acetylglucosamine-L-serine, N-acetylglucosamine-L-threonine, and O-phosphotyrosine. In addition, the term "unnatural amino acid" includes, but is not limited to, amino acids that do not occur naturally and that are synthetically obtained or that can be obtained by modification of unnatural amino acids.
As used herein, the term "nucleic acid" refers to deoxynucleotides, deoxynucleosides, nucleosides or nucleotides, and polymers thereof in single-or double-stranded form. By way of example only, these nucleic acids and nucleic acid polymers include, but are not limited to (i) analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides; (ii) Oligonucleotide analogs including, but not limited to, PNA (peptide-based nucleic acids), analogs of DNA used in antisense technology (phosphorothioates, phosphoramidates, and analogs thereof); (iii) Conservatively modified variants thereof (including but not limited to degenerate codon substitutions), complementary sequences, and the sequence explicitly indicated. For example, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed bases and/or deoxyinosine residues (Batzer et al, nucleic Acid Res.19:5081 (1991); ohtsuka et al, J.biol. Chem.260:2605-2608 (1985); and Rossolini et al, mol. Cell. Probes 8:91-98 (1994)).
As used herein, the term "oxidizing agent" refers to a compound or substance that is capable of removing electrons from an oxidized compound. For example, oxidizing agents include, but are not limited to, oxidized glutathione, cystine, cystamine, oxidized dithiothreitol, oxidized erythritol (oxidized erythreitol), and oxygen. A variety of oxidizing agents are suitable for use in the methods and compositions described herein.
As used herein, the term "pharmaceutically acceptable" refers to a substance comprising, but not limited to, a salt, carrier, or diluent that does not abrogate the biological activity or properties of the compound, and that is relatively non-toxic, i.e., the substance can be administered to a subject without causing undue biological effects or interacting in a deleterious manner with any of the components of the composition in which it is comprised.
As used herein, the term "photoaffinity label" refers to a label having a group that forms a bond with a molecule for which the label has affinity after exposure to light. By way of example only, such linkage may be covalent or non-covalent linkage.
As used herein, the term "photocell moiety" refers to a group that binds, covalently or non-covalently, to other ions or molecules after irradiation at a particular wavelength.
As used herein, the term "photocleavable group" refers to a group that breaks upon exposure to light.
As used herein, the term "photocrosslinker" refers to a compound that includes two or more functional groups that can react with two or more monomers or polymeric molecules and form covalent or non-covalent linkages upon exposure to light.
As used herein, the term "photoisomerizable moiety" refers to a group in which one isomeric form changes to another isomeric form upon irradiation with light.
As used herein, the term "polyalkylene glycol" refers to a linear or branched polymeric polyether polyol. These polyalkylene glycols include, but are not limited to, polyethylene glycol, polypropylene glycol, polytetramethylene glycol, and derivatives thereof. Other illustrative embodiments are listed, for example, in commercial supplier catalogs such as the catalog "polyethylene glycol and derivatives for biomedical applications" ("Polyethylene Glycol and Derivatives for Biomedical Applications") (2001). By way of example only, these polymeric polyether polyols have an average molecular weight of between about 0.1kDa and about 100 kDa. For example, these polymeric polyether polyols include, but are not limited to, between about 100Da and about 100,000Da or higher. The molecular weight of the polymer may be between about 100Da and about 100,000Da, including, but not limited to, 100,000Da, 95,000Da, 90,000Da, 85,000Da, 80,000Da, 75,000Da, 70,000Da, 65,000Da, 60,000Da, 55,000Da, 50,000Da, 45,000Da, 40,000Da, 35,000Da, 30,000Da, 25,000Da, 20,000Da, 15,000Da, 10,000Da, 9,000Da, 8,000Da, 7,000Da, 6,000Da, 5,000Da, 4,000Da, 3,000Da, 2,000Da, 1,000Da, 900Da, 800Da, 700Da, 600Da, 500Da, 400Da, 300Da, 200Da, and 100Da. In some embodiments, the molecular weight of the polymer is between about 100Da and 50,000 Da. In some embodiments, the molecular weight of the polymer is between about 100Da and 40,000 Da. In some embodiments, the molecular weight of the polymer is between about 1,000da and 40,000 da. In some embodiments, the molecular weight of the polymer is between about 5,000da and 40,000 da. In some embodiments, the molecular weight of the polymer is between about 10,000da and 40,000 da. In some embodiments, the polyethylene glycol molecule is a branched polymer. The molecular weight of the branched PEG may be between about 1,000da and about 100,000da, including, but not limited to, 100,000da, 95,000da, 90,000da, 85,000da, 80,000da, 75,000da, 70,000da, 65,000da, 60,000da, 55,000da, 50,000da, 45,000da, 40,000da, 35,000da, 30,000da, 25,000da, 20,000da, 15,000da, 10,000da, 9,000da, 8,000da, 7,000da, 6,000da, 5,000da, 4,000da, 3,000da, 2,000da and 1,000da. In some embodiments, the branched PEG has a molecular weight between about 1,000da and 50,000 da. In some embodiments, the branched PEG has a molecular weight between about 1,000da and 40,000 da. In some embodiments, the branched PEG has a molecular weight between about 5,000da and 40,000 da. In some embodiments, the branched PEG has a molecular weight between about 5,000da and 20,000 da.
As used herein, the term "polymer" refers to a molecule consisting of repeating subunits. Such molecules include, but are not limited to, polypeptides, polynucleotides or polysaccharides or polyalkylene glycols.
The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. That is, the description for polypeptides applies equally to the description for peptides and the description for proteins, and vice versa. These terms apply to naturally occurring amino acid polymers and amino acid polymers in which one or more amino acid residues are unnatural amino acids. In addition, these "polypeptides", "peptides" and "proteins" comprise amino acid chains of any length, including full length proteins, in which the amino acid residues are linked by covalent peptide bonds.
The term "post-translational modification" refers to any modification of a natural or unnatural amino acid that occurs after the amino acid has been translationally incorporated into a polypeptide chain. Such modifications include, but are not limited to, co-translational in vivo modifications, co-translational in vitro modifications (such as in a cell-free translation system), post-translational in vivo modifications, and post-translational in vitro modifications.
As used herein, the term "prodrug" or "pharmaceutically acceptable prodrug" refers to an agent that is converted to the parent drug in vivo or in vitro, wherein it does not abrogate the biological activity or properties of the drug, and is relatively non-toxic, i.e., the agent can be administered to a subject without undue biological effects or interactions in a deleterious manner with any of the components of the composition in which it is contained. Prodrugs are typically prodrugs that, upon administration to a subject and subsequent absorption, are converted by certain processes (such as transformation by metabolic pathways) to substances that are active or more active. Some prodrugs have chemical groups present on the prodrug that make the prodrug less active and/or impart solubility or some other property to the drug. Once the chemical groups are cleaved and/or modified, the active agent is produced from the prodrug. Prodrugs are converted in vivo to the active drug by enzymatic or non-enzymatic reactions. Prodrugs can provide improved physicochemical properties such as better solubility, enhanced delivery characteristics (such as specific targeting to a particular cell, tissue, organ or ligand), and improved therapeutic value of the drug. Benefits of these prodrugs include (but are not limited to): (i) ease of administration compared to the parent drug; (ii) Prodrugs can be bioavailable by oral administration, whereas the parent is not; and (iii) the prodrug may also have improved solubility in the pharmaceutical composition compared to the parent drug. Prodrugs include pharmacologically inactive or reduced activity derivatives of the active agent. Prodrugs can be designed to modulate the amount of a drug or bioactive molecule by manipulating properties of the drug (such as physicochemical, biomedical, or pharmacokinetic properties) to a desired site of action. An example of a prodrug, without limitation, may be an unnatural amino acid polypeptide that is administered in the form of an ester ("prodrug") to facilitate transport across a cell membrane, where water solubility is detrimental to mobility, but which is subsequently metabolically hydrolyzed to a carboxylic acid (active entity) once inside the cell where water solubility is beneficial. Prodrugs can be designed as reversible drug derivatives to act as modifiers to enhance drug delivery to site-specific tissues.
As used herein, the term "prophylactically effective amount" refers to an amount of a composition containing at least one unnatural amino acid polypeptide or at least one modified unnatural amino acid polypeptide that is prophylactically applied to a patient, which will alleviate to some extent the symptoms of one or more of the diseases, conditions, or disorders treated. In these prophylactic applications, the amount will depend on the health of the patient, the weight and the like. It is fully believed that those skilled in the art can determine such prophylactically effective amounts by routine experimentation, including but not limited to dose escalation clinical trials.
As used herein, the term "protected" refers to the presence of a "protecting group" or moiety that prevents a chemically reactive functional group from reacting under certain reaction conditions. The protecting group will vary depending on the type of chemically reactive group that is protected. By way of example only, (i) if the chemically reactive group is an amine or a hydrazide, the protecting group may be selected from t-butoxycarbonyl (t-Boc) and 9-fluorenylmethoxycarbonyl (Fmoc); (ii) If the chemically reactive group is a thiol, the protecting group may be an ortho-pyridyl disulfide; and (iii) if the chemically reactive group is a carboxylic acid (such as butyric acid or propionic acid) or a hydroxyl group, the protecting group may be benzyl or alkyl, such as methyl, ethyl or t-butyl.
By way of example only, blocking/protecting groups may also be selected from:
Figure BDA0001546710350000281
in addition, protecting groups include, but are not limited to, groups that include photolabile groups such as Nvoc and MeNvoc, as well as other protecting groups known in the art. Other protecting groups are described in Greene and Wuts, protective Groups in Organic Synthesis, 3 rd edition, john Wiley & Sons, new York, NY,1999, which is incorporated herein by reference in its entirety.
As used herein, the term "radioactive moiety" refers to a group whose core spontaneously emits nuclear radiation (such as alpha particles, beta particles, or gamma particles); wherein the alpha particles are helium nuclei, the beta particles are electrons, and the gamma particles are high-energy photons.
As used herein, the term "reactive compound" refers to a compound that is reactive with another atom, molecule, or compound under appropriate conditions.
The term "recombinant host cell" (also referred to as a "host cell") refers to a cell comprising an exogenous polynucleotide, wherein the method for inserting the exogenous polynucleotide into the cell includes, but is not limited to, direct uptake, transduction, f-pairing, or other methods known in the art of producing recombinant host cells. By way of example only, the exogenous polynucleotide may be a non-integrative vector (including, but not limited to, a plasmid), or may be integrated into the host genome.
As used herein, the term "redox active agent" refers to a molecule that oxidizes or reduces another molecule whereby the redox active agent becomes reduced or oxidized. Examples of redox active agents include, but are not limited to, ferrocene, quinone, ru2+/3+ complexes, co2+/3+ complexes, and os2+/3+ complexes.
As used herein, the term "reducing agent" refers to a compound or substance that is capable of adding electrons to a reduced compound. For example, reducing agents include, but are not limited to, dithiothreitol (DTT), 2-mercaptoethanol, dithioerythritol, cysteine, cysteamine (2-aminoethanethiol), and reduced glutathione. These reducing agents may be used, for example only, to maintain sulfhydryl groups in a reduced state and reduce intramolecular or intermolecular disulfide bonds.
"refolding" as used herein describes any process, reaction, or method of converting an improperly folded or unfolded state into a natural or properly folded conformation. Merely by way of example, refolding converts a disulfide-containing polypeptide from an improperly folded or unfolded state to a natural or properly folded conformation with respect to disulfide bonds. These disulfide bond containing polypeptides may be natural amino acid polypeptides or non-natural amino acid polypeptides.
As used herein, the term "resin" refers to high molecular weight insoluble polymer beads. By way of example only, these beads may be used as carriers for solid phase peptide synthesis, or as sites for attaching molecules prior to purification.
As used herein, the term "saccharide" refers to a range of carbohydrates including, but not limited to, sugars, monosaccharides, oligosaccharides, and polysaccharides.
As used herein, the term "safety" or "safety profile" refers to side effects that may be associated with the administration of a drug relative to the number of times the drug has been administered. For example, drugs that have been administered multiple times and only mildly produce side effects or no side effects are referred to as having an excellent safety profile. A non-limiting example of a method of evaluating a security profile is given in example 92. Such methods can be used to assess the safety profile of any polypeptide.
As used herein, the phrase "selective hybridization" or "specific hybridization" refers to the binding, duplex, or hybridization of a molecule to a specific nucleotide sequence under stringent hybridization conditions when that sequence is present in a complex mixture, including but not limited to total cell or pool DNA or RNA.
As used herein, the term "spin label" refers to a molecule containing an atom or group of atoms (i.e., a stable paramagnetic group) exhibiting unpaired electron spin that is detectable by electron spin resonance spectroscopy and that is connectable to another molecule. These spin labeling molecules include, but are not limited to, nitroxyl and nitroxide, and can be single spin labeling or dual spin labeling.
As used herein, the term "stoichiometric" refers to a molar ratio of compounds that participate in a chemical reaction of about 0.9 to about 1.1.
As used herein, the term "stoichiometric-like" refers to a chemical reaction that becomes stoichiometric or near stoichiometric upon a change in reaction conditions or in the presence of an additive. These changes in reaction conditions include, but are not limited to, temperature increases or pH changes. These additives include, but are not limited to, accelerators.
The phrase "stringent hybridization conditions" refers to hybridization of sequences of DNA, RNA, PNA or other nucleic acid mimics or combinations thereof under conditions of low ion concentration and high temperature. For example, a probe hybridizes under stringent conditions to its target subsequence in a complex mixture of nucleic acids, including but not limited to total cell or pool DNA or RNA, but not to other sequences in the complex mixture. Stringent conditions are sequence-dependent and will be different in different situations. For example, longer sequences hybridize specifically at higher temperatures. Stringent hybridization conditions include (but are not limited to): (i) About 5 ℃ to about 10 ℃ lower than the thermal melting point (Tm) of the specific sequence at a determined ion concentration and pH; (ii) A salt concentration of about 0.01M to about 1.0M at about pH 7.0 to about pH 8.3, and a temperature of at least about 30 ℃ for short probes (including but not limited to 10 to 50 nucleotides), and at least about 60 ℃ for long probes (including but not limited to more than 50 nucleotides); (iii) Adding a destabilizing agent, including but not limited to formamide; (iv) 50% formamide, 5 XSSC, and 1% SDS, at 42℃or 5 XSSC, 1% SDS, at 65℃with washing at 65℃in 0.2 XSSC and 0.1% SDS for between about 5 minutes to about 120 minutes. By way of example only, detection of selective or specific hybridization includes, but is not limited to, a positive signal at least twice background. An exhaustive guidance for nucleic acid hybridization is found in Tijssen, laboratory Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Probes, "Overview of principles of hybridization and the strategy of nucleic acid assays" (1993).
As used herein, the term "subject" refers to an animal that is the subject of treatment, observation or experiment. By way of example only, the subject may be, but is not limited to, a mammal, including, but not limited to, a human.
As used herein, the term "substantially purified" refers to a component of interest that may be substantially or essentially free of other components that typically accompany or interact with the component of interest prior to purification. By way of example only, a component of interest may be "substantially purified" when a formulation of the component of interest contains 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 less than about 1% (by dry weight) of contaminating components. Thus, a "substantially purified" component of interest may have a purity level of about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or more. By way of example only, in the case of recombinantly produced natural or unnatural amino acid polypeptides, the natural or unnatural amino acid polypeptide can be purified from a natural cell or host cell. For example, a preparation of a natural amino acid polypeptide or a non-natural amino acid polypeptide may be "substantially purified" when the preparation contains 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 less than about 1% (by dry weight) of contaminating substances. For example, when the natural or unnatural amino acid polypeptide is recombinantly produced by a host cell, the natural or unnatural amino acid polypeptide can be present in an amount of about 30%, about 25%, about 20%, about 15%, about 10%, about 5%, about 4%, about 3%, about 2%, or about 1% or less of the dry weight of the cell. For example, when the natural or unnatural amino acid polypeptide is recombinantly produced by a host cell, the natural or unnatural amino acid polypeptide can be present in the culture medium in an amount of about 5g/L, about 4g/L, about 3g/L, about 2g/L, about 1g/L, about 750mg/L, about 500mg/L, about 250mg/L, about L00mg/L, about 50mg/L, about 10mg/L, or about 1mg/L or less of the dry weight of the cell. For example, a "substantially purified" natural amino acid polypeptide or non-natural amino acid polypeptide can have a purity level of 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 more as determined by a suitable method, including, but not limited to, SDS/PAGE analysis, RP-HPLC, SEC, and capillary electrophoresis.
The term "substituent" (also referred to as a "non-interfering substituent") refers to a group that can be used to displace another group on a molecule. These groups include, but are not limited to, halo, C 1 -C 10 Alkyl, C 2 -C 10 Alkenyl, C 2 -C 10 Alkynyl, C 1 -C 10 Alkoxy, C 5 -C 12 Aralkyl, C 3 -C 12 Cycloalkyl, C 4 -C 12 Cycloalkenyl, phenyl, substituted phenyl, tolyl, xylyl, biphenyl, C 2 -C 12 Alkoxyalkyl, C 5 -C 12 Alkoxyaryl, C 5 -C 12 Aryloxyalkyl, C 7 -C 12 Oxyaryl, C 1 -C 6 Alkylsulfinyl, C 1 -C 10 Alkylsulfonyl, - (CH) 2 ) m -O-(C 1 -C 10 Alkyl) (wherein m is 1 to 8), aryl, substituted alkoxy, fluoroalkyl, heterocyclyl, substituted heterocyclyl, nitroalkyl, -NO 2 、-CN、-NRC(O)-(C 1 -C 10 Alkyl), -C (O) - (C) 1 -C 10 Alkyl group, C 2 -C 10 Alkylthioalkyl, -C (O) O- (C) 1 -C 10 Alkyl), -OH, -SO 2 、＝S、-COOH、-NR 2 Carbonyl, -C (O) - (C) 1 -C 10 Alkyl) -CF 3 、-C(O)-CF 3 、-C(O)NR 2 、-(C 1 -C 10 Aryl) -S- (C 6 -C 10 Aryl), -C (O) - (C) 6 -C 10 Aryl) - (CH) 2 ) m -O-(CH 2 ) m -O-(C 1 -C 10 Alkyl) (wherein each m is 1 to 8), -C (O) NR 2 、-C(S)NR 2 、-SO 2 NR 2 、-NRC(O)NR 2 、-NRC(S)NR 2 Salts thereof, and the like. Each R group in the foregoing list includes, but is not limited to, H, alkyl or substituted alkyl, aryl or substituted aryl, or alkylaryl. When the substituents are referred to by their conventional formulas written from left to rightTiming, which likewise encompasses chemically identical substituents which may result from writing structures from right to left, e.g., -CH 2 O-equals-OCH 2 -。
By way of example only, substituents for alkyl and heteroalkyl groups (including those groups referred to as alkylene, alkenyl, heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl) include, but are not limited to: -OR, =o, =nr, =n-OR, -NR 2 -SR, -halogen, -SiR 3 、-OC(O)R、-C(O)R、-CO 2 R、-CONR 2 、-OC(O)NR 2 、-NRC(O)R、-NRC(O)NR 2 、-NR(O) 2 R、-NR-C(NR 2 )＝NR、-S(O)R、-S(O) 2 R、-S(O) 2 NR 2 、-NRSO 2 R, -CN and-NO 2 . Each R group in the foregoing list includes, but is not limited to, hydrogen, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl (including, but not limited to, aryl substituted with 1-3 halogens), substituted or unsubstituted alkyl, alkoxy, or thioalkoxy, or aralkyl. When two R groups are attached to the same nitrogen atom, they may be combined with the nitrogen atom to form a 5-membered ring, a 6-membered ring, or a 7-membered ring. For example, -NR 2 Meaning including but not limited to 1-pyrrolidinyl and 4-morpholinyl.
For example, substituents for aryl and heteroaryl groups include (but are not limited to) -OR, =o, =nr, =n-OR, -NR 2 -SR, -halogen, -SiR 3 、-OC(O)R、-C(O)R、-CO 2 R、-CONR 2 、-OC(O)NR 2 、-NRC(O)R、-NRC(O)NR 2 、-NR(O) 2 R、-NR-C(NR 2 )＝NR、-S(O)R、-S(O) 2 R、-S(O) 2 NR 2 、-NRSO 2 R、-CN、-NO 2 、-R、-N 3 、-CH(Ph) 2 Fluorine (C) 1 -C 4 ) Alkoxy and fluoro (C) 1 -C 4 ) Alkyl groups, the number ranging from zero to the total number of open valencies on the aromatic ring system; and wherein each R group in the foregoing list includes, but is not limited to, hydrogen, alkyl, heteroalkyl, aryl, and heteroaryl.
As used herein, the term "therapeutically effective amount" refers to an amount of a composition containing at least one unnatural amino acid polypeptide and/or at least one modified unnatural amino acid polypeptide that is administered to a patient who has had a disease, condition, or disorder, sufficient to cure or at least partially arrest or alleviate to some extent the symptoms of at least one treated disease, condition, or disorder. The efficacy of these compositions depends on conditions including, but not limited to, the severity and course of the disease, disorder or condition, previous treatments, the health status of the patient and the response to the drug, and the discretion of the attendant physician. By way of example only, a therapeutically effective amount may be determined by routine experimentation, including but not limited to, a dose escalation clinical trial.
As used herein, the term "thioalkoxy" refers to a thioalkyl group bonded to a molecule through an oxygen atom.
The term "thermal melting point" or Tm is the temperature at which 50% of the probe complementary to the target hybridizes to the target sequence at equilibrium (at a defined ion concentration, pH, and nucleic acid concentration).
As used herein, the term "toxic moiety" refers to a compound that can cause injury or death.
As used herein, the term "treating" includes alleviating, or ameliorating a symptom of a disease or condition, preventing other symptoms, ameliorating or preventing the underlying metabolic etiology of a symptom, inhibiting the disease or condition, e.g., arresting the development of the disease or condition, alleviating the disease or condition, allowing the disease or condition to return, alleviating the condition caused by the disease or condition, or terminating the symptom of the disease or condition. The term "treatment" includes, but is not limited to, prophylactic and/or therapeutic treatment.
As used herein, the term "water-soluble polymer" refers to any polymer that is soluble in an aqueous solvent. These water-soluble polymers include, but are not limited to, polyethylene glycol propionaldehyde, mono-C thereof 1 -C 10 Alkoxy or aryloxy derivatives (described in U.S. Pat. No. 5,252,714, incorporated herein by reference), monomethoxy-polyethylene glycol, polyvinylpyrrolidone, polyvinyl alcohol, polyamino acids, divinylEther maleic anhydride, N- (2-hydroxypropyl) -methacrylamide, dextran derivatives (including dextran sulfate), polypropylene glycol, polyoxypropylene/ethylene oxide copolymers, polyoxyethylated polyols, heparin fragments, polysaccharides, oligosaccharides, glycans, cellulose and cellulose derivatives (including but not limited to methylcellulose and carboxymethylcellulose), serum albumin, starch and starch derivatives, polypeptides, polyalkylene glycols and derivatives thereof, copolymers of polyalkylene glycols and derivatives thereof, polyvinyl ethyl ether, and alpha-beta-poly [ (2-hydroxyethyl) -DL-asparagine, and analogs or mixtures thereof. By way of example only, coupling of these water-soluble polymers to natural amino acid polypeptides or non-natural polypeptides may result in alterations including, but not limited to, increased water solubility, increased or modulated serum half-life, increased or modulated therapeutic half-life, increased bioavailability, modulated biological activity, prolonged circulation time, modulated immunogenicity, modulated physical association properties, including, but not limited to, aggregation and multimerization, altered receptor binding, altered binding to one or more binding partners, and altered receptor dimerization or multimerization relative to the unmodified form. In addition, these water-soluble polymers may or may not have their own biological activity.
Unless otherwise indicated, conventional methods of mass spectrometry, NMR, HPLC, protein chemistry, biochemistry, recombinant DNA techniques, and pharmacology in the art are used.
The compounds set forth herein, including but not limited to unnatural amino acids, unnatural amino acid polypeptides, and modified unnatural amino acid polypeptides, as well as reagents for making the foregoing compounds, comprise isotopically-labeled compounds that are identical to those recited in the various formulae and structures set forth herein, but for the substitution of one or more atoms with an atom having an atomic mass or mass number that is different from the atomic mass or mass number typically found in nature. Examples of isotopes that can be incorporated into compounds of the invention include isotopes of hydrogen, carbon, nitrogen, oxygen, fluorine, and chlorine, such as respectively 2 H、 3 H、 13 C、 14 C、 15 N、 18 O、 17 O、 35 S、 15 F、 36 Cl. Certain isotopically-labeled compounds described herein (e.g., such as 3 H and 14 those compounds into which the radioisotope of C is incorporated) are suitable for pharmaceutical and/or substrate tissue distribution assays. In addition, the use of a metal such as deuterium (i.e 2 H) Isotope substitution of (c) may provide certain therapeutic advantages resulting from higher metabolic stability, such as increased in vivo half-life or reduced dosage requirements.
Some of the compounds herein (including, but not limited to, unnatural amino acids, unnatural amino acid polypeptides, and modified unnatural amino acid polypeptides, as well as reagents for making the foregoing compounds) have asymmetric carbon atoms and may therefore exist in enantiomeric or diastereoisomeric forms. Based on their physicochemical differences, the diastereomeric mixtures can be separated into their individual diastereomers by known methods (e.g., chromatography and/or fractional crystallization). Enantiomers can be separated by converting an enantiomeric mixture into a diastereomeric mixture by reaction with an appropriate optically active compound (e.g., alcohol), separating the diastereomers and converting (e.g., hydrolyzing) the individual diastereomers to the corresponding pure enantiomers. All such isomers, including diastereomers, enantiomers, and mixtures thereof, are considered as part of the compositions described herein.
In additional or other embodiments, the compounds described herein (including, but not limited to, unnatural amino acids, unnatural amino acid polypeptides, and modified unnatural amino acid polypeptides, as well as reagents for making the foregoing compounds) are used in the form of prodrugs. In additional or other embodiments, the compounds described herein (including, but not limited to, unnatural amino acids, unnatural amino acid polypeptides, and modified unnatural amino acid polypeptides, as well as reagents for making the foregoing compounds) are metabolized following administration to an organism in need of metabolite production, which is subsequently used to produce the desired effect, which comprises the desired therapeutic effect. In other or additional embodiments are active metabolites of unnatural amino acids and "modified or unmodified" unnatural amino acid polypeptides.
The methods and formulations described herein comprise the use of unnatural amino acids, unnatural amino acid polypeptides, and modified N-oxides, crystalline forms (also referred to as polymorphs) or pharmaceutically acceptable salts of unnatural amino acid polypeptides. In certain embodiments, the unnatural amino acid, unnatural amino acid polypeptide, and modified unnatural amino acid polypeptide can exist in tautomeric forms. All tautomers are included within the scope of the unnatural amino acids, unnatural amino acid polypeptides, and modified unnatural amino acid polypeptides set forth herein. In addition, the unnatural amino acids, unnatural amino acid polypeptides, and modified unnatural amino acid polypeptides described herein can exist in non-solvate form as well as in solvate form with pharmaceutically acceptable solvents (such as water, ethanol, and the like). The unnatural amino acids, unnatural amino acid polypeptides, and solvate forms of modified unnatural amino acid polypeptides presented herein are also considered disclosed herein.
Some of the compounds herein (including, but not limited to, unnatural amino acids, unnatural amino acid polypeptides, and modified unnatural amino acid polypeptides) can exist in several tautomeric forms as reagents for preparing the foregoing compounds. All of these tautomeric forms are considered as part of the compositions described herein. Also, for example, all enol-ketone forms of any of the compounds herein, including, but not limited to, unnatural amino acids, unnatural amino acid polypeptides, and modified unnatural amino acid polypeptides, as well as reagents for making the foregoing compounds, are considered as part of the compositions described herein.
Some of the compounds herein, including but not limited to unnatural amino acids, unnatural amino acid polypeptides, and modified unnatural amino acid polypeptides, as well as reagents for preparing any of the foregoing compounds, are acidic and can form salts with pharmaceutically acceptable cations. Some of the compounds herein, including but not limited to unnatural amino acids, unnatural amino acid polypeptides, and modified unnatural amino acid polypeptides, as well as reagents for making the foregoing compounds, can be basic and thus form salts with pharmaceutically acceptable anions. All such salts, including di-salts, are within the scope of the compositions described herein and can be prepared by conventional methods. For example, salts can be prepared by contacting an acidic entity with a basic entity in an aqueous, non-aqueous, or partially aqueous medium. Recovering the salt by using at least one of the following techniques: filtering; precipitating with non-solvent, and filtering; evaporating the solvent; or (in the case of aqueous solutions) freeze-drying.
Pharmaceutically acceptable salts of the unnatural amino acid polypeptides disclosed herein can be formed when acidic protons present in the parent unnatural amino acid polypeptide are replaced with metal ions (e.g., alkali metal ions, alkaline earth metal ions, or aluminum ions), or otherwise complexed with an organic base. In addition, salt forms of the disclosed unnatural amino acid polypeptides can be prepared using salts of starting materials or intermediates. The unnatural amino acid polypeptides described herein can be prepared as pharmaceutically acceptable acid addition salts (which are types of pharmaceutically acceptable salts) by reacting the free base form of the unnatural amino acid polypeptide described herein with a pharmaceutically acceptable inorganic or organic acid. Alternatively, the unnatural amino acid polypeptides described herein can be prepared as pharmaceutically acceptable base addition salts (which are types of pharmaceutically acceptable salts) by reacting the free acid form of the unnatural amino acid polypeptide described herein with a pharmaceutically acceptable inorganic or organic base.
Types of pharmaceutically acceptable salts include (but are not limited to): (1) Acid addition salts with inorganic or organic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like; such as acetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3- (4-hydroxybenzoyl) benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1, 2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, 2-naphthalenesulfonic acid, 4-methylbicyclo- [2.2.2] oct-2-ene-1-carboxylic acid, glucoheptonic acid, 4' -methylenebis- (3-hydroxy-2-ene-1-carboxylic acid), 3-phenylpropionic acid, trimethylacetic acid, t-butylacetic acid, lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid, and the like; (2) When acidic protons present in the parent compound are replaced with metal ions (e.g., alkali metal ions, alkaline earth metal ions, or aluminum ions); or a salt formed upon complexation with an organic base. Acceptable organic bases include ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, and the like. Acceptable inorganic bases include aluminum hydroxide, calcium hydroxide, potassium hydroxide, sodium carbonate, sodium hydroxide, and the like.
The corresponding counter ions of the pharmaceutically acceptable salts of the unnatural amino acid polypeptides can be analyzed and identified using a variety of methods, including, but not limited to, ion exchange chromatography, ion chromatography, capillary electrophoresis, inductively coupled plasma, atomic absorption spectroscopy, mass spectrometry, or any combination thereof. In addition, the therapeutic activity of pharmaceutically acceptable salts of these unnatural amino acid polypeptides can be tested using the techniques and methods described in examples 87-91.
It is to be understood that reference to a salt includes solvent-addition forms or crystalline forms thereof, particularly solvates or polymorphs. Solvates contain stoichiometric or non-stoichiometric amounts of solvent and are typically formed during crystallization with pharmaceutically acceptable solvents such as water, ethanol, and the like. Hydrates are formed when the solvent is water, or alcoholates are formed when the solvent is an alcohol. Polymorphs comprise different crystal combinations arrangements of a compound of the same elemental composition. Polymorphs typically have different X-ray diffraction patterns, infrared spectra, melting points, densities, hardness, crystal shape, optical and electrical properties, stability and solubility. Various factors such as recrystallization solvent, crystallization rate, and storage temperature may dominate the single crystal form.
Screening and characterization of pharmaceutically acceptable salt polymorphs and/or solvates of the unnatural amino acid polypeptide can be accomplished using a variety of techniques, including, but not limited to, thermal analysis, X-ray diffraction, spectroscopy, vapor adsorption, and microscopy. Thermal analysis methods focus on thermochemical degradation or thermophysical processes (including but not limited to polymorph conversion), and these methods are used to analyze relationships between polymorphic forms, determine weight loss to find glass transition temperatures, or for excipient compatibility studies. Such methods include, but are not limited to, differential Scanning Calorimetry (DSC), modulated Differential Scanning Calorimetry (MDSC), thermogravimetric analysis (TGA), and thermogravimetric and infrared analysis (TG/IR). X-ray diffraction methods include, but are not limited to, single crystal and powder diffractometers and synchrotron sources. Various spectroscopic techniques are used including, but not limited to, raman, FTIR, UVIS and NMR (liquid and solid). Various microscopy techniques include, but are not limited to, polarized light microscopy, scanning Electron Microscopy (SEM) with energy dispersive X-ray analysis (EDX), ambient scanning electron microscopy (in a gas or water vapor atmosphere) with EDX, IR microscopy, and raman microscopy.
Drawings
A better understanding of the features and advantages of the methods and compositions of the present invention may be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of our methods, compositions, apparatus and devices are utilized, the accompanying drawings of which:
FIG. 1 presents a schematic representation of the relationship of certain aspects of the methods, compositions, strategies, and techniques described herein.
Fig. 2 presents an illustrative, non-limiting example of the types of unnatural amino acids described herein. These unnatural amino acids can be used or incorporated into any of the methods, compositions, techniques, and strategies for preparing, purifying, characterizing, and using the unnatural amino acids, unnatural amino acid polypeptides, and modified unnatural amino acid polypeptides described herein.
Fig. 3 presents an illustrative, non-limiting example of the types of unnatural amino acids described herein. These unnatural amino acids can be used or incorporated into any of the methods, compositions, techniques, and strategies for preparing, purifying, characterizing, and using the unnatural amino acids, unnatural amino acid polypeptides, and modified unnatural amino acid polypeptides described herein.
FIG. 4 presents an illustrative, non-limiting example of a synthetic method for preparing the unnatural amino acids described herein. These unnatural amino acids can be used or incorporated into any of the methods, compositions, techniques, and strategies for preparing, purifying, characterizing, and using the unnatural amino acids, unnatural amino acid polypeptides, and modified unnatural amino acid polypeptides described herein.
FIG. 5 presents an illustrative, non-limiting example of a synthetic method for preparing the unnatural amino acids described herein. These unnatural amino acids can be used or incorporated into any of the methods, compositions, techniques, and strategies for preparing, purifying, characterizing, and using the unnatural amino acids, unnatural amino acid polypeptides, and modified unnatural amino acid polypeptides described herein.
FIG. 6 presents an illustrative, non-limiting example of a synthetic method for preparing the unnatural amino acids described herein. These unnatural amino acids can be used or incorporated into any of the methods, compositions, techniques, and strategies for preparing, purifying, characterizing, and using the unnatural amino acids, unnatural amino acid polypeptides, and modified unnatural amino acid polypeptides described herein.
FIG. 7 presents an illustrative, non-limiting example of post-translational modification of a carbonyl-containing unnatural amino acid polypeptide with a hydroxylamine-containing reagent to form a modified oxime-containing unnatural amino acid polypeptide. These unnatural amino acid polypeptides can be used or incorporated into any of the methods, compositions, techniques, and strategies for preparing, purifying, characterizing, and using the unnatural amino acids, unnatural amino acid polypeptides, and modified unnatural amino acid polypeptides described herein.
FIG. 8 presents an illustrative, non-limiting example of an additive that can be used to enhance the reaction of a carbonyl-containing unnatural amino acid polypeptide with a hydroxylamine-containing reagent to form a modified oxime-containing unnatural amino acid polypeptide.
FIG. 9 presents an illustrative, non-limiting example of post-translational modification of an oxime-containing unnatural amino acid polypeptide with a carbonyl-containing reagent to form a modified oxime-containing unnatural amino acid polypeptide. These unnatural amino acid polypeptides can be used or incorporated into any of the methods, compositions, techniques, and strategies for preparing, purifying, characterizing, and using the unnatural amino acids, unnatural amino acid polypeptides, and modified unnatural amino acid polypeptides described herein.
FIG. 10 presents an illustrative, non-limiting example of post-translational modification of a hydroxylamine-containing unnatural amino acid polypeptide with a carbonyl-containing reagent to form a modified oxime-containing unnatural amino acid polypeptide. These unnatural amino acid polypeptides can be used or incorporated into any of the methods, compositions, techniques, and strategies for preparing, purifying, characterizing, and using the unnatural amino acids, unnatural amino acid polypeptides, and modified unnatural amino acid polypeptides described herein.
FIG. 11 presents an illustrative, non-limiting example of a PEG-containing reagent that can be used to modify a non-natural amino acid polypeptide to form a PEG-containing oxime-linked non-natural amino acid polypeptide.
FIG. 12 presents an illustrative, non-limiting example of the synthesis of a PEG-containing reagent that can be used to modify a non-natural amino acid polypeptide to form a PEG-containing oxime-linked non-natural amino acid polypeptide.
FIG. 13 presents an illustrative, non-limiting example of synthesis of an amide-containing PEG reagent that can be used to modify an unnatural amino acid polypeptide to form a PEG-containing oxime-linked unnatural amino acid polypeptide.
FIG. 14 presents an illustrative, non-limiting example of synthesis of carbamate group-containing PEG reagents that can be used to modify an unnatural amino acid polypeptide to form a PEG-containing oxime-linked unnatural amino acid polypeptide.
FIG. 15 presents an illustrative, non-limiting example of synthesis of carbamate group-containing PEG reagents that can be used to modify an unnatural amino acid polypeptide to form a PEG-containing oxime-linked unnatural amino acid polypeptide.
FIG. 16 presents an illustrative, non-limiting example of the synthesis of simple PEG-containing reagents that can be used to modify an unnatural amino acid polypeptide to form a PEG-containing oxime-linked unnatural amino acid polypeptide.
FIG. 17 presents illustrative, non-limiting examples of branched PEG-containing reagents that can be used to modify a non-natural amino acid polypeptide to form a PEG-containing oxime-linked non-natural amino acid polypeptide, and the use of one such reagent for modifying a carbonyl-based non-natural amino acid polypeptide.
FIG. 18 presents an illustrative, non-limiting example of synthesis of a bifunctional linker group useful for modifying and linking a non-natural amino acid polypeptide.
FIG. 19 presents an illustrative, non-limiting example of a multifunctional linker group that can be used to modify and bind a non-natural amino acid polypeptide.
FIG. 20 presents an illustrative, non-limiting representation of the use of bifunctional linker groups for modifying and bonding a non-natural amino acid polypeptide to a PEG group.
FIG. 21 presents an illustrative, non-limiting example of the use of a difunctional linker group for modifying and bonding a non-natural amino acid polypeptide to a PEG group.
FIG. 22 presents an illustrative, non-limiting representation of the use of a difunctional linker group to link two unnatural amino acid polypeptides together to form a homodimer.
FIG. 23 presents an illustrative, non-limiting representation of the use of a difunctional linker group to link two different unnatural amino acid polypeptides together to form a heterodimer.
FIG. 24 presents an illustrative, non-limiting representative view of the synthesis of carbonyl-containing unnatural amino acids. These unnatural amino acids can be used or incorporated into any of the methods, compositions, techniques, and strategies for preparing, purifying, characterizing, and using the unnatural amino acids, unnatural amino acid polypeptides, and modified unnatural amino acid polypeptides described herein.
FIG. 25 presents an illustrative, non-limiting representative view of the synthesis of a dicarbonyl-containing unnatural amino acid. These unnatural amino acids can be used or incorporated into any of the methods, compositions, techniques, and strategies for preparing, purifying, characterizing, and using the unnatural amino acids, unnatural amino acid polypeptides, and modified unnatural amino acid polypeptides described herein.
FIG. 26 presents an illustrative, non-limiting representative view of the synthesis of a dicarbonyl-containing unnatural amino acid.
FIG. 27 presents an illustrative, non-limiting representative view of the synthesis of a dicarbonyl-containing unnatural amino acid. These unnatural amino acids can be used or incorporated into any of the methods, compositions, techniques, and strategies for preparing, purifying, characterizing, and using the unnatural amino acids, unnatural amino acid polypeptides, and modified unnatural amino acid polypeptides described herein.
FIG. 28 presents an illustrative, non-limiting representative view of the synthesis of a dicarbonyl-containing unnatural amino acid. These unnatural amino acids can be used or incorporated into any of the methods, compositions, techniques, and strategies for preparing, purifying, characterizing, and using the unnatural amino acids, unnatural amino acid polypeptides, and modified unnatural amino acid polypeptides described herein.
FIG. 29 presents an illustrative, non-limiting representative view of the synthesis of a dicarbonyl-containing unnatural amino acid. These unnatural amino acids can be used or incorporated into any of the methods, compositions, techniques, and strategies for preparing, purifying, characterizing, and using the unnatural amino acids, unnatural amino acid polypeptides, and modified unnatural amino acid polypeptides described herein.
FIG. 30 presents an illustrative, non-limiting representative view of the synthesis of a dicarbonyl-containing unnatural amino acid. These unnatural amino acids can be used or incorporated into any of the methods, compositions, techniques, and strategies for preparing, purifying, characterizing, and using the unnatural amino acids, unnatural amino acid polypeptides, and modified unnatural amino acid polypeptides described herein.
FIG. 31 presents an illustrative, non-limiting representative diagram of the synthesis of a dicarbonyl-containing unnatural amino acid. These unnatural amino acids can be used or incorporated into any of the methods, compositions, techniques, and strategies for preparing, purifying, characterizing, and using the unnatural amino acids, unnatural amino acid polypeptides, and modified unnatural amino acid polypeptides described herein.
FIG. 32 presents an illustrative, non-limiting representative view of the synthesis of a dicarbonyl-containing unnatural amino acid. These unnatural amino acids can be used or incorporated into any of the methods, compositions, techniques, and strategies for preparing, purifying, characterizing, and using the unnatural amino acids, unnatural amino acid polypeptides, and modified unnatural amino acid polypeptides described herein.
FIG. 33 presents an illustrative, non-limiting representative view of the synthesis of a dicarbonyl-containing unnatural amino acid. These unnatural amino acids can be used or incorporated into any of the methods, compositions, techniques, and strategies for preparing, purifying, characterizing, and using the unnatural amino acids, unnatural amino acid polypeptides, and modified unnatural amino acid polypeptides described herein.
FIG. 34 presents an illustrative, non-limiting representative view of the synthesis of a dicarbonyl-containing unnatural amino acid. These unnatural amino acids can be used or incorporated into any of the methods, compositions, techniques, and strategies for preparing, purifying, characterizing, and using the unnatural amino acids, unnatural amino acid polypeptides, and modified unnatural amino acid polypeptides described herein.
FIG. 35 presents an illustrative, non-limiting representation of carbonyl-containing unnatural amino acids and dicarbonyl-containing unnatural amino acids. These unnatural amino acids can be used or incorporated into any of the methods, compositions, techniques, and strategies for preparing, purifying, characterizing, and using the unnatural amino acids, unnatural amino acid polypeptides, and modified unnatural amino acid polypeptides described herein.
FIG. 36 presents an illustrative, non-limiting representation of the synthesis of unnatural amino acids. These unnatural amino acids can be used or incorporated into any of the methods, compositions, techniques, and strategies for preparing, purifying, characterizing, and using the unnatural amino acids, unnatural amino acid polypeptides, and modified unnatural amino acid polypeptides described herein.
FIG. 37 presents an illustrative, non-limiting representative diagram of the synthesis of carbonyl-containing unnatural amino acids and dicarbonyl-containing unnatural amino acids. These unnatural amino acids can be used or incorporated into any of the methods, compositions, techniques, and strategies for preparing, purifying, characterizing, and using the unnatural amino acids, unnatural amino acid polypeptides, and modified unnatural amino acid polypeptides described herein.
FIG. 38 presents an illustrative, non-limiting representative diagram of the synthesis of carbonyl-containing unnatural amino acids and dicarbonyl-containing unnatural amino acids. These unnatural amino acids can be used or incorporated into any of the methods, compositions, techniques, and strategies for preparing, purifying, characterizing, and using the unnatural amino acids, unnatural amino acid polypeptides, and modified unnatural amino acid polypeptides described herein.
FIG. 39 presents an illustrative, non-limiting representative view of the synthesis of carbonyl-containing unnatural amino acids and dicarbonyl-containing unnatural amino acids. These unnatural amino acids can be used or incorporated into any of the methods, compositions, techniques, and strategies for preparing, purifying, characterizing, and using the unnatural amino acids, unnatural amino acid polypeptides, and modified unnatural amino acid polypeptides described herein.
FIG. 40 presents an illustrative, non-limiting representation of a dicarbonyl-containing unnatural amino acid. These unnatural amino acids can be used or incorporated into any of the methods, compositions, techniques, and strategies for preparing, purifying, characterizing, and using the unnatural amino acids, unnatural amino acid polypeptides, and modified unnatural amino acid polypeptides described herein.
FIG. 41 presents an illustrative, non-limiting representation of a dicarbonyl-containing unnatural amino acid. These unnatural amino acids can be used or incorporated into any of the methods, compositions, techniques, and strategies for preparing, purifying, characterizing, and using the unnatural amino acids, unnatural amino acid polypeptides, and modified unnatural amino acid polypeptides described herein.
FIG. 42 presents an illustrative, non-limiting representative view of a dicarbonyl-containing unnatural amino acid. These unnatural amino acids can be used or incorporated into any of the methods, compositions, techniques, and strategies for preparing, purifying, characterizing, and using the unnatural amino acids, unnatural amino acid polypeptides, and modified unnatural amino acid polypeptides described herein.
FIG. 43 presents an illustrative, non-limiting representation of (a) a protected or unprotected 1, 3-ketoaldehyde-containing unnatural amino acid, and (b) a 1-3-ketocarboxy (thio) ester-containing unnatural amino acid. These unnatural amino acids can be used or incorporated into any of the methods, compositions, techniques, and strategies for preparing, purifying, characterizing, and using the unnatural amino acids, unnatural amino acid polypeptides, and modified unnatural amino acid polypeptides described herein.
FIG. 44 presents an illustrative, non-limiting representation of a hydrazide-containing unnatural amino acid. These unnatural amino acids can be used or incorporated into any of the methods, compositions, techniques, and strategies for preparing, purifying, characterizing, and using the unnatural amino acids, unnatural amino acid polypeptides, and modified unnatural amino acid polypeptides described herein.
FIG. 45 presents an illustrative, non-limiting representation of a hydrazide-containing unnatural amino acid. These unnatural amino acids can be used or incorporated into any of the methods, compositions, techniques, and strategies for preparing, purifying, characterizing, and using the unnatural amino acids, unnatural amino acid polypeptides, and modified unnatural amino acid polypeptides described herein.
Fig. 46A and 46B present illustrative, non-limiting representative diagrams of oxime-containing unnatural amino acids, and fig. 46C presents illustrative, non-limiting representative diagrams of hydrazine-containing unnatural amino acids. These unnatural amino acids can be used or incorporated into any of the methods, compositions, techniques, and strategies for preparing, purifying, characterizing, and using the unnatural amino acids, unnatural amino acid polypeptides, and modified unnatural amino acid polypeptides described herein.
FIG. 47 presents an illustrative, non-limiting representation of one-step engagement to a non-natural amino acid polypeptide and two-step engagement to a non-natural amino acid polypeptide. For example, these splices comprise PEGylation of unnatural amino acid polypeptides.
FIG. 48 presents an illustrative, non-limiting representation of the synthesis of mPEG-hydroxylamine compounds. These unnatural amino acids can be used or incorporated into any of the methods, compositions, techniques, and strategies for preparing, purifying, characterizing, and using the unnatural amino acids, unnatural amino acid polypeptides, and modified unnatural amino acid polypeptides described herein.
FIG. 49 presents an illustrative, non-limiting representation of the synthesis of mPEG-hydroxylamine compounds. These unnatural amino acids can be used or incorporated into any of the methods, compositions, techniques, and strategies for preparing, purifying, characterizing, and using the unnatural amino acids, unnatural amino acid polypeptides, and modified unnatural amino acid polypeptides described herein.
FIG. 50 presents an illustrative, non-limiting representation of the synthesis of mPEG-hydroxylamine compounds. These unnatural amino acids can be used or incorporated into any of the methods, compositions, techniques, and strategies for preparing, purifying, characterizing, and using the unnatural amino acids, unnatural amino acid polypeptides, and modified unnatural amino acid polypeptides described herein.
FIG. 51 presents an illustrative, non-limiting representation of the synthesis of mPEG-hydroxylamine compounds. These unnatural amino acids can be used or incorporated into any of the methods, compositions, techniques, and strategies for preparing, purifying, characterizing, and using the unnatural amino acids, unnatural amino acid polypeptides, and modified unnatural amino acid polypeptides described herein.
FIG. 52 presents an illustrative, non-limiting representation of the synthesis of mPEG-hydroxylamine compounds. These unnatural amino acids can be used or incorporated into any of the methods, compositions, techniques, and strategies for preparing, purifying, characterizing, and using the unnatural amino acids, unnatural amino acid polypeptides, and modified unnatural amino acid polypeptides described herein.
FIG. 53 presents an illustrative, non-limiting representation of the synthesis of mPEG-hydroxylamine compounds. These unnatural amino acids can be used or incorporated into any of the methods, compositions, techniques, and strategies for preparing, purifying, characterizing, and using the unnatural amino acids, unnatural amino acid polypeptides, and modified unnatural amino acid polypeptides described herein.
FIG. 54 presents an illustrative, non-limiting representation of the synthesis of mPEG-hydroxylamine compounds. These unnatural amino acids can be used or incorporated into any of the methods, compositions, techniques, and strategies for preparing, purifying, characterizing, and using the unnatural amino acids, unnatural amino acid polypeptides, and modified unnatural amino acid polypeptides described herein.
FIG. 55 presents an illustrative, non-limiting representation of the synthesis of mPEG-hydroxylamine compounds. These unnatural amino acids can be used or incorporated into any of the methods, compositions, techniques, and strategies for preparing, purifying, characterizing, and using the unnatural amino acids, unnatural amino acid polypeptides, and modified unnatural amino acid polypeptides described herein.
FIG. 56 presents an illustrative, non-limiting representation of the synthesis of mPEG-hydroxylamine compounds. These unnatural amino acids can be used or incorporated into any of the methods, compositions, techniques, and strategies for preparing, purifying, characterizing, and using the unnatural amino acids, unnatural amino acid polypeptides, and modified unnatural amino acid polypeptides described herein.
FIG. 57 presents an illustrative, non-limiting representation of the synthesis of mPEG-hydroxylamine compounds. These unnatural amino acids can be used or incorporated into any of the methods, compositions, techniques, and strategies for preparing, purifying, characterizing, and using the unnatural amino acids, unnatural amino acid polypeptides, and modified unnatural amino acid polypeptides described herein.
FIG. 58 presents an illustrative, non-limiting representation of the synthesis of mPEG-hydroxylamine compounds. These unnatural amino acids can be used or incorporated into any of the methods, compositions, techniques, and strategies for preparing, purifying, characterizing, and using the unnatural amino acids, unnatural amino acid polypeptides, and modified unnatural amino acid polypeptides described herein.
FIG. 59 presents an illustrative, non-limiting representation of the synthesis of mPEG-hydroxylamine compounds. These unnatural amino acids can be used or incorporated into any of the methods, compositions, techniques, and strategies for preparing, purifying, characterizing, and using the unnatural amino acids, unnatural amino acid polypeptides, and modified unnatural amino acid polypeptides described herein.
FIG. 60 presents an illustrative, non-limiting representation of the synthesis of hydroxylamine compounds. These unnatural amino acids can be used or incorporated into any of the methods, compositions, techniques, and strategies for preparing, purifying, characterizing, and using the unnatural amino acids, unnatural amino acid polypeptides, and modified unnatural amino acid polypeptides described herein.
FIG. 61 presents an illustrative, non-limiting representation of the synthesis of mPEG-hydroxylamine compounds. These unnatural amino acids can be used or incorporated into any of the methods, compositions, techniques, and strategies for preparing, purifying, characterizing, and using the unnatural amino acids, unnatural amino acid polypeptides, and modified unnatural amino acid polypeptides described herein.
FIG. 62A presents an illustrative, non-limiting representative diagram of the synthesis of hydroxylamine compounds; FIG. 62B presents an illustrative, non-limiting representative view of the synthesis of mPEG compounds. These unnatural amino acids can be used or incorporated into any of the methods, compositions, techniques, and strategies for preparing, purifying, characterizing, and using the unnatural amino acids, unnatural amino acid polypeptides, and modified unnatural amino acid polypeptides described herein.
Fig. 63 presents illustrative, non-limiting examples of (a) modification of an unnatural amino acid polypeptide by chemical conversion to a carbonyl-containing (including dicarbonyl-containing) unnatural amino acid polypeptide and (B) modification of an unnatural amino acid polypeptide by chemical conversion to a hydroxylamine-containing unnatural amino acid polypeptide. These unnatural amino acid polypeptides can be used or incorporated into any of the methods, compositions, techniques, and strategies for preparing, purifying, characterizing, and using the unnatural amino acids, unnatural amino acid polypeptides, and modified unnatural amino acid polypeptides described herein.
FIG. 64 presents an illustrative, non-limiting representation of the synthesis of PEG-hydroxylamine compounds. These unnatural amino acids can be used or incorporated into any of the methods, compositions, techniques, and strategies for preparing, purifying, characterizing, and using the unnatural amino acids, unnatural amino acid polypeptides, and modified unnatural amino acid polypeptides described herein.
FIG. 65 presents an illustrative, non-limiting representation of the synthesis of PEG-hydroxylamine compounds. These unnatural amino acids can be used or incorporated into any of the methods, compositions, techniques, and strategies for preparing, purifying, characterizing, and using the unnatural amino acids, unnatural amino acid polypeptides, and modified unnatural amino acid polypeptides described herein.
Detailed Description
I. Introduction to the invention
In recent years, entirely new techniques in protein science have been reported that are intended to overcome many of the limitations associated with site-specific modification of proteins. In particular, new components have been added to the prokaryotes E.coli (e.coli) (e.g., L.Wang et al, (2001),Science292:498-500) and eukaryotic Saccharomyces cerevisiae (Sacchromyces cerevisiae) (S.cerevisiae) (e.g., J.Chin et al,Science301:964-7 (2003)) which enables incorporation of unnatural amino acids into proteins in vivo. Using this approach, many new amino acids (including photoaffinity labels and photoisomerizable amino acids, keto amino acids, and glycosylated amino acids) with novel chemical, physical, or biological properties have been incorporated into proteins in e.coli as well as yeast with high fidelity in response to the amber codon TAG. See, for example, j.w.chip et al, (2002),Journal of the American Chemical Society124:9026-9027 (incorporated by reference in its entirety); J.W.Chin and P.G.Schultz, (2002),ChemBioChem3 (11) 1135-1137 (incorporated by reference in its entirety); j.w.chip, et al, (2002),PNAS United States of America99 (17) 11020-11024 (incorporated by reference in its entirety); and, L.Wang and P.G.Schultz, (2002), Chem.Comm.,1-11 (incorporated by reference in their entirety). These studies have demonstrated that it is possible to selectively and routinely introduce chemical functional groups that are not present in proteins, which are chemically inert to all functional groups present in the 20 common genetically encoded amino acids, and which can be used to effectively and selectively react to form stable covalent bonds.
Overview of
Fig. 1 presents an overview of the compositions, methods, and techniques described herein. To some extent, described herein are means (methods, compositions, techniques) for producing and using polypeptides comprising at least one unnatural amino acid or an unnatural amino acid modified with a carbonyl, dicarbonyl, oxime or hydroxylamine group. These unnatural amino acids can contain additional functional groups, which include (but are not limited to): marking; a dye; a polymer; a water-soluble polymer; derivatives of polyethylene glycol; a photocrosslinking agent; a cytotoxic compound; a drug; affinity labeling; a photoaffinity label; a reactive compound; a resin; a second protein or polypeptide analog; an antibody or antibody fragment; a metal chelator; a cofactor; a fatty acid; a carbohydrate; a polynucleotide; DNA; RNA; an antisense polynucleotide; saccharides, water-soluble dendrimers, cyclodextrins, biomaterials; a nanoparticle; spin labeling; a fluorophore; a metal-containing portion; a radioactive portion; novel functional groups; groups that interact covalently or non-covalently with other molecules; a light-shielding portion; an actinic radiation excitable moiety; a ligand; a photoisomerizable moiety; biotin; biotin analogues; a moiety incorporating a heavy atom; a chemically cleavable group; a photocleavable group; an extended side chain; a carbon-bonded sugar; a redox active agent; an aminothiopropionic acid; a toxic moiety; isotopically labeled moieties; a biophysical probe; phosphorescent groups; a chemiluminescent group; an electron dense group; a magnetic group; an insertion group; a chromophore; an energy transfer agent; bioactive agents (in which case the bioactive agent may comprise an agent having therapeutic activity and the unnatural amino acid polypeptide or modified unnatural amino acid may serve as a co-therapeutic with the adjunct therapeutic agent or as a means of delivering the therapeutic agent to a desired site in the organism); a detectable label; a small molecule; inhibitory ribonucleic acid; a radionucleotide; a neutron capture agent; derivatives of biotin; a quantum dot; nano transitorin; a radiotransferrin; abzymes, activating complex activators, viruses, adjuvants, aglycones, allergens, angiostatin, anti-hormones, antioxidants, aptamers, guide RNAs, saponins, shuttle vectors, macromolecules, pseudoepitopes, receptors, reverse micelles, and any combination thereof. It should be noted that the various foregoing functional groups are not meant to imply that members of one functional group are not classifiable as members of another functional group. Of course, they may overlap according to the particular situation. By way of example only, the water-soluble polymer overlaps in scope with the derivative of polyethylene glycol, however the overlap is not complete and thus both functional groups are listed above.
As shown in fig. 1, one aspect is a method of selecting and designing a polypeptide to be modified using the methods, compositions and techniques described herein. Novel polypeptides can be designed de novo, including, by way of example only, as part of a high throughput screening method (in which case numerous polypeptides can be designed, synthesized, characterized, and/or tested) or designed according to the interest of the researcher. The novel polypeptides may also be designed based on the structure of known or partially characterized polypeptides. By way of example only, the growth hormone gene superfamily (Growth Hormone Gene Superfamily) (see below) has been the subject of extensive research by the scientific community; novel polypeptides may be designed based on the structure of members of this gene superfamily. The principle of which amino acid to select for substitution and/or modification is described herein alone. The choice of which modification to use is also described herein and can be used to meet the needs of the experimenter or end user. These requirements may include, but are not limited to, manipulating the therapeutic efficacy of the polypeptide; improving the safety profile of the polypeptide; modulating the pharmacokinetics, pharmacology, and/or potency of the polypeptide; such as, for example only, increasing water solubility and bioavailability; increasing serum half-life; increase the half-life of treatment; modulating immunogenicity; modulating biological activity; or to extend the cycle time. In addition, these modifications include, by way of example only, providing the polypeptide with other functional groups; incorporating a tag, label or detectable signal into the polypeptide; facilitating the isolation properties of the polypeptide, and any combination of the foregoing modifications.
Also described herein are unnatural amino acids that have been modified or can be modified to contain an oxime, carbonyl, dicarbonyl, or hydroxylamine group. This aspect includes methods for preparing, purifying, characterizing, and using these unnatural amino acids. Another aspect described herein is methods, strategies, and techniques for incorporating at least one of the unnatural amino acids into polypeptides. This aspect also includes methods for preparing, purifying, characterizing, and using these polypeptides containing at least one of the unnatural amino acids. This aspect also includes compositions and methods for preparing, purifying, characterizing, and using oligonucleotides (including DNA and RNA) useful for preparing polypeptides that contain (at least in part) at least one unnatural amino acid. This aspect also includes compositions and methods for preparing, purifying, characterizing, and using cells that express such oligonucleotides that can be used to make polypeptides that contain at least one unnatural amino acid (at least in part).
Accordingly, provided and described herein are polypeptides comprising at least one unnatural amino acid or an unnatural amino acid modified with a carbonyl, dicarbonyl, oxime or hydroxylamine group. In certain embodiments, a polypeptide having at least one unnatural amino acid or unnatural amino acid modified with a carbonyl, dicarbonyl, oxime, or hydroxylamine group comprises at least one post-translational modification at some position on the polypeptide. In some embodiments, the co-translational or post-translational modifications occur through cellular mechanisms (e.g., glycosylation, acetylation, acylation, lipid-modification, palmitoylation, palmitate addition, phosphorylation, glycolipid-bond modification, and the like), and in many cases, these co-translational or post-translational modifications based on cellular mechanisms occur at naturally occurring amino acid sites on the polypeptide, however, in some embodiments, the co-translational or post-translational modifications based on cellular mechanisms occur at non-natural amino acid sites on the polypeptide.
In other embodiments, the post-translational modification does not utilize cellular mechanisms, but rather the molecules comprising the second reactive group (including, but not limited to, labels) by utilizing the chemical methods described herein, or other methods applicable to the particular reactive group; a dye; a polymer; water soluble polymers, derivatives of polyethylene glycol, photocrosslinkers, cytotoxic compounds, drugs, affinity labels, photoaffinity labels, reactive compounds, resins, second proteins or polypeptides or polypeptide analogues, antibodies or antibody fragments, metal chelators, cofactors, fatty acids, carbohydrates, polynucleotides, DNA, RNA, antisense polynucleotides, saccharides, water soluble dendrimers, cyclodextrins, biological materials, nanoparticles, spin labels, fluorophores, metal-containing moieties, radioactive moieties, novel functional groups, groups that interact covalently or non-covalently with other molecules, photocage moieties, actinic radiation-excited moieties, ligands, photoisomerizable moieties, biotin analogues, moieties bound to heavy atoms, chemically cleavable groups, photocleavable groups, elongated side chains, carbon-linked sugars, redox active agents, aminothioacids, toxic moieties, isotopically labeled moieties, biophotonic groups, chemiluminescent groups, electron-dense groups, magnetic groups, intercalating groups, transfer groups, fluorescent groups, nuclear transfer agents, radioactive radiation-excited moieties, ligands, photoisomerizable moieties, biotin analogues, heavy atom-bound moieties, chemically cleavable groups, extended side chains, carbon-linked sugars, redox active agents, redox agents, amino acids, fluorescent agents, nuclear transfer agents, nucleic acid-active agents, dye-transfer agents, dye-transfer agents, and dye-transfer agents, dye-transfer agents, dye, and dye, and dye-transfer, and dye, and dye, aglycone, allergen, angiostatin, anti-hormone, antioxidant, aptamer, guide RNA, saponin, shuttle vector, macromolecule, epitope, receptor, reverse micelle, and any combination thereof) to at least one unnatural amino acid comprising a first reactive group (including, but not limited to, an unnatural amino acid comprising a ketone, aldehyde, acetal, hemi-acetal, oxime, or hydroxylamine functional group). In certain embodiments, the co-translation or post-translational modification is performed in vivo in a eukaryotic organism or a non-eukaryotic organism. In certain embodiments, the post-translational modification is performed in vitro without using cellular mechanisms. This aspect also includes methods for preparing, purifying, characterizing, and using such polypeptides that contain at least one of such co-translated or post-translationally modified unnatural amino acids.
Also included within the scope of the methods, compositions, strategies, and techniques described herein are reagents that are capable of reacting with an unnatural amino acid (containing a carbonyl or dicarbonyl group, an oxime group, a hydroxylamine group, or a masked or protected form thereof) as part of a polypeptide to produce any of the foregoing post-translational modifications. In general, the resulting post-translationally modified unnatural amino acid should contain at least one oxime group; the resulting modified oxime-containing unnatural amino acid can be subjected to subsequent modification reactions. This aspect also includes methods for preparing, purifying, characterizing, and using the agents capable of performing any of the post-translational modifications of the unnatural amino acid.
In certain embodiments, the polypeptide comprises at least one co-translational or post-translational modification performed in vivo by one host cell, wherein the post-translational modification is not typically performed by another host cell type. In certain embodiments, the polypeptide comprises at least one co-translational or post-translational modification made by a eukaryotic organism in vivo, wherein the co-translational or post-translational modification is not typically made by a non-eukaryotic organism. Examples of such co-translational or post-translational modifications include, but are not limited to, glycosylation, acetylation, acylation, lipid-modification, palmitoylation, palmitate addition, phosphorylation, glycolipid-linkage modification, and the like. In one embodiment, co-translational or post-translational modification includes attachment of an oligosaccharide to an asparagine (including, but not limited to), by GlcNAc-asparagine linkage, where the oligosaccharide includes (GlcNAc-Man) 2 Man-GlcNAc, and the like). In another embodiment, co-translation or post-translational modification includes attachment of an oligosaccharide (including, but not limited to, gal-GalNAc, gal-GlcNAc, etc.) to serine or threonine by GalNAc-serine, galNAc-threonine, glcNAc-serine, or GlcNAc-threonine linkage. In certain embodiments, the protein or polypeptide may comprise a secretion or localization sequence, an epitope tag, a FLAG tag, a polyhistidine tag, a GST fusion, and/or an analog thereof. This aspect also includes methods for preparing, purifying, characterizing, and using these polypeptides that contain at least one such co-translational or post-translational modification. In other embodiments, the glycosylated unnatural amino acid polypeptide is prepared in a non-glycosylated form. Such non-glycosylated forms of glycosylated non-natural amino acids may be produced by a method comprising chemically or enzymatically removing oligosaccharide groups from an isolated or substantially purified or unpurified glycosylated non-natural amino acid polypeptide, or a combination of any of these methods; in non-glycosylationThe unnatural amino acid is produced in a host of the unnatural amino acid polypeptide, which host comprises a prokaryote or eukaryote engineered or mutated to not glycosylate the polypeptide, into which the unnatural amino acid polypeptide is introduced into a cell culture medium produced by a eukaryote that normally glycosylates the polypeptide. Also described herein are such non-glycosylated forms of a normally glycosylated non-natural amino acid polypeptide (normally glycosylated means a polypeptide that is glycosylated when the polypeptide is produced under conditions in which it is glycosylated). Of course, these non-glycosylated forms of normally glycosylated non-natural amino acid polypeptides (or indeed any of the polypeptides described herein) may be in an unpurified form, a substantially purified form, or an isolated form.
In certain embodiments, the unnatural amino acid polypeptide comprises at least one post-translational modification in the presence of the promoter, where the post-translational modification is stoichiometric, stoichiometrically-like, or near stoichiometric. In other embodiments, the polypeptide is contacted with an agent of formula (XIX) in the presence of a promoter. In other embodiments, the accelerator is selected from the group consisting of:
Figure BDA0001546710350000461
。
the polypeptide comprising an unnatural amino acid can comprise at least one, 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 ten or more unnatural amino acids that comprise a carbonyl or dicarbonyl group, an oxime group, a hydroxylamine group, or protected form thereof. The unnatural amino acids can be the same or different, e.g., there can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more than 20 different sites in the protein that comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more than 20 different unnatural amino acids. In certain embodiments, at least one (but less than all) of the particular amino acids present in the naturally occurring form of the protein are substituted with unnatural amino acids.
The methods and compositions provided and described herein comprise polypeptides comprising at least one unnatural amino acid that comprises a carbonyl or dicarbonyl group, an oxime group, a hydroxylamine group, or protected or masked forms thereof. The introduction of at least one unnatural amino acid into a polypeptide can allow for applications including conjugation chemistry that involve specific chemical reactions, including but not limited to reactions with one or more unnatural amino acids, but not with the 20 commonly found amino acids. Once incorporated, non-naturally occurring amino acid side chains can also be modified using chemical methods described herein or applicable to the particular functional groups or substituents present in the naturally encoded amino acid.
The unnatural amino acid methods and compositions described herein provide conjugates of materials having various functional groups, substituents, or moieties with other materials, including (but not limited to): marking; a dye; a polymer; a water-soluble polymer; derivatives of polyethylene glycol; a photocrosslinking agent; a cytotoxic compound; a drug; affinity labeling; a photoaffinity label; a reactive compound; a resin; a second protein or polypeptide analog; an antibody or antibody fragment; a metal chelator; a cofactor; a fatty acid; a carbohydrate; a polynucleotide; DNA; RNA; an antisense polynucleotide; saccharides, water-soluble dendrimers, cyclodextrins, biomaterials; a nanoparticle; spin labeling; a fluorophore; a metal-containing portion; a radioactive portion; novel functional groups; groups that interact covalently or non-covalently with other molecules; a light-shielding portion; an actinic radiation excitable moiety; a ligand; a photoisomerizable moiety; biotin; biotin analogues; a moiety incorporating a heavy atom; a chemically cleavable group; a photocleavable group; an extended side chain; a carbon-bonded sugar; a redox active agent; an aminothiopropionic acid; a toxic moiety; isotopically labeled moieties; a biophysical probe; phosphorescent groups; a chemiluminescent group; an electron dense group; a magnetic group; an insertion group; a chromophore; an energy transfer agent; a bioactive agent; a detectable label; a small molecule; inhibitory ribonucleic acid; a radionucleotide; a neutron capture agent; derivatives of biotin; a quantum dot; nano transitorin; a radiotransferrin; abzymes, activating complex activators, viruses, adjuvants, aglycones, allergens, angiostatin, anti-hormones, antioxidants, aptamers, guide RNAs, saponins, shuttle vectors, macromolecules, pseudoepitopes, receptors, reverse micelles, and any combination thereof.
In certain embodiments, the unnatural amino acids, unnatural amino acid polypeptides, linkers, and reagents described herein comprising compounds of formulas (I) - (XXXIII) are stable in aqueous solutions under moderately acidic conditions, including, but not limited to, pH 2-8. In other embodiments, these compounds are stable under moderately acidic conditions for at least one month. In other embodiments, these compounds are stable under moderately acidic conditions for at least 2 weeks. In other embodiments, these compounds are stable under moderately acidic conditions for at least 5 days.
Another aspect of the compositions, methods, techniques and strategies described herein is a method for studying or using any of the foregoing "modified or unmodified" unnatural amino acid polypeptides. Included in this aspect are, by way of example only, therapeutic, diagnostic, assay-based, industrial, cosmetic, plant biology, environmental, energy production, consumer products, and/or military uses that would benefit from polypeptides including "modified or unmodified" non-natural amino acid polypeptides or proteins.
III localization of unnatural amino acids in polypeptides
The methods and compositions described herein comprise incorporating one or more unnatural amino acids into polypeptides. One or more unnatural amino acids can be incorporated at one or more specific positions that do not disrupt the activity of the polypeptide. This may be accomplished by making "conservative" substitutions (including, but not limited to, substitution of hydrophobic amino acids with unnatural or natural hydrophobic amino acids, substitution of bulky amino acids with unnatural or natural bulky amino acids, substitution of hydrophilic amino acids with unnatural or natural hydrophilic amino acids) and/or inserting unnatural amino acids into positions where activity is not desired.
Various biochemical and structural methods can be used to select for desired sites within a polypeptide that are substituted with unnatural amino acids. Any position of the polypeptide chain is suitable for selection to incorporate unnatural amino acids, and can be selected for any or no particular desired purpose, either by rational design or by random selection. The selection of the desired site may be based on the creation of a polypeptide having any desired property or activity (including, but not limited to, an agonist, a super-agonist, a partial agonist, a reverse agonist, an antagonist, a receptor binding modulator, a receptor activity modulator, a modulator that binds to a binding partner, a modulator of binding partner activity, a modulator of binding partner conformation, dimer or multimer formation), a non-natural amino acid polypeptide that is inactive or altered in nature compared to the native molecule (which may be further modified or remain unmodified), or any physical or chemical property of the manipulated polypeptide, such as solubility, aggregation or stability, for example, a method that includes, but is not limited to, a point mutation assay, alanine scanning or homolog scanning method may be used to identify the position in the polypeptide where biological activity of the polypeptide is desired.
The structure and activity of naturally occurring mutants containing deleted polypeptides can also be studied to determine protein regions that may be susceptible to substitution with unnatural amino acids. Once residues that may allow substitution with unnatural amino acids are removed, methods involving, but not limited to, the three-dimensional structure of the relevant polypeptide and any associated ligands or binding proteins can be used to study the effect of the proposed substitution at each remaining position. In the protein database (Protein Data Bank) (PDB,www.rcsb.org) (a centralized database of three-dimensional structural data for macromolecules containing proteins as well as nucleic acids) many X-ray crystalline and NMR structures of polypeptides are available which can be used to identify amino acid positions that can be substituted with unnatural amino acids. In addition, if three-dimensional structural data is not available, a model can be made to study the secondary and tertiary structure of the polypeptide. Thus, the amino acid position itself, which can be substituted with an unnatural amino acid, can be readily obtained.
Exemplary sites for incorporation of unnatural amino acids include, but are not limited to, those that exclude potential receptor binding regions, or regions that bind to binding proteins or ligands may be fully or partially solvent exposed, have minimal or no hydrogen bond interactions with adjacent residues, may be minimally exposed to adjacent reactive residues, and/or may be in regions that are predicted to be highly flexible as by the three-dimensional crystal structure of the receptor, ligand, or binding protein with which a particular polypeptide is associated.
A variety of unnatural amino acids can be substituted or incorporated into a polypeptide at a given position. For example, particular unnatural amino acids for incorporation can be selected for investigation of the three-dimensional crystal structure of the polypeptide with its associated ligand, receptor, and/or binding protein, with conservative substitutions being preferred.
In one embodiment, the methods described herein comprise incorporating an unnatural amino acid into a polypeptide, where the unnatural amino acid comprises a first reactive group; and labeling the polypeptide with a molecule comprising a second reactive group (including but not limited to; a dye; a polymer; water soluble polymers, derivatives of polyethylene glycol, photocrosslinkers, cytotoxic compounds, drugs, affinity labels, photoaffinity labels, reactive compounds, resins, second proteins or polypeptides or polypeptide analogues, antibodies or antibody fragments, metal chelators, cofactors, fatty acids, carbohydrates, polynucleotides, DNA, RNA, antisense polynucleotides, saccharides, water soluble dendrimers, cyclodextrins, biological materials, nanoparticles, spin labels, fluorophores, metal-containing moieties, radioactive moieties, novel functional groups, groups that interact covalently or non-covalently with other molecules, photocage moieties, actinic radiation-excitable moieties, ligands, photoisomerizable moieties, biotin analogues, moieties bound to heavy atoms, chemically cleavable groups, photocleavable groups, elongated side chains, carbon-linked sugars, redox active agents, aminothioacids, toxic moieties, isotopically labeled moieties, biophysical probes, phosphorescent groups, chemiluminescent groups, electron-dense groups, magnetic groups, intercalating groups, fluorescent groups, metal-containing moieties, radioactive moieties, fluorescent moieties, dye transfer agents, fluorescent moieties, dye-transfer agents, dye-pair ligands, dye-transfer inhibitors, fluorescent moieties, dye-transfer inhibitors, fluorescent ligands, dye-transfer inhibitors, fluorescent ligands, dye-transfer-pair, dye pair, and dye pair, and dye pair, dye, and, receptors, reverse micelles, and any combination thereof). In certain embodiments, the first reactive group is a carbonyl or dicarbonyl moiety and the second reactive group is a hydroxylamine moiety, thereby forming an oxime bond. In certain embodiments, the first reactive group is a hydroxylamine moiety and the second reactive group is a carbonyl or dicarbonyl moiety, thereby forming an oxime bond. In certain embodiments, the first reactive group is a carbonyl or dicarbonyl moiety and the second reactive group is an oxime moiety, whereby an oxime exchange reaction occurs. In certain embodiments, the first reactive group is an oxime moiety and the second reactive group is a carbonyl or dicarbonyl moiety, whereby an oxime exchange reaction occurs.
In some cases, unnatural amino acid substitutions or incorporation will be combined with other additions, substitutions, or deletions within polypeptides to affect other chemical, physical, pharmacological, and/or biological properties. In some cases, other additions, substitutions, or deletions may increase the stability of the polypeptide (including, but not limited to, resistance to proteolytic degradation) or increase the affinity of the polypeptide for its appropriate receptor, ligand, and/or binding protein. In some cases, other additions, substitutions, or deletions may increase the solubility of the polypeptide (including, but not limited to, when expressed in E.coli or other host cells). In some embodiments, a site is selected for substitution with a naturally encoded or unnatural amino acid, and in addition, another site is selected for incorporation of the unnatural amino acid for the purpose of increasing the solubility of the polypeptide after expression in E.coli or other recombinant host cells. In some embodiments, the polypeptide includes another addition, substitution, or deletion that modulates affinity for an associated ligand, binding protein, and/or receptor, modulates (including but not limited to increasing or decreasing) receptor dimerization, stabilizes receptor dimers, modulates circulatory half-life, modulates release or bioavailability, facilitates purification, or modifies or alters a particular route of administration. Similarly, the unnatural amino acid polypeptide can comprise a detection (including but not limited to GFP), purification, delivery through tissue or cell membranes, prodrug release or activation, chemical or enzymatic cleavage sequences of reduced size or other properties, protease cleavage sequences, reactive groups, antibody binding domains (including but not limited to FLAG or poly-His), or other affinity-based sequences (including but not limited to FLAG, poly-His, GST, etc.), or a binding molecule (including but not limited to biotin).
Exemplary growth hormone supergene family
The methods, compositions, strategies, and techniques described herein are not limited to a particular polypeptide or protein type, class, or family. Indeed, virtually any polypeptide may be designed or modified to include at least one "modified or unmodified" unnatural amino acid described herein. By way of example only, the polypeptide may be homologous to a therapeutic protein selected from the group consisting of: alpha-1 antitrypsin, angiostatin, anti-hemolytic factor, antibody fragment, apolipoprotein, apoprotein, atrial natriuretic factor, atrial natriuretic polypeptide, atrial peptide, C-X-C chemokine, T39765, NAP-2, ENA-78, gro-a, gro-b, gro-C, IP-10, GCP-2, NAP-4, SDF-1, PF4, MIG, calcitonin, C-kit ligand, cytokine, CC chemokine, monocyte chemotactic protein-1, monocyte chemotactic protein-2, monocyte chemotactic protein-3, monocyte inflammatory protein-1 alpha, monocyte inflammatory protein-iβ, RANTES, 1309, R83915, R91733, HCC1, T58847, D31065, T64262, CD40 ligand, C-kit ligand collagen, colony Stimulating Factor (CSF), complement factor 5a, complement inhibitor, complement receptor 1, cytokine, epithelial neutrophil activating peptide-78, MIP-16, MCP-1, epidermal Growth Factor (EGF), epithelial neutrophil activating peptide, erythropoietin (EPO), epidermolysis toxin, factor IX, factor VII, factor VIII, factor X, fibroblast Growth Factor (FGF), fibrinogen, fibronectin, four-helix bundle protein, G-CSF, glp-1, GM-CSF, glucocerebrosidase, gonadotropin, growth factor receptor, grf, hedgehog protein, hemoglobin, hepatocyte growth factor (hGF), hirudin, human growth hormone (hGH), human serum albumin, ICAM-1 receptor, LFA-1 receptor, insulin-like growth factor (IGF), IGF-I, IGF-II, interferon (IFN), IFN-alpha, IFN-beta, IFN-gamma, interleukin (IL), IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, keratinocyte Growth Factor (KGF), lactoferrin, leukemia inhibitory factor, luciferase, neurogenin, neutrophil Inhibitory Factor (NIF), oncostatin M, osteoblast, oncogene product, paracillin, parathyroid hormone, PD-ECSF, PDGF, peptide hormones, pleiotrophin, protein A, protein G, pth, pyrogenic exotoxin A, the therapeutic agents include, but are not limited to, caloric exotoxin B, caloric exotoxin C, pyy, relaxin, renin, SCF, small biosynthetic protein, soluble complement receptor I, soluble I-CAM 1, soluble interleukin receptor, soluble TNF receptor, somatostatin, growth hormone (somatoropin), streptokinase, superantigen, staphylococcal enterotoxin, SEA, SEB, SEC1, SEC2, SEC3, SED, SEE, steroid hormone receptor, superoxide dismutase, toxic shock syndrome toxin, thymosin alpha 1, tissue plasminogen activator, tumor Growth Factor (TGF), tumor necrosis factor alpha, tumor necrosis factor beta, tumor Necrosis Factor Receptor (TNFR), VLA-4 protein, VCAM-1 protein, vascular Endothelial Growth Factor (VEGF), urokinase, mos, ras, raf, met, p, tat, fos, myc, jun, myb, rel, estrogen receptor, progesterone receptor, testosterone receptor, aldosterone receptor, LDL receptor, and corticosterone.
Thus, the following description of the Growth Hormone (GH) supergene family is provided for illustrative purposes and by way of example only, and is not intended as a limitation on the scope of the methods, compositions, strategies, and techniques described herein. Furthermore, references to GH polypeptides in this application are intended to use general terminology as an example of any member of the GH supergene family. Thus, it will be appreciated that the modifications and chemistries described herein with respect to GH polypeptides or proteins are equally applicable to any member of the GH supergene family, including those specifically listed herein.
The following proteins include those encoded by genes of the Growth Hormone (GH) super gene 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); silvennenoin, O. And Ihle, J.N., signalling by the Hematopoietic Cytokine Receptors (1996)): growth hormone, lactation hormone, placental lactogen, erythropoietin (EPO), thrombopoietin (TPO), interleukin-2 (IL-2), IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-10, IL-11, IL-12 (p 35 subunit), IL-13, IL-15, oncostatin M, ciliary neurotrophic factor, leukemia inhibitory factor, interferon-alpha, interferon-beta, interferon-epsilon, interferon-gamma, interferon omega, tau, granulocyte-colony stimulating factor (G-CSF), granulocyte-macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF), and cardiotrophin-1 (CT-1) ("GH supergene family"). Other members of this gene family are expected to be identified in the future by gene cloning and sequencing. Members of the GH supergene family have similar secondary and tertiary structures, although they generally have limited amino acid or DNA sequence identity. The common structural features allow new members of the gene family to be readily identified and similarly applied to the unnatural amino acid methods and compositions described herein.
Including G-CSF (Zink et al, FEBS Lett.314:435 (1992), zink et al, biochemistry 33:8453 (1994), hill et al, proc. Natl. Acad. Sci. USA 90:5167 (1993)), GM-CSF (Diederichs, K. Et al, science 154:1779-1782 (1991), walter et al, J.mol. Biol.224:1075-1085 (1992)), IL-2 (Bazan, J.F. and McKay, D.B., science 257:410-413 (1992)); the structure of many cytokines of IL-4 (Redfield et al, biochemistry 30:11029-11035 (1991); powers et al, science 256:1673-1677 (1992)) and IL-5 (Milburn et al, nature 363:172-176 (1993)) have been determined by X-ray diffraction and NMR studies and exhibit surprising conservation with GH structure, but lack significant primary sequence homology. IFN is considered to be a member of this family according to models and other studies (Lee et al, J. Interferon Cytokine Res.15:341 (1995); murgolo et al, proteins 17:62 (1993); radhakrishanan et al, structure 4:1453 (1996); klaus et al, J. Mol. Biol.274:661 (1997)). A number of other cytokines and growth factors including ciliary neurotrophic factor (CNTF), leukemia Inhibitory Factor (LIF), thrombopoietin (TPO), oncostatin M, macrophage colony stimulating factor (M-CSF), IL-3, IL-6, IL-7, IL-9, IL-12, IL-13, IL-15 and granulocyte colony stimulating factor (G-CSF) and IFN such as alpha, beta, omega, tau, epsilon and gamma interferon belong to this family (reviewed in Mott and Campbell, current Opinion in Structural Biology 5:114-121 (1995); silvennnoinen and Ihle (1996) Signalling by the Hematopoietic Cytokine Receptors). All of the above cytokines and growth factors are now considered to comprise a large gene family.
In addition to sharing similar secondary and tertiary structures, members of this family share the property that they must oligomerize cell surface receptors to activate intracellular signal transduction pathways. Some GH family members, including but not limited to GH and EPO, bind to and form homodimers with a single type of receptor. Other family members, including but not limited to IL-2, IL4, and IL-6, bind to more than one type of receptor and cause these receptors to form heterodimers or higher order aggregates (Davis et al, (1993) Science 260:1805-1808; paonessa et al, (1995) EMBO J.14:1942-1951; mott and Campbell, current Opinion in Structural Biology 5:114-121 (1995)). Mutagenesis studies have shown that these other cytokines and growth factors, like GH, contain multiple receptor binding sites (usually two) and bind in turn to their cognate receptors (Mott and Campbell, current Opinion in Structural Biology 5:114-121 (1995); matthews et al, (1996) Proc. Natl. Acad. Sci. USA 93:9471-9476). Like GH, the primary receptor binding sites of these other family members are found predominantly in the four alpha helices and the a-B loop. The specific amino acids in the helix bundle that are involved in receptor binding differ among family members. Most cell surface receptors that interact with members of the GH supergene family are structurally related and constitute the second largest polygene family. See, for example, U.S. patent No. 6,608,183, which is incorporated by reference herein in its entirety.
The general conclusion from mutational studies of various GH supergene family members is that loops linking the alpha helices generally tend not to participate in receptor binding. In particular, a short B-C ring appears to be unnecessary for receptor binding in most, if not all, family members. For this reason, in members of the GH supergene family, the B-C loop may be substituted with unnatural amino acids as described herein. The A-B ring, C-D ring (and the D-E ring of the IL-10 like member of the interferon/GH superfamily) may also be substituted with unnatural amino acids. Amino acids closest to helix a and further from the last helix also tend not to participate in receptor binding and may also be sites for introducing unnatural amino acids. In some embodiments, the unnatural amino acid is substituted at any position within the ring structure that comprises, but is not limited to, the first 1, 2, 3, 4, 5, 6, 7, or more than 7 amino acids of the A-B, B-C, C-D or D-E ring. In some embodiments, the unnatural amino acid is substituted within the last 1, 2, 3, 4, 5, 6, 7, or more than 7 amino acids of the A-B, B-C, C-D or D-E loop.
Certain members of the GH family, including but not limited to EPO, IL-2, IL-3, IL-4, IL-6, IFN, GM-CSF, TPO, IL-10, IL-12p35, IL-13, IL-15, and interferon-beta, contain N-linked and/or O-linked saccharides. Glycosylation sites in proteins are almost entirely present in the loop region and not in the alpha helix bundle. Because the loop region is generally not involved in receptor binding and because it is a site for covalent attachment of a sugar group, it may be a suitable site for introducing unnatural amino acid substitutions into proteins. Amino acids in proteins that include N-linked glycosylation sites as well as O-linked glycosylation sites can be sites for unnatural amino acid substitution, as these amino acids are surface exposed. Thus, native proteins may allow for attachment to bulky sugar groups on the protein at these sites and the glycosylation sites tend to be located away from the receptor binding site.
It is possible to find other members of the GH gene family in the future. Novel members of the GH supergene family can be identified by computer-aided secondary and tertiary structural analysis of predicted protein sequences, and by selection techniques designed to identify molecules that bind to a particular target. Members of the GH supergene family typically have four or five helices of amphipathic molecules joined by non-helical amino acids (loop regions). The protein may contain a hydrophobic signal sequence at its N-terminus to facilitate secretion from the cell. These later discovered GH supergene family members are also encompassed in the methods and compositions described herein.
V. unnatural amino acids
The unnatural amino acids used in the methods and compositions described herein have at least one of four properties: (1) The functional group on the side chain of at least one unnatural amino acid has at least one feature and/or activity and/or reactivity that is orthogonal to the chemical reactivity of 20 common genetically encoded amino acids (i.e., alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine), or at least orthogonal to the chemical reactivity of naturally occurring amino acids present in polypeptides comprising unnatural amino acids; (2) The introduced unnatural amino acid is substantially chemically inert to the 20 common genetically encoded amino acids; (3) Unnatural amino acids can be stably incorporated into polypeptides, preferably with stability comparable to naturally occurring amino acids or under typical physiological conditions, and further preferably, the incorporation can occur through in vivo systems; and (4) the unnatural amino acid comprises an oxime functional group or a functional group that can be converted to an oxime group by reaction with a reagent, preferably under conditions that do not disrupt the biological properties of the polypeptide comprising the unnatural amino acid (unless of course the disruption of such biological properties is for modification/conversion purposes), or where the conversion can occur under aqueous conditions at a pH of between about 4 and about 8, or where the reactive site on the unnatural amino acid is an electrophilic site. Illustrative, non-limiting examples of amino acids satisfying the four properties for unnatural amino acids that can be used in the compositions and methods described herein are presented in fig. 2, 3, 35, and 40-43. Any number of unnatural amino acids can be introduced into a polypeptide. The unnatural amino acid can also comprise a protected or masked oxime or a protected or masked group that can be converted to an oxime group after the protecting group is deprotected or the masking group is unmasked. Unnatural amino acids can also include protected or masked carbonyl or dicarbonyl groups, which can be converted to carbonyl or dicarbonyl groups after deprotection of the protecting group or unmasking of the masking group and can in turn be used to react with hydroxylamine or oxime to form an oxime group.
Unnatural amino acids useful in the methods and compositions described herein include, but are not limited to, amino acids including photoactivatable crosslinkers, spin-labeled amino acids, fluorescent amino acids, metal-binding amino acids, metal-containing amino acids, radioactive amino acids, amino acids with novel functional groups, amino acids that interact covalently or non-covalently with other molecules, photocaged and/or photoisomerizable amino acids, amino acids including biotin or biotin analogs, glycosylated amino acids (such as sugar-substituted serine), other carbohydrate-modified amino acids, keto-containing amino acids, aldehyde-containing amino acids, amino acids including polyethylene glycol or other polyethers, heavy atom-substituted amino acids, chemically cleavable and/or photocleavable amino acids, amino acids with side chains that are elongated as compared to natural amino acids (including, but not limited to, polyethers or long chain hydrocarbons that include, but are not limited to, greater than about 5 carbons or greater than about 10 carbons), carbon-linked sugar-containing amino acids, redox-active amino acids, amino-sulfur-containing amino acids, and amino acids that include one or more toxic moieties.
In some embodiments, the unnatural amino acid comprises a saccharide moiety. Examples of such amino acids include N-acetyl-L-glucosamine-L-serine, N-acetyl-L-galactosamine-L-serine, N-acetyl-L-glucosamine-L-threonine, N-acetyl-L-glucosamine-L-asparagine, and O-mannosamine-L-serine. Examples of these amino acids also include examples in which the naturally occurring N-bond or O-bond between the amino acid and the sugar is replaced by a covalent bond that does not normally exist in nature, including but not limited to olefins, oximes, thioethers, amides, and the like. Examples of these amino acids also include saccharides that are not normally present in naturally occurring proteins, such as 2-deoxy-glucose, 2-deoxy galactose, and the like.
The incorporation of chemical moieties into polypeptides by incorporating unnatural amino acids into such polypeptides provides a variety of advantages and manipulation of the polypeptides. For example, the unique reactivity of carbonyl or dicarbonyl functional groups (including ketone functional groups or aldehyde functional groups) allows for selective modification of proteins with any of a number of hydrazine-containing or hydroxylamine-containing reagents, both in vivo and in vitro. Heavy atom unnatural amino acids, for example, can be useful for phasing X-ray structural data. The use of unnatural amino acid site-specific introduction of heavy atoms also provides selectivity and flexibility in selecting the position of the heavy atoms. Photoreactive unnatural amino acids, including but not limited to amino acids having benzophenone and aryl azide (including but not limited to phenyl azide) side chains, for example, render photocrosslinking of polypeptides effective in vivo and in vitro. Examples of photoreactive unnatural amino acids include, but are not limited to, para-azido-phenylalanine and para-benzoyl-phenylalanine. The polypeptide with the photoreactive unnatural amino acid can then be optionally crosslinked by excitation of the photoreactive group (providing transient control). In a non-limiting example, methyl groups of unnatural amino acids can be isotopically substituted (including, but not limited to, methyl substitution), as probes for local structure and dynamics (including, but not limited to, by means of nuclear magnetic resonance and vibrational spectroscopy).
A. Unnatural amino acid: carbonyl, carbonyl-like, masked carbonyl, and protected carbonyl structures and synthesis
Amino acids having electrophilic reactive groups allow for a variety of reactions to bind molecules through a variety of chemical reactions, including, but not limited to, nucleophilic addition reactions. These electrophilic reactive groups include carbonyl or dicarbonyl (including keto or aldehyde groups), carbonyl-like or dicarbonyl-like groups (which have a reactivity similar to and are similar in structure to carbonyl or dicarbonyl groups), masked carbonyl or masked dicarbonyl groups (which can be readily converted to carbonyl or dicarbonyl groups), or protected carbonyl or protected dicarbonyl groups (which have a reactivity similar to carbonyl or dicarbonyl groups after deprotection). These amino acids comprise amino acids having the structure of formula (I):
Figure BDA0001546710350000551
wherein:
a is optional and when present is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkyl, substituted lower heterocycloalkyl, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene;
B is optional and when present is a linker selected from the group consisting of: lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O- (alkylene or substituted alkylene) -, -S- (alkylene or substituted alkylene) -, -S (O) k - (wherein k is 1, 2 or 3), -S (O) k (alkylene or substituted alkylene) -, -C (O) -, -NS (O) 2 -、-OS(O) 2 -C (O) - (alkylene or substituted alkylene) -, -C (S) - (alkylene or substituted alkylene) -, -N (R '), -NR' - (alkylene or substituted alkylene) -, -C (O) N (R '), -CON (R') - (alkylene or substituted alkylene) -, -CSN (R ')') -, -CSN (R ') - (alkylene or substituted alkylene) -, -N (R ') CO- (alkylene or substituted alkylene) -, -N (R ') C (O) O-, -S (O) k N(R')-、-N(R')C(O)N(R')-、-N(R')C(S)N(R')-、-N(R')S(O) k N(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 ') -, wherein each R' is independently H, alkyl or substituted alkyl;
j is
Figure BDA0001546710350000552
R is H, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl;
each R "is independently H, alkyl, substituted alkyl, or a protecting group, or when more than one R" group is present, two R "optionally form a heterocycloalkyl;
R 1 Is H, an amino protecting group, a resin, an amino acid, a polypeptide or a polynucleotide; and is also provided with
R 2 Is OH, an ester protecting group, a resin, an amino acid, a polypeptide or a polynucleotide;
R 3 and R is R 4 Each independently is H, halogen, lower alkyl or substituted lower alkyl, or R 3 And R is R 4 Or two R 3 The radicals optionally form cycloalkyl or heterocycloalkyl;
or-a-B-J-R groups together form a bicyclic or tricyclic cycloalkyl or heterocycloalkyl group including at least one carbonyl (including dicarbonyl), protected carbonyl (including protected dicarbonyl), or masked carbonyl (including masked dicarbonyl);
or-J-R groups together form a mono-or bi-cyclic cycloalkyl or heterocycloalkyl group comprising at least one carbonyl group (including dicarbonyl), protected carbonyl group (including protected dicarbonyl), or masked carbonyl group (including masked dicarbonyl);
with the proviso that when A is phenylene and R 3 When each is H, B exists; and when A is- (CH) 2 ) 4 -and R 3 When each is H, B is not-NHC (O) (CH 2 CH 2 ) -; and when A and BIs absent and R 3 When each is H, R is not methyl. These unnatural amino acids can be in the form of salts, or can be incorporated into unnatural amino acid polypeptides, polymers, polysaccharides, or polynucleotides and optionally post-translationally modified.
In certain embodiments, the compound of formula (I) is stable in aqueous solution under moderately acidic conditions for at least 1 month. In certain embodiments, the compound of formula (I) is stable under moderately acidic conditions for at least 2 weeks. In certain embodiments, the compound of formula (I) is stable under moderately acidic conditions for at least 5 days. In certain embodiments, the acidic condition is a pH of 2 to 8.
In certain embodiments of the compounds of formula (I), B is lower alkylene, substituted lower alkylene, -O- (alkylene or substituted alkylene) -, -C (R ') =n-N (R ') -, -N (R ') CO-, -C (O) -, -C (R ') =n-, -C (O) - (alkylene or substituted alkylene) -, -CON (R ') - (alkylene or substituted alkylene) -, -S (O) (alkylene or substituted alkylene) -or-S (O) 2 (alkylene or substituted alkylene) -. In certain embodiments of the compounds of formula (I), B is-O (CH) 2 )-、-CH＝N-、-CH＝N-NH-、-NHCH 2 -、-NHCO-、-C(O)-、-C(O)-(CH 2 )-、-CONH-(CH 2 )-、-SCH 2 -、-S(＝O)CH 2 -or-S (O) 2 CH 2 -. In certain embodiments of the compounds of formula (I), R is C 1-6 Alkyl or cycloalkyl. In certain embodiments of the compounds of formula (I), R is-CH 3 、-CH(CH 3 ) 2 Or cyclopropyl. In certain embodiments of the compounds of formula (I), R 1 Is H, t-butoxycarbonyl (Boc), 9-fluorenylmethoxycarbonyl (Fmoc), N-acetyl, tetrafluoroacetyl (TFA) or benzyloxycarbonyl (Cbz). In certain embodiments of the compounds of formula (I), R 1 Is a resin, amino acid, polypeptide or polynucleotide. In certain embodiments of the compounds of formula (I), R 2 Is OH, O-methyl, O-ethyl or O-tert-butyl. In certain embodiments of the compounds of formula (I), R 2 Is a resin, amino acid, polypeptide or polynucleotide. In certain embodiments of the compounds of formula (I), R 2 Is a polynucleotide. In certain embodiments of the compounds of formula (I)In the example, R 2 Is ribonucleic acid (RNA). In certain embodiments of the compounds of formula (I), R 2 Is tRNA. In certain embodiments of the compounds of formula (I), the tRNA specifically recognizes a selector codon. In certain embodiments of the compounds of formula (I), the selector codon is selected from the group consisting of an amber codon, an ocher codon, an opal codon, a unique codon, a rare codon, a non-natural codon, a five base codon, and a four base codon. In certain embodiments of the compounds of formula (I), R 2 To suppress tRNA.
In certain embodiments of the compounds of formula (I),
Figure BDA0001546710350000571
is selected from the group consisting of:
(i) A is a substituted lower alkylene group, C 4 -arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene or substituted aralkylene;
B is optional and when present is a divalent linker selected from the group consisting of: lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, -O- (alkylene or substituted alkylene) -, -S (O) 2 -、-NS(O) 2 -、-OS(O) 2 -C (O) -, -C (O) - (alkylene or substituted alkylene) -, -C (S) -, -N (R '), -C (O) N (R'), -CON (R ') - (alkylene or substituted alkylene) -, -CSN (R'), -N (R ') CO- (alkylene or substituted alkylene) -, -N (R') C (O) O-, -N (R ') C (S) -, -S (O) N (R'), -S (O) 2 N(R')、-N(R')C(O)N(R')-、-N(R')C(S)N(R')-、-N(R')S(O)N(R')-、-N(R')S(O) 2 N(R')-、-N(R')-N＝、-C(R')＝N-N(R')-、-C(R')＝N-N＝、-C(R') 2 -n=n-and-C (R') 2 -N(R')-N(R')-；
(ii) A is optional and when present is a substituted lower alkylene, C 4 -arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene or substituted aralkyleneAn alkyl group;
b is a divalent linker selected from the group consisting of: lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, -O- (alkylene or substituted alkylene) -, -S (O) 2 -、-NS(O) 2 -、-OS(O) 2 -C (O) -, -C (O) - (alkylene or substituted alkylene) -, -C (S) -, -N (R '), -C (O) N (R'), -CON (R ') - (alkylene or substituted alkylene) -, -CSN (R'), -N (R ') CO- (alkylene or substituted alkylene) -, -N (R') C (O) O-, -N (R ') C (S) -, -S (O) N (R'), -S (O) 2 N(R')、-N(R')C(O)N(R')-、-N(R')C(S)N(R')-、-N(R')S(O)N(R')-、-N(R')S(O) 2 N(R')-、-N(R')-N＝、-C(R')＝N-N(R')-、-C(R')＝N-N＝、-C(R') 2 -n=n-and-C (R') 2 -N(R')-N(R')-；
(iii) A is lower alkylene;
b is optional and when present is a divalent linker selected from the group consisting of: lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, -O- (alkylene or substituted alkylene) -, -S (O) 2 -、-NS(O) 2 -、-OS(O) 2 -C (O) -, -C (O) - (alkylene or substituted alkylene) -, -C (S) -, -N (R '), -C (O) N (R '), -CSN (R '), -CON (R ') - (alkylene or substituted alkylene) -, -N (R ') C (O) O-, -N (R ') C (S) -, -S (O) N (R '), -S (O) 2 N(R')、-N(R')C(O)N(R')-、-N(R')C(S)N(R')-、-N(R')S(O)N(R')-、-N(R')S(O) 2 N(R')-、-N(R')-N＝、-C(R')＝N-N(R')-、-C(R')＝N-N＝、-C(R') 2 -n=n-and-C (R') 2 -N (R') -; and
(iv) A is phenylene;
b is a divalent linker selected from the group consisting of: lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, -O- (alkylene or substituted alkylene) -, -S (O) 2 -、-NS(O) 2 -、-OS(O) 2 -C (O) -, -C (O) - (alkylene or substituted alkylene) -, -C (S) -, -N (R') -,-C (O) N (R '), -CON (R ') - (alkylene or substituted alkylene) -, -CSN (R '), -N (R ') CO- (alkylene or substituted alkylene) -, -N (R ') C (O) O-, -N (R ') C (S) -, -S (O) N (R '), -S (O) 2 N(R')、-N(R')C(O)N(R')-、-N(R')C(S)N(R')、-N(R')S(O)N(R')-、-N(R')S(O) 2 N(R')-、-N(R')-N＝、-C(R')＝N-N(R')-、-C(R')＝N-N＝、-C(R') 2 -n=n-and-C (R') 2 -N(R')-N(R')-；
J is
Figure BDA0001546710350000581
Each R' is independently H, alkyl or substituted alkyl;
R 1 is optional and when present is H, an amino protecting group, a resin, an amino acid, a polypeptide or a polynucleotide; and is also provided with
R 2 Is optional and when present is an OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; and is also provided with
R 3 And R is R 4 Each independently is H, halogen, lower alkyl or substituted lower alkyl;
r is H, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl;
in addition, an amino acid comprising a structure having formula (II):
Figure BDA0001546710350000582
wherein:
a is optional and when present is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkyl, substituted lower heterocycloalkyl, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene;
b is optional and when presentAnd at the time a linker selected from the group consisting of: lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O- (alkylene or substituted alkylene) -, -S- (alkylene or substituted alkylene) -, -S (O) k - (wherein k is 1, 2 or 3), -S (O) k (alkylene or substituted alkylene) -, -C (O) -, -NS (O) 2 -、-OS(O) 2 -C (O) - (alkylene or substituted alkylene) -, -C (S) - (alkylene or substituted alkylene) -, -N (R '), -NR ' - (alkylene or substituted alkylene) -, -C (O) N (R '), -CON (R ') - (alkylene or substituted alkylene) -, -CSN (R ') - (alkylene or substituted alkylene) -, -N (R ') CO- (alkylene or substituted alkylene) -, -N (R ') C (O) O-, -S (O) k N(R')-、-N(R')C(O)N(R')-、-N(R')C(S)N(R')-、-N(R')S(O) k N(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 ') -, wherein each R' is independently H, alkyl or substituted alkyl;
r is H, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl;
R 1 is H, an amino protecting group, a resin, an amino acid, a polypeptide or a polynucleotide; and is also provided with
R 2 Is OH, an ester protecting group, a resin, an amino acid, a polypeptide or a polynucleotide;
with the proviso that when A is phenylene, B is present; and when A is- (CH) 2 ) 4 When B is not-NHC (O) (CH 2 CH 2 ) -; and when A and B are absent, R is not methyl. These unnatural amino acids can be in the form of salts, or can be incorporated into unnatural amino acid polypeptides, polymers, polysaccharides, or polynucleotides and optionally post-translationally modified.
In addition, an amino acid comprising a structure having formula (III):
Figure BDA0001546710350000591
Wherein:
b is a linker selected from the group consisting of: lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O- (alkylene or substituted alkylene) -, -S- (alkylene or substituted alkylene) -, -S (O) k - (wherein k is 1, 2 or 3), -S (O) k (alkylene or substituted alkylene) -, -C (O) -, -NS (O) 2 -、-OS(O) 2 -C (O) - (alkylene or substituted alkylene) -, -C (S) - (alkylene or substituted alkylene) -, -N (R '), -NR ' - (alkylene or substituted alkylene) -, -C (O) N (R '), -CON (R ') - (alkylene or substituted alkylene) -, -CSN (R ') - (alkylene or substituted alkylene) -, -N (R ') CO- (alkylene or substituted alkylene) -, -N (R ') C (O) O-, -S (O) k N(R')-、-N(R')C(O)N(R')-、-N(R')C(S)N(R')-、-N(R')S(O) k N(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 ') -, wherein each R' is independently H, alkyl or substituted alkyl;
r is H, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl;
R 1 is H, an amino protecting group, a resin, an amino acid, a polypeptide or a polynucleotide; and is also provided with
R 2 Is OH, an ester protecting group, a resin, an amino acid, a polypeptide or a polynucleotide;
R a each independently selected from the group consisting of: H. halogen, alkyl, substituted alkyl, -N (R') 2 、-C(O) k R '(wherein k is 1, 2 or 3), -C (O) N (R') 2 -OR' and-S (O) k R ', wherein R' are each independently H, alkyl or substituted alkyl. These unnatural amino acids can be in the form of salts, or can be incorporated into unnatural amino acid polypeptides, polymers, polysaccharides, or polynucleotides and optionally post-translationally modified.
In addition, the following amino acids are included:
Figure BDA0001546710350000601
these unnatural amino acids can be in the form of amino protected groups, carboxyl protected groups, and/or salts, as appropriate, or can be incorporated into unnatural amino acid polypeptides, polymers, polysaccharides, or polynucleotides, and as appropriate post-translationally modified.
In addition, an amino acid having the structure of formula (IV) below is included:
Figure BDA0001546710350000602
wherein the method comprises the steps of
-NS(O) 2 -、-OS(O) 2 -optionally, and when present, a linker selected from the group consisting of: lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O- (alkylene or substituted alkylene) -, -S- (alkylene or substituted alkylene) -, -S (O) k - (wherein k is 1, 2 or 3), -S (O) k (alkylene or substituted alkylene) -, -C (O) - (alkylene or substituted alkylene) -, -C (S) - (alkylene or substituted alkylene) -, -N (R ') -, -NR ' - (alkylene or substituted alkylene) -, -C (O) N (R ') -, CON (R ') - (alkylene or substituted alkylene) -, -CSN (R ') - (alkylene or substituted alkylene) -, -N (R ') CO- (alkylene or substituted alkylene) -, -N (R ') C (O) O-, -S (O) k N(R')-、-N(R')C(O)N(R')-、-N(R')C(S)N(R')-、-N(R')S(O) k N(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 ') -, wherein each R' is independently H, alkyl or substituted alkyl;
r is H, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl;
R 1 is H, amino protecting group, resin, amino acid, polypeptide or polynuclearA glycoside acid; and is also provided with
R 2 Is OH, an ester protecting group, a resin, an amino acid, a polypeptide or a polynucleotide;
R a each independently selected from the group consisting of: H. halogen, alkyl, substituted alkyl, -N (R') 2 、-C(O) k R '(wherein k is 1, 2 or 3), -C (O) N (R') 2 -OR' and-S (O) k R ', wherein R' are each independently H, alkyl, or substituted alkyl; and n is 0 to 8;
with the proviso that when A is- (CH) 2 ) 4 When B is not-NHC (O) (CH 2 CH 2 ) -. These unnatural amino acids can be in the form of salts, or can be incorporated into unnatural amino acid polypeptides, polymers, polysaccharides, or polynucleotides and optionally post-translationally modified.
In addition, the following amino acids are included:
Figure BDA0001546710350000611
wherein these compounds are optionally amino protected, optionally carboxyl protected, optionally amino protected and carboxyl protected, or salts thereof, or can be incorporated into non-natural amino acid polypeptides, polymers, polysaccharides or polynucleotides and optionally post-translationally modified.
In addition, an amino acid having the structure of formula (VIII) below is included:
Figure BDA0001546710350000621
wherein, the liquid crystal display device comprises a liquid crystal display device,
a is optional and when present is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkyl, substituted lower heterocycloalkyl, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene;
b is optional and when present is a linker selected from the group consisting of: lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O- (alkylene or substituted alkylene) -, -S- (alkylene or substituted alkylene) -, -S (O) k - (wherein k is 1, 2 or 3), -S (O) k (alkylene or substituted alkylene) -, -C (O) -, -NS (O) 2 -、-OS(O) 2 -C (O) - (alkylene or substituted alkylene) -, -C (S) - (alkylene or substituted alkylene) -, -N (R '), -NR ' - (alkylene or substituted alkylene) -, -C (O) N (R '), -CON (R ') - (alkylene or substituted alkylene) -, -CSN (R ') - (alkylene or substituted alkylene) -, -N (R ') CO- (alkylene or substituted alkylene) -, -N (R ') C (O) O-, -S (O) k N(R')-、-N(R')C(O)N(R')-、-N(R')C(S)N(R')-、-N(R')S(O) k N(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 ') -, wherein each R' is independently H, alkyl or substituted alkyl;
R 1 is H, an amino protecting group, a resin, an amino acid, a polypeptide or a polynucleotide; and is also provided with
R 2 Is OH, an ester protecting group, a resin, an amino acid, a polypeptide or a polynucleotide.
These unnatural amino acids can be in the form of salts, or can be incorporated into unnatural amino acid polypeptides, polymers, polysaccharides, or polynucleotides and optionally post-translationally modified.
In addition, an amino acid having the structure of formula (IX) below is included:
Figure BDA0001546710350000631
wherein, the liquid crystal display device comprises a liquid crystal display device,
b is optional and when present is a linker selected from the group consisting of:lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O- (alkylene or substituted alkylene) -, -S- (alkylene or substituted alkylene) -, -S (O) k - (wherein k is 1, 2 or 3), -S (O) k (alkylene or substituted alkylene) -, -C (O) -, -NS (O) 2 -、-OS(C)) 2 -C (O) - (alkylene or substituted alkylene) -, -C (S) - (alkylene or substituted alkylene) -, -N (R '), -NR ' - (alkylene or substituted alkylene) -, -C (O) N (R '), -CON (R ') - (alkylene or substituted alkylene) -, -CSN (R ') - (alkylene or substituted alkylene) -, -N (R ') CO- (alkylene or substituted alkylene) -, -N (R ') C (O) O-, -S (O) k N(R')-、-N(R')C(O)N(R')-、-N(R')C(S)N(R')-、-N(R')S(O) k N(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 ') -, wherein each R' is independently H, alkyl or substituted alkyl;
r is H, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl;
R 1 is H, an amino protecting group, a resin, an amino acid, a polypeptide or a polynucleotide; and is also provided with
R 2 Is OH, an ester protecting group, a resin, an amino acid, a polypeptide or a polynucleotide;
wherein R is a Each independently selected from the group consisting of: H. halogen, alkyl, substituted alkyl, -N (R') 2 、-C(O) k R '(wherein k is 1, 2 or 3), -C (O) N (R') 2 -OR' and-S (O) k R ', wherein R' are each independently H, alkyl or substituted alkyl.
These unnatural amino acids can be in the form of salts, or can be incorporated into unnatural amino acid polypeptides, polymers, polysaccharides, or polynucleotides and optionally post-translationally modified.
In addition, the following amino acids are included:
Figure BDA0001546710350000641
wherein these compounds are optionally amino protected, optionally carboxyl protected, optionally amino protected and carboxyl protected, or salts thereof, or can be incorporated into non-natural amino acid polypeptides, polymers, polysaccharides or polynucleotides and optionally post-translationally modified.
In addition, an amino acid having the structure of formula (X) below is included:
Figure BDA0001546710350000642
Wherein, the liquid crystal display device comprises a liquid crystal display device,
b is optional and when present is a linker selected from the group consisting of: lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O- (alkylene or substituted alkylene) -, -S- (alkylene or substituted alkylene) -, -S (O) k - (wherein k is 1, 2 or 3), -S (O) k (alkylene or substituted alkylene) -, -C (O) -, -NS (O) 2 -、-OS(O) 2 -C (O) - (alkylene or substituted alkylene) -, -C (S) - (alkylene or substituted alkylene) -, -N (R '), -NR ' - (alkylene or substituted alkylene) -, -C (O) N (R '), -CON (R ') - (alkylene or substituted alkylene) -, -CSN (R ') - (alkylene or substituted alkylene) -, -N (R ') CO- (alkylene or substituted alkylene) -, -N (R ') C (O) O-, -S (O) k N(R')-、-N(R')C(O)N(R')-、-N(R')C(S)N(R')-、-N(R')S(O) k N(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 ') -, wherein each R' is independently H, alkyl or substituted alkyl;
r is H, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl;
R 1 is H, an amino protecting group, a resin, an amino acid, a polypeptide or a polynucleotide; and is also provided with
R 2 Is OHAn ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide;
R a Each independently selected from the group consisting of: H. halogen, alkyl, substituted alkyl, -N (R') 2 、-C(O) k R '(wherein k is 1, 2 or 3), -C (O) N (R') 2 -OR' and-S (O) k R ', wherein R' are each independently H, alkyl, or substituted alkyl; and n is 0 to 8.
These unnatural amino acids can be in the form of salts, or can be incorporated into unnatural amino acid polypeptides, polymers, polysaccharides, or polynucleotides and optionally post-translationally modified.
In addition, the following amino acids are included:
Figure BDA0001546710350000651
wherein these compounds are optionally amino protected, optionally carboxyl protected, optionally amino protected and carboxyl protected, or salts thereof, or can be incorporated into non-natural amino acid polypeptides, polymers, polysaccharides or polynucleotides and optionally post-translationally modified.
In addition to the monocarbonyl structure, the unnatural amino acids described herein can include groups such as dicarbonyl, dicarbonyl-like, masked dicarbonyl, and protected dicarbonyl.
For example, an amino acid comprising the following structure having formula (V):
Figure BDA0001546710350000652
wherein, the liquid crystal display device comprises a liquid crystal display device,
a is optional and when present is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkyl, substituted lower heterocycloalkyl, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene;
B is optional and when present is a linker selected from the group consisting of: lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O- (alkylene or substituted alkylene) -, -S- (alkylene or substituted alkylene) -, -S (O) k - (wherein k is 1, 2 or 3), -S (O) k (alkylene or substituted alkylene) -, -C (O) -, -NS (O) 2 -、-OS(O) 2 -C (O) - (alkylene or substituted alkylene) -, -C (S) - (alkylene or substituted alkylene) -, -N (R '), -NR ' - (alkylene or substituted alkylene) -, -C (O) N (R '), -CON (R ') - (alkylene or substituted alkylene) -, -CSN (R ') - (alkylene or substituted alkylene) -, -N (R ') CO- (alkylene or substituted alkylene) -, -N (R ') C (O) O-, -S (O) k N(R')-、-N(R')C(O)N(R')-、-N(R')C(S)N(R')-、-N(R')S(O) k N(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 ') -, wherein each R' is independently H, alkyl or substituted alkyl;
r is H, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl;
R 1 is H, an amino protecting group, a resin, an amino acid, a polypeptide or a polynucleotide; and is also provided with
R 2 Is OH, an ester protecting group, a resin, an amino acid, a polypeptide or a polynucleotide.
These unnatural amino acids can be in the form of salts, or can be incorporated into unnatural amino acid polypeptides, polymers, polysaccharides, or polynucleotides and optionally post-translationally modified.
In addition, an amino acid having the structure of formula (VI) below is included:
Figure BDA0001546710350000661
wherein, the liquid crystal display device comprises a liquid crystal display device,
b is optional and when present is a linker selected from the group consisting of: lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O- (alkylene or substituted alkylene) -, -S- (alkylene or substituted alkylene) -, -S (O) k - (wherein k is 1, 2 or 3), -S (O) k (alkylene or substituted alkylene) -, -C (O) -, -NS (O) 2 -、-OS(O) 2 -C (O) - (alkylene or substituted alkylene) -, -C (S) - (alkylene or substituted alkylene) -, -N (R '), -NR ' - (alkylene or substituted alkylene) -, -C (O) N (R '), -CON (R ') - (alkylene or substituted alkylene) -, -CSN (R ') - (alkylene or substituted alkylene) -, -N (R ') CO- (alkylene or substituted alkylene) -, -N (R ') C (O) O-, -S (O) k N(R')-、-N(R')C(O)N(R')-、-N(R')C(S)N(R')-、-N(R')S(O) k N(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 ') -, wherein each R' is independently H, alkyl or substituted alkyl;
R is H, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl;
R 1 is H, an amino protecting group, a resin, an amino acid, a polypeptide or a polynucleotide; and is also provided with
R 2 Is OH, an ester protecting group, a resin, an amino acid, a polypeptide or a polynucleotide;
wherein R is a Each independently selected from the group consisting of: H. halogen, alkyl, substituted alkyl, -N (R') 2 、-C(O) k R '(wherein k is 1, 2 or 3), -C (O) N (R') 2 -OR' and-S (O) k R ', wherein R' are each independently H, alkyl or substituted alkyl.
These unnatural amino acids can be in the form of salts, or can be incorporated into unnatural amino acid polypeptides, polymers, polysaccharides, or polynucleotides and optionally post-translationally modified.
In addition, the following amino acids are included:
Figure BDA0001546710350000671
wherein these compounds are optionally amino-protected and carboxyl-protected, or salts thereof. These unnatural amino acids can be in the form of salts, or can be incorporated into unnatural amino acid polypeptides, polymers, polysaccharides, or polynucleotides and optionally post-translationally modified.
In addition, an amino acid having the structure of formula (VII) below is included:
Figure BDA0001546710350000672
wherein, the liquid crystal display device comprises a liquid crystal display device,
b is optional and when present is a linker selected from the group consisting of: lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O- (alkylene or substituted alkylene) -, -S- (alkylene or substituted alkylene) -, -S (O) k - (wherein k is 1, 2 or 3), -S (O) k (alkylene or substituted alkylene) -, -C (O) -, -NS (O) 2 -、-OS(O) 2 -C (O) - (alkylene or substituted alkylene) -, -C (S) - (alkylene or substituted alkylene) -, -N (R '), -NR ' - (alkylene or substituted alkylene) -, -C (O) N (R '), -CON (R ') - (alkylene or substituted alkylene) -, -CSN (R ') - (alkylene or substituted alkylene) -, -N (R ') CO- (alkylene or substituted alkylene) -, -N (R ') C (O) O-, -S (O) k N(R')-、-N(R')C(O)N(R')-、-N(R')C(S)N(R')-、-N(R')S(O) k N(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 ') -, wherein each R' is independently H, alkyl or substituted alkyl;
r is H, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl;
R 1 is HAmino protecting groups, resins, amino acids, polypeptides or polynucleotides; and is also provided with
R 2 Is OH, an ester protecting group, a resin, an amino acid, a polypeptide or a polynucleotide;
R a each independently selected from the group consisting of: H. halogen, alkyl, substituted alkyl, -N (R') 2 、-C(O) k R '(wherein k is 1, 2 or 3), -C (O) N (R') 2 -OR' and-S (O) k R ', wherein R' are each independently H, alkyl, or substituted alkyl; and n is 0 to 8.
These unnatural amino acids can be in the form of salts, or can be incorporated into unnatural amino acid polypeptides, polymers, polysaccharides, or polynucleotides and optionally post-translationally modified.
In addition, the following amino acids are included:
Figure BDA0001546710350000681
wherein these compounds are optionally amino-protected and carboxy-protected, or are salts thereof, or can be incorporated into unnatural amino acid polypeptides, polymers, polysaccharides, or polynucleotides and optionally post-translationally modified.
In addition, an amino acid having the structure of formula (XXX) below is included:
Figure BDA0001546710350000691
wherein:
a is optional and when present is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkyl, substituted lower heterocycloalkyl, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene;
r is H, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl;
R 1 is H, an amino protecting group, a resin, an amino acid, a polypeptide or a polynucleotide; and is also provided with
R 2 Is OH, an ester protecting group, a resin, an amino acid, a polypeptide or a polynucleotide;
X 1 c, S or S (O); and L is alkylene, substituted alkylene, N (R ') (alkylene), or N (R ') (substituted alkylene), wherein R ' is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl.
These unnatural amino acids can be in the form of salts, or can be incorporated into unnatural amino acid polypeptides, polymers, polysaccharides, or polynucleotides and optionally post-translationally modified.
In addition, an amino acid having the structure of formula (XXX-A) below is included:
Figure BDA0001546710350000692
wherein:
a is optional and when present is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkyl, substituted lower heterocycloalkyl, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene;
r is H, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl;
R 1 is H, an amino protecting group, a resin, an amino acid, a polypeptide or a polynucleotide; and is also provided with
R 2 Is OH, an ester protecting group, a resin, an amino acid, a polypeptide or a polynucleotide;
l is alkylene, substituted alkylene, N (R ') (alkylene), or N (R ') (substituted alkylene), wherein R ' is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl.
These unnatural amino acids can be in the form of salts, or can be incorporated into unnatural amino acid polypeptides, polymers, polysaccharides, or polynucleotides and optionally post-translationally modified.
In addition, an amino acid having the structure of formula (XXX-B) below is included:
Figure BDA0001546710350000701
wherein:
a is optional and when present is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkyl, substituted lower heterocycloalkyl, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene;
r is H, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl;
R 1 is H, an amino protecting group, a resin, an amino acid, a polypeptide or a polynucleotide;
and R is 2 Is OH, an ester protecting group, a resin, an amino acid, a polypeptide or a polynucleotide;
l is alkylene, substituted alkylene, N (R ') (alkylene), or N (R ') (substituted alkylene), wherein R ' is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl.
These unnatural amino acids can be in the form of salts, or can be incorporated into unnatural amino acid polypeptides, polymers, polysaccharides, or polynucleotides and optionally post-translationally modified.
In addition, the amino acid having the structure of formula (XXXI) below:
Figure BDA0001546710350000711
wherein:
a is optional and when present is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkyl, substituted lower heterocycloalkyl, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene;
r is H, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl;
R 1 is H, an amino protecting group, a resin, an amino acid, a polypeptide or a polynucleotide; and is also provided with
R 2 Is OH, an ester protecting group, a resin, an amino acid, a polypeptide or a polynucleotide;
X 1 c, S or S (O); and n is 0, 1, 2, 3, 4 or 5; and each CR 8 R 9 R on a group 8 And R is 9 Each independently selected from the group consisting of H, alkoxy, alkylamine, halogen, alkyl, aryl, or any R 8 And R is 9 Can be taken together to form =o or cycloalkyl, or any and adjacent R 8 The groups may together form cycloalkyl.
These unnatural amino acids can be in the form of salts, or can be incorporated into unnatural amino acid polypeptides, polymers, polysaccharides, or polynucleotides and optionally post-translationally modified.
In addition, an amino acid having the structure of formula (XXXI-A) below is contained:
Figure BDA0001546710350000712
wherein:
a is optional and when present is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkyl, substituted lower heterocycloalkyl, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene;
r is H, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl;
R 1 is H, an amino protecting group, a resin, an amino acid, a polypeptide or a polynucleotide; and is also provided with
R 2 Is OH, an ester protecting group, a resin, an amino acid, a polypeptide or a polynucleotide;
n is 0, 1, 2, 3, 4 or 5; and each CR 8 R 9 R on a group 8 And R is 9 Each independently selected from the group consisting of H, alkoxy, alkylamine, halogen, alkyl, aryl, or any R 8 And R is 9 Can be taken together to form =o or cycloalkyl, or any and adjacent R 8 The groups may together form cycloalkyl.
These unnatural amino acids can be in the form of salts, or can be incorporated into unnatural amino acid polypeptides, polymers, polysaccharides, or polynucleotides and optionally post-translationally modified.
In addition, an amino acid having the structure of formula (XXXI-B) below is contained:
Figure BDA0001546710350000721
wherein:
a is optional and when present is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkyl, substituted lower heterocycloalkyl, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene;
r is H, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl;
R 1 is H, an amino protecting group, a resin, an amino acid, a polypeptide or a polynucleotide; and is also provided with
R 2 Is OH, an ester protecting group, a resin, an amino acid, a polypeptide or a polynucleotide;
n is 0, 1, 2, 3, 4 or 5; and each CR 8 R 9 R on a group 8 And R is 9 Each independently selected from the group consisting of H, alkoxy, alkylamine, halogen, alkyl, aryl, or any R 8 And R is 9 Can be taken together to form =o or cycloalkyl, or any and adjacent R 8 The groups may together form cycloalkyl.
These unnatural amino acids can be in the form of salts, or can be incorporated into unnatural amino acid polypeptides, polymers, polysaccharides, or polynucleotides and optionally post-translationally modified.
In addition, an amino acid having the structure of formula (XXXII) below is included:
Figure BDA0001546710350000731
wherein:
a is optional and when present is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkyl, substituted lower heterocycloalkyl, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene;
r is H, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl;
R 1 is H, an amino protecting group, a resin, an amino acid, a polypeptide or a polynucleotide; and is also provided with
R 2 Is OH, an ester protecting group, a resin, an amino acid, a polypeptide or a polynucleotide;
X 1 c, S or S (O); and L is alkylene, substituted alkylene, N (R ') (alkylene) or N (R') (takenSubstituted alkylene), wherein R' is H, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl.
These unnatural amino acids can be in the form of salts, or can be incorporated into unnatural amino acid polypeptides, polymers, polysaccharides, or polynucleotides and optionally post-translationally modified.
In addition, an amino acid having the structure of formula (XXXII-A) below is included:
Figure BDA0001546710350000732
wherein:
a is optional and when present is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkyl, substituted lower heterocycloalkyl, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene;
r is H, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl;
R 1 is H, an amino protecting group, a resin, an amino acid, a polypeptide or a polynucleotide; and is also provided with
R 2 Is OH, an ester protecting group, a resin, an amino acid, a polypeptide or a polynucleotide;
l is alkylene, substituted alkylene, N (R ') (alkylene), or N (R ') (substituted alkylene), wherein R ' is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl.
These unnatural amino acids can be in the form of salts, or can be incorporated into unnatural amino acid polypeptides, polymers, polysaccharides, or polynucleotides and optionally post-translationally modified.
In addition, an amino acid having the structure of formula (XXXII-B) below is included:
Figure BDA0001546710350000741
wherein:
a is optional and when present is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkyl, substituted lower heterocycloalkyl, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene;
r is H, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl;
R 1 is H, an amino protecting group, a resin, an amino acid, a polypeptide or a polynucleotide; and is also provided with
R 2 Is OH, an ester protecting group, a resin, an amino acid, a polypeptide or a polynucleotide;
l is alkylene, substituted alkylene, N (R ') (alkylene), or N (R ') (substituted alkylene), wherein R ' is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl.
These unnatural amino acids can be in the form of salts, or can be incorporated into unnatural amino acid polypeptides, polymers, polysaccharides, or polynucleotides and optionally post-translationally modified.
In addition, an amino acid comprising a structure having formula (XXXX):
Figure BDA0001546710350000751
wherein:
a is optional and when present is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkyl, substituted lower heterocycloalkyl, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene;
m is-C (R) 3 )-，
Figure BDA0001546710350000752
Figure BDA0001546710350000753
/>
Wherein (a) indicates bonding to the A group and (b) indicates bonding to the respective carbonyl group, R 3 And R is R 4 Independently selected from H, halogen, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl, or R 3 And R is R 4 Or two R 3 Radicals or two R 4 The radicals optionally form cycloalkyl or heterocycloalkyl;
r is H, halogen, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl;
T 3 is a bond, C (R) (R), O, or S, and R is H, halogen, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;
R 1 is H, an amino protecting group, a resin, an amino acid, a polypeptide or a polynucleotide; and is also provided with
R 2 Is OH, an ester protecting group, a resin, an amino acid, a polypeptide or a polynucleotide.
These unnatural amino acids can be in the form of salts, or can be incorporated into unnatural amino acid polypeptides, polymers, polysaccharides, or polynucleotides and optionally post-translationally modified.
In addition, an amino acid comprising a structure having formula (XXXXI):
Figure BDA0001546710350000761
wherein:
m is-C (R) 3 )-，
Figure BDA0001546710350000762
Figure BDA0001546710350000763
Wherein (a) indicates bonding to the A group and (b) indicates bonding to the respective carbonyl group, R 3 And R is R 4 Independently selected from H, halogen, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl, or R 3 And R is R 4 Or two R 3 Radicals or two R 4 The radicals optionally form cycloalkyl or heterocycloalkyl;
r is H, halogen, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl;
T 3 is a bond, C (R) (R), O, or S, and R is H, halogen, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;
R 1 Is H, an amino protecting group, a resin, an amino acid, a polypeptide or a polynucleotide; and is also provided with
R 2 Is OH, an ester protecting group, a resin, an amino acid, a polypeptide or a polynucleotide;
R a each independently selected from the group consisting of: H. halogen, alkyl, substituted alkyl, -N (R') 2 、-C(O) k R '(wherein k is 1, 2 or 3), -C (O) N (R') 2 -OR' and-S (O) k R ', wherein R' are each independently H, alkyl or substituted alkyl.
These unnatural amino acids can be in the form of salts, or can be incorporated into unnatural amino acid polypeptides, polymers, polysaccharides, or polynucleotides and optionally post-translationally modified.
In addition, an amino acid comprising a structure having formula (xxxviii):
Figure BDA0001546710350000771
wherein:
r is H, halogen, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl; and is also provided with
T 3 Is O or S.
These unnatural amino acids can be in the form of salts, or can be incorporated into unnatural amino acid polypeptides, polymers, polysaccharides, or polynucleotides and optionally post-translationally modified.
In addition, an amino acid comprising a structure having formula (xxxiii):
Figure BDA0001546710350000772
wherein:
r is H, halogen, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl.
In addition, an amino acid having the structure of formula (XXXXIII) below is included:
Figure BDA0001546710350000773
These unnatural amino acids can be in the form of salts, or can be incorporated into unnatural amino acid polypeptides, polymers, polysaccharides, or polynucleotides and optionally post-translationally modified.
The carbonyl or dicarbonyl functional groups can be selectively reacted with hydroxylamine-containing reagents in aqueous solution under mild conditions to form the corresponding oxime bonds that are stable under physiological conditions. See, e.g., jencks, W.P., J.Am.Chem.Soc.81,475-481 (1959); shao, J. And Tam, J.P., J.Am.Chem.Soc.117 (14): 3893-3899 (1995). Furthermore, the unique reactivity of carbonyl or dicarbonyl groups allows for selective modification in the presence of other amino acid side chains. See, for example, cornish, V.W., et al, J.Am.chem.Soc.118:8150-8151 (1996); geoghegan, K.F. and Stroh, J.G., bioconjug.Chem.3:138-146 (1992); mahal, L.K. et al Science 276:1125-1128 (1997).
The synthesis of p-acetyl- (+/-) -phenylalanine and m-acetyl- (+/-) -phenylalanine is described in Zhang, Z.et al, biochemistry 42:6735-6746 (2003), incorporated by reference. Other carbonyl or dicarbonyl containing amino acids may be similarly prepared. Further, non-limiting exemplary syntheses of the unnatural amino acids contained herein are presented in fig. 4, 24-34, and 36-39.
In some embodiments, polypeptides comprising unnatural amino acids are chemically modified to produce reactive carbonyl or dicarbonyl functionalities. For example, aldehyde functional groups suitable for use in the conjugation reaction may be generated from functional groups having adjacent amino and hydroxyl groups. Where the biologically active molecule is a polypeptide, for example, the aldehyde functionality can be produced using an N-terminal serine or threonine (which may typically be present or may be exposed by chemical or enzymatic digestion) under mild oxidative cleavage conditions using periodate. See, for example, gaertner et al, bioconjug. Chem.3:262-268 (1992); geoghegan, K. And Stroh, J., bioconjug. Chem.3:138-146 (1992); gaertner et al, J.biol. Chem.269:7224-7230 (1994). However, the methods known in the art are limited to amino acids at the N-terminus of peptides or proteins.
In addition, unnatural amino acids, e.g., with adjacent hydroxyl and amino groups, can be incorporated into polypeptides in the form of "masked" aldehyde functional groups. For example, 5-hydroxylysine has a hydroxyl group adjacent to epsilon amine. Reaction conditions for producing aldehydes typically include adding a molar excess of sodium metaperiodate under mild conditions to avoid oxidation at other sites within the polypeptide. The pH of the oxidation reaction is typically about 7.0. A typical reaction involves adding about 1.5 molar excess of sodium metaperiodate to a buffered solution of the polypeptide followed by incubation in the dark for about 10 minutes. See, for example, U.S. patent No. 6,423,685.
B. Unnatural amino acid: structure and synthesis of hydroxylamine-containing amino acids
Unnatural amino acids containing hydroxylamine (also known as aminooxy) groups are susceptible to reaction with a variety of electrophilic groups to form conjugates, including but not limited to, with PEG or other water-soluble polymers. As with hydrazine, hydrazide and hemi-carbazide, the enhanced nucleophilicity of the aminooxy groups allows them to effectively and selectively react with a variety of molecules containing carbonyl or dicarbonyl groups, including but not limited to ketones, aldehydes or other functional groups having similar chemical reactivity. See, for example, shao, J. And Tarn, J., J.Am.chem.Soc.117:3893-3899 (1995); H.Hang and C.Bertozzi, ace.Chem.Res.34 (9): 727-736 (2001). Whereas the reaction with hydrazine groups results in the corresponding hydrazones, oximes are typically produced by the reaction of aminooxy groups with carbonyl-or dicarbonyl-containing groups such as ketones, aldehydes or other functional groups having similar chemical reactivity.
Thus, in certain embodiments described herein are unnatural amino acids having side chains that include hydroxylamine groups, hydroxylamino-like groups (which have reactivity similar to hydroxylamine groups and are structurally similar to hydroxylamine groups), masked hydroxylamine groups (which can be readily converted to hydroxylamine groups), or protected hydroxylamine groups (which have reactivity similar to hydroxylamine groups after deprotection). These amino acids comprise amino acids having the structure:
Figure BDA0001546710350000791
Wherein:
a is optional and when present is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkyl, substituted lower heterocycloalkyl, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene;
b is optional and when present is a linker selected from the group consisting of: lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O- (alkylene or substituted alkylene) -, -S- (alkylene or substituted alkylene) -, -S (O) k - (wherein k is 1, 2 or 3), -S (O) k (alkylene or substituted alkylene) -, -C (O) -, -NS (O) 2 -、-OS(O) 2 -C (O) - (alkylene or substituted alkylene) -, -C (S) - (alkylene or substituted alkylene) -, -N (R '), -NR ' - (alkylene or substituted alkylene) -, -C (O) N (R '), -CON (R ') - (alkylene or substituted alkylene) -, -CSN (R ') - (alkylene) Alkyl or substituted alkylene) -, -N (R ') CO- (alkylene or substituted alkylene) -, -N (R') C (O) O-, -S (O) k N(R')-、-N(R')C(O)N(R')-、-N(R')C(S)N(R')-、-N(R')S(O) k N(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 ') -, wherein each R' is independently H, alkyl or substituted alkyl;
k is-NR 6 R 7 Or-n=cr 6 R 7 ；
R 1 Is H, an amino protecting group, a resin, an amino acid, a polypeptide or a polynucleotide; and is also provided with
R 2 Is OH, an ester protecting group, a resin, an amino acid, a polypeptide or a polynucleotide;
R 3 and R is R 4 Each independently is H, halogen, lower alkyl or substituted lower alkyl, or R 3 And R is R 4 Or two R 3 The radicals optionally form cycloalkyl or heterocycloalkyl;
R 6 and R is R 7 Each independently selected from the group consisting of: H. alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, polyoxyalkylene, substituted polyoxyalkylene, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkylaryl, substituted alkylaryl, arylalkyl, and substituted arylalkyl, -C (O) R', -C (O) 2 R"、-C(O)N(R") 2 Wherein each R "is independently hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, alkylaryl, substituted alkylaryl, arylalkyl, or substituted arylalkyl; or R is 6 Or R is 7 Is L-X, wherein
X is selected from the group consisting of: marking; a dye; a polymer; a water-soluble polymer; derivatives of polyethylene glycol; a photocrosslinking agent; a cytotoxic compound; a drug; affinity labeling; a photoaffinity label; a reactive compound; a resin; a second protein or polypeptide analog; an antibody or antibody fragment; a metal chelator; a cofactor; a fatty acid; a carbohydrate; a polynucleotide; DNA; RNA; an antisense polynucleotide; saccharides, water-soluble dendrimers, cyclodextrins, biomaterials; a nanoparticle; spin labeling; a fluorophore-containing moiety; a radioactive portion; novel functional groups; groups that interact covalently or non-covalently with other molecules; a light-shielding portion; a photoisomerizable moiety; biotin; biotin analogues; a moiety incorporating a heavy atom; a chemically cleavable group; a photocleavable group; an extended side chain; a carbon-bonded sugar; a redox active agent; an aminothiopropionic acid; a toxic moiety; isotopically labeled moieties; a biophysical probe; phosphorescent groups; a chemiluminescent group; an electron dense group; a magnetic group; an insertion group; a chromophore; an energy transfer agent; a bioactive agent; a detectable label; and any combination thereof; and is also provided with
L is optional and when present is a linker selected from the group consisting of: alkylene, substituted alkylene, alkenylene, substituted alkenylene, -O- (alkylene or substituted alkylene) -, -S- (alkylene or substituted alkylene) -, -S (O) k - (wherein k is 1, 2 or 3), -S (O) k (alkylene or substituted alkylene) -, -C (O) - (alkylene or substituted alkylene) -, -C (S) - (alkylene or substituted alkylene) -, -N (R ') -, -NR ' - (alkylene or substituted alkylene) -, -C (O) N (R ') -, CON (R ') - (alkylene or substituted alkylene) -, -CSN (R ') - (alkylene or substituted alkylene) -, -N (R ') CO- (alkylene or substituted alkylene) -, -N (R ') C (O) O-, -S (O) k N(R')-、-N(R')C(O)N(R')-、-N(R')C(S)N(R')-、-N(R')S(O) k N(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 ') -, wherein each R' is independently H, alkyl or substituted alkyl.
These unnatural amino acids can be in the form of salts, or can be incorporated into unnatural amino acid polypeptides, polymers, polysaccharides, or polynucleotides and optionally post-translationally modified.
In certain embodiments of the compounds of formula (XIV), A is phenylene or viaSubstituted phenylene. In certain embodiments of the compounds of formula (XIV), B is- (alkylene or substituted alkylene) -, -O- (alkylene or substituted alkylene) -, -S- (alkylene or substituted alkylene) -or-C (O) - (alkylene or substituted alkylene) -. In certain embodiments of the compounds of formula (XIV), B is-O (CH) 2 ) 2 、-S(CH 2 ) 2 -、-NH(CH 2 ) 2 -、-CO(CH 2 ) 2 -or- (CH) 2 ) n -, wherein n is 1 to 4. In certain embodiments of the compounds of formula (XIV), R 1 Is H, t-butoxycarbonyl (Boc), 9-fluorenylmethoxycarbonyl (Fmoc), N-acetyl, tetrafluoroacetyl (TFA) or benzyloxycarbonyl (Cbz). In certain embodiments of the compounds of formula (XIV), wherein R 1 Is a resin, amino acid, polypeptide or polynucleotide. In certain embodiments of the compounds of formula (XIV), wherein R 2 Is OH, O-methyl, O-ethyl or O-tert-butyl. In certain embodiments of the compounds of formula (XIV), wherein R 2 Is a resin, amino acid, polypeptide or polynucleotide. In certain embodiments of the compounds of formula (XIV), wherein R 2 Is a polynucleotide. In certain embodiments of the compounds of formula (XIV), wherein R 2 Is ribonucleic acid (RNA). In certain embodiments of the compounds of formula (XIV), wherein R 2 Is tRNA. In certain embodiments of the compound of formula (XIV), wherein the tRNA specifically recognizes a selector codon. In certain embodiments of the compounds of formula (XIV), wherein the selector codon is selected from the group consisting of an amber codon, an ocher codon, an opal codon, a unique codon, a rare codon, an unnatural codon, a five base codon, and a four base codon. In certain embodiments of the compounds of formula (XIV), wherein R 2 To suppress tRNA. In certain embodiments of the compounds of formula (XIV), R 6 And R is R 7 Each independently selected from the group consisting of: H. alkyl, substituted alkyl, alkoxy, substituted alkoxy, polyoxyalkylene, substituted polyoxyalkylene, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkylaryl, substituted alkylaryl, arylalkyl, and substituted arylalkyl. In certain embodiments of the compounds of formula (XIV), R 6 And R is R 7 Each independently selected from the group consisting of: H. methyl, phenyl and- [ (alkylene or substituted alkylene) -O- (hydrogen, alkyl or substituted alkyl)] x Wherein x is 1-50. In certain embodiments of the compounds of formula (XIV), K is-NR 6 R 7 。
In certain embodiments of the compounds of formula (XIV), X is a bioactive agent selected from the group consisting of peptides, proteins, enzymes, antibodies, drugs, dyes, lipids, nucleosides, oligonucleotides, cells, viruses, liposomes, microparticles, and micelles. In certain embodiments of the compounds of formula (XIV), X is a drug selected from the group consisting of antibiotics, fungicides, antiviral agents, anti-inflammatory agents, antitumor agents, cardiovascular agents, anxiolytic agents, hormones, growth factors, and steroid agents. In certain embodiments of the compounds of formula (XIV), X is an enzyme selected from the group consisting of horseradish peroxidase, alkaline phosphatase, beta-galactosidase, and glucose oxidase. In certain embodiments of the compounds of formula (XIV), X is a detectable label selected from the group consisting of a fluorescent moiety, a phosphorescent moiety, a chemiluminescent moiety, a chelating moiety, an electron dense moiety, a magnetic moiety, an intercalating moiety, a radioactive moiety, a chromophore moiety, and an energy transfer moiety.
In certain embodiments, the compound of formula (XIV) is stable in aqueous solution under moderately acidic conditions for at least 1 month. In certain embodiments, the compound of formula (XIV) is stable under moderately acidic conditions for at least 2 weeks. In certain embodiments, the compound of formula (XIV) is stable under moderately acidic conditions for at least 5 days. In certain embodiments, the acidic condition is a pH of 2 to 8.
These amino acids comprise amino acids having the structure of formula (XV):
Figure BDA0001546710350000821
wherein the method comprises the steps of
A is optional and when present is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkyl, substituted lower heterocycloalkyl, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene;
b is optional and when present is a linker selected from the group consisting of: lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O- (alkylene or substituted alkylene) -, -S- (alkylene or substituted alkylene) -, -S (O) k - (wherein k is 1, 2 or 3), -S (O) k (alkylene or substituted alkylene) -, -C (O) -, -NS (O) 2 -、-OS(O) 2 -C (O) - (alkylene or substituted alkylene) -, -C (S) - (alkylene or substituted alkylene) -, -N (R '), -NR ' - (alkylene or substituted alkylene) -, -C (O) N (R '), -CON (R ') - (alkylene or substituted alkylene) -, -CSN (R ') - (alkylene or substituted alkylene) -, -N (R ') CO- (alkylene or substituted alkylene) -, -N (R ') C (O) O-, -S (O) k N(R')-、-N(R')C(O)N(R')-、-N(R')C(S)N(R')-、-N(R')S(O) k N(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 ') -, wherein each R' is independently H, alkyl or substituted alkyl;
R 1 is H, an amino protecting group, a resin, an amino acid, a polypeptide or a polynucleotide; and is also provided with
R 2 Is OH, an ester protecting group, a resin, an amino acid, a polypeptide or a polynucleotide;
R 3 and R is R 4 Each independently is H, halogen, lower alkyl or substituted lower alkyl, or R 3 And R is R 4 Or two R 3 The groups optionally form cycloalkyl or heterocycloalkyl.
These unnatural amino acids can be in the form of salts, or can be incorporated into unnatural amino acid polypeptides, polymers, polysaccharides, or polynucleotides and optionally post-translationally modified.
Non-limiting, representative amino acids have the following structure:
Figure BDA0001546710350000831
these unnatural amino acids can be in the form of salts, or can be incorporated into unnatural amino acid polypeptides, polymers, polysaccharides, or polynucleotides and optionally post-translationally modified.
Amino acids containing aminooxy groups can be prepared from readily available amino acid precursors (homoserine, serine, and threonine). See, for example, M.Carrasco and R.Brown, J.Org.Chem.68:8853-8858 (2003). Certain amino acids containing aminooxy groups, such as L-2-amino-4- (aminooxy) butanoic acid, have been isolated from natural sources (Rosenthal, G.et al, life Sci.60:1635-1641 (1997)). Other amino acids containing aminooxy groups can be similarly prepared. Furthermore, a non-limiting exemplary synthesis of the unnatural amino acids described herein is presented in fig. 5.
C. Unnatural amino acid: chemical synthesis of oxime-containing amino acids
The oxime-containing unnatural amino acids are susceptible to reaction with a variety of reagents containing certain reactive carbonyl or dicarbonyl groups, including but not limited to ketones, aldehydes, or other groups with similar reactivity, to form new unnatural amino acids that include new oxime groups. This oxime exchange reaction allows for further functionalization of the unnatural amino acid polypeptide. Furthermore, the original unnatural amino acid that contains an oxime group can itself be suitable, as long as the oxime linkage is stable under the conditions necessary to incorporate the amino acid into the polypeptide (e.g., in vivo, in vitro, and chemical synthesis methods described herein).
Thus, in certain embodiments described herein are unnatural amino acids having side chains that include oxime groups, oxime-like groups (which have a reactivity similar to oxime groups and are structurally similar to oxime groups), masked oxime groups (which can be readily converted to oxime groups), or protected oxime groups (which have a reactivity similar to oxime groups after deprotection). These amino acids comprise amino acids having the structure of formula (XI):
Figure BDA0001546710350000832
wherein:
a is optional and when present is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkyl, substituted lower heterocycloalkyl, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene;
b is optional and when present is a linker selected from the group consisting of: lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O- (alkylene or substituted alkylene) -, -S- (alkylene or substituted alkylene) -, -S (O) k - (wherein k is 1, 2 or 3), -S (O) k (alkylene or substituted alkylene) -, -C (O) -, -NS (O) 2 -、-OS(O) 2 -C (O) - (alkylene or substituted alkylene) -, -C (S) - (alkylene or substituted alkylene) -, -N (R '), -NR ' - (alkylene or substituted alkylene) -, -C (O) N (R '), -CON (R ') - (alkylene or substituted alkylene) -, -CSN (R ') - (alkylene or substituted alkylene) -, -N (R ') CO- (alkylene or substituted alkylene) -, -N (R ') C (O) O-, -S (O) k N(R')-、-N(R')C(O)N(R')-、-N(R')C(S)N(R')-、-N(R')S(O) k N(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 ') -, wherein each R' is independently H, alkyl or substituted alkyl;
r is H, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl;
R 1 is H, amino protecting group, resin and aminoAn acid, polypeptide or polynucleotide; and is also provided with
R 2 Is OH, an ester protecting group, a resin, an amino acid, a polypeptide or a polynucleotide;
R 3 and R is R 4 Each independently is H, halogen, lower alkyl or substituted lower alkyl, or R 3 And R is R 4 Or two R 3 The radicals optionally form cycloalkyl or heterocycloalkyl;
R 5 is H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, alkylalkoxy, substituted alkylalkoxy, polyoxyalkylene, substituted polyoxyalkylene, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkylaryl, substituted alkylaryl, arylalkyl, substituted arylalkyl, - (alkylene or substituted alkylene) -ON (R') 2 (alkylene or substituted alkylene) -C (O) SR ', - (alkylene or substituted alkylene) -S-S- (aryl or substituted aryl), -C (O) R', -C (O) 2 R 'or-C (O) N (R') 2 Wherein each R "is independently hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, alkylaryl, substituted alkylaryl, arylalkyl, or substituted arylalkyl;
or R is 5 Is L-X, wherein
X is selected from the group consisting of: marking; a dye; a polymer; a water-soluble polymer; derivatives of polyethylene glycol; a photocrosslinking agent; a cytotoxic compound; a drug; affinity labeling; a photoaffinity label; a reactive compound; a resin; a second protein or polypeptide analog; an antibody or antibody fragment; a metal chelator; a cofactor; a fatty acid; a carbohydrate; a polynucleotide; DNA; RNA; an antisense polynucleotide; saccharides, water-soluble dendrimers, cyclodextrins, biomaterials; a nanoparticle; spin labeling; a fluorophore-containing moiety; a radioactive portion; novel functional groups; groups that interact covalently or non-covalently with other molecules; a light-shielding portion; a photoisomerizable moiety; biotin; biotin analogues; bonding of A moiety having a heavy atom; a chemically cleavable group; a photocleavable group; an extended side chain; a carbon-bonded sugar; a redox active agent; an aminothiopropionic acid; a toxic moiety; isotopically labeled moieties; a biophysical probe; phosphorescent groups; a chemiluminescent group; an electron dense group; a magnetic group; an insertion group; a chromophore; an energy transfer agent; a bioactive agent; a detectable label; and any combination thereof; and L is optional and when present is a linker selected from the group consisting of: alkylene, substituted alkylene, alkenylene, substituted alkenylene, -O- (alkylene or substituted alkylene) -, -S- (alkylene or substituted alkylene) -, -S (O) k - (wherein k is 1, 2 or 3), -S (O) k (alkylene or substituted alkylene) -, -C (O) - (alkylene or substituted alkylene) -, -C (S) - (alkylene or substituted alkylene) -, -N (R ') -, NR' - (alkylene or substituted alkylene) -, -C (O) N (R ') -, CON (R') - (alkylene or substituted alkylene) -, -CSN (R ') -, CSN (R') - (alkylene or substituted alkylene) -, -N (R ') CO- (alkylene or substituted alkylene) -, -N (R') C (O) O-, alkylene or substituted alkylene) -O-n=cr '-, - (alkylene or substituted alkylene) -C (O) NR' - (alkylene or substituted alkylene) -, - (alkylene or substituted alkylene) -S (O) k (alkylene or substituted alkylene) -S-, - (alkylene or substituted alkylene) -S-S-, -S (O) k N(R')-、-N(R')C(O)N(R')-、-N(R')C(S)N(R')-、-N(R')S(O) k N(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 ') -, wherein each R' is independently H, alkyl or substituted alkyl;
with the proviso that when A and B are absent, R is not methyl.
These unnatural amino acids can be in the form of salts, or can be incorporated into unnatural amino acid polypeptides, polymers, polysaccharides, or polynucleotides and optionally post-translationally modified.
In certain embodiments of the compounds of formula (XI), B is-O- (alkylene or substituted alkylene) -. In certain embodiments of the compounds of formula (XI)B is-O (CH) 2 ) -. In certain embodiments of the compounds of formula (XI), R is C 1-6 An alkyl group. In certain embodiments of the compounds of formula (XI), R is-CH 3 . In certain embodiments of the compounds of formula (XI), R 1 Is H, t-butoxycarbonyl (Boc), 9-fluorenylmethoxycarbonyl (Fmoc), N-acetyl, tetrafluoroacetyl (TFA) or benzyloxycarbonyl (Cbz). In certain embodiments of the compounds of formula (XI), R 1 Is a resin, amino acid, polypeptide or polynucleotide. In certain embodiments of the compounds of formula (XI), R 2 Is OH, O-methyl, O-ethyl or O-tert-butyl. In certain embodiments of the compounds of formula (XI), R 2 Is a resin, amino acid, polypeptide or polynucleotide. In certain embodiments of the compounds of formula (XI), R 2 Is a polynucleotide.
In certain embodiments of the compounds of formula (XI), R 2 Is ribonucleic acid (RNA). In certain embodiments of the compounds of formula (XI), R 2 Is tRNA. In certain embodiments of the compound of formula (XI), the tRNA specifically recognizes a selector codon. In certain embodiments of the compound of formula (XI), the selector codon is selected from the group consisting of an amber codon, an ocher codon, an opal codon, a unique codon, a rare codon, a non-natural codon, a five base codon, and a four base codon. In certain embodiments of the compounds of formula (XI), R 2 To suppress tRNA. In certain embodiments of the compounds of formula (XI), R 5 Is an alkylalkoxy, a substituted alkylalkoxy, a polyoxyalkylene, a substituted polyoxyalkylene or-C (O) 2 R is as follows. In certain embodiments of the compounds of formula (XI), R 5 Is- [ (alkylene or substituted alkylene) -O- (hydrogen, alkyl or substituted alkyl)] x Wherein x is 1-50. In certain embodiments of the compounds of formula (XI), R 5 Is- (CH) 2 CH 2 )-O-CH 3 or-COOH.
In certain embodiments, the compound of formula (I) is stable in aqueous solution under moderately acidic conditions for at least 1 month. In certain embodiments, the compound of formula (I) is stable under moderately acidic conditions for at least 2 weeks. In certain embodiments, the compound of formula (I) is stable under moderately acidic conditions for at least 5 days. In certain embodiments, the acidic condition is a pH of 2 to 8.
The amino acid of formula (XI) comprises an amino acid having the structure of formula (XII):
Figure BDA0001546710350000861
wherein, the liquid crystal display device comprises a liquid crystal display device,
b is optional and when present is a linker selected from the group consisting of: lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O- (alkylene or substituted alkylene) -, -S- (alkylene or substituted alkylene) -, -S (O) k - (wherein k is 1, 2 or 3), -S (O) k (alkylene or substituted alkylene) -, -C (O) -, -NS (O) 2 -、-OS(O) 2 -C (O) - (alkylene or substituted alkylene) -, -C (S) - (alkylene or substituted alkylene) -, -N (R '), -NR ' - (alkylene or substituted alkylene) -, -C (O) N (R '), -CON (R ') - (alkylene or substituted alkylene) -, -CSN (R ') - (alkylene or substituted alkylene) -, -N (R ') CO- (alkylene or substituted alkylene) -, -N (R ') C (O) O-, -S (O) k N(R')-、-N(R')C(O)N(R')-、-N(R')C(S)N(R')-、-N(R')S(O) k N(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 ') -, wherein each R' is independently H, alkyl or substituted alkyl;
r is H, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl;
R 1 is H, an amino protecting group, a resin, an amino acid, a polypeptide or a polynucleotide; and is also provided with
R 2 Is OH, an ester protecting group, a resin, an amino acid, a polypeptide or a polynucleotide;
R 5 is H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, alkylalkoxy, substituted alkylalkaneOxy, polyoxyalkylene, substituted polyoxyalkylene, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkylaryl, substituted alkylaryl, arylalkyl, substituted arylalkyl, - (alkylene or substituted alkylene) -ON (R') 2 (alkylene or substituted alkylene) -C (O) SR ', - (alkylene or substituted alkylene) -S-S- (aryl or substituted aryl), -C (O) R', -C (O) 2 R 'or-C (O) N (R') 2 The method comprises the steps of carrying out a first treatment on the surface of the Wherein each R "is independently hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, alkylaryl, substituted alkylaryl, arylalkyl, or substituted arylalkyl;
or R is 5 Is L-X, wherein
X is selected from the group consisting of: marking; a dye; a polymer; a water-soluble polymer; derivatives of polyethylene glycol; a photocrosslinking agent; a cytotoxic compound; a drug; affinity labeling; a photoaffinity label; a reactive compound; a resin; a second protein or polypeptide analog; an antibody or antibody fragment; a metal chelator; a cofactor; a fatty acid; a carbohydrate; a polynucleotide; DNA; RNA; an antisense polynucleotide; saccharides, water-soluble dendrimers, cyclodextrins, biomaterials; a nanoparticle; spin labeling; a fluorophore-containing moiety; a radioactive portion; novel functional groups; groups that interact covalently or non-covalently with other molecules; a light-shielding portion; a photoisomerizable moiety; biotin; biotin analogues; a moiety incorporating a heavy atom; a chemically cleavable group; a photocleavable group; an extended side chain; a carbon-bonded sugar; a redox active agent; an aminothiopropionic acid; a toxic moiety; isotopically labeled moieties; a biophysical probe; phosphorescent groups; a chemiluminescent group; an electron dense group; a magnetic group; an insertion group; a chromophore; an energy transfer agent; a bioactive agent; a detectable label; and any combination thereof; and L is optional and when present is a linker selected from the group consisting of: alkylene, substituted alkylene, alkenylene, substituted alkenylene, -O- (alkylene, or substituted alkylene ) -, -S- (alkylene or substituted alkylene) -, -S (O) k - (wherein k is 1, 2 or 3), -S (O) k (alkylene or substituted alkylene) -, -C (O) - (alkylene or substituted alkylene) -, -C (S) - (alkylene or substituted alkylene) -, -N (R ') -, NR' - (alkylene or substituted alkylene) -, -C (O) N (R ') -, CON (R') - (alkylene or substituted alkylene) -, -CSN (R ') -, CSN (R') - (alkylene or substituted alkylene) -, -N (R ') CO- (alkylene or substituted alkylene) -, -N (R') C (O) O-, alkylene or substituted alkylene) -O-n=cr '-, - (alkylene or substituted alkylene) -C (O) NR' - (alkylene or substituted alkylene) -, - (alkylene or substituted alkylene) -S (O) k - (alkylene or substituted alkylene) -S-, - (alkylene or substituted alkylene) -S-S-, -S (O) k N(R')-、-N(R')C(O)N(R')-、-N(R')C(S)N(R')-、-N(R')S(O) k N(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 ') -, wherein each R' is independently H, alkyl or substituted alkyl.
These unnatural amino acids can be in the form of salts, or can be incorporated into unnatural amino acid polypeptides, polymers, polysaccharides, or polynucleotides and optionally post-translationally modified.
These amino acids comprise amino acids having the structure of formula (XIII):
Figure BDA0001546710350000881
wherein, the liquid crystal display device comprises a liquid crystal display device,
r is H, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl;
R 1 Is H, an amino protecting group, a resin, an amino acid, a polypeptide or a polynucleotide; and is also provided with
R 2 Is OH, an ester protecting group, a resin, an amino acid, a polypeptide or a polynucleotide;
R 5 is H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, alkylAlkoxy, substituted alkylalkoxy, polyoxyalkylene, substituted polyoxyalkylene, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkylaryl, substituted alkylaryl, arylalkyl, substituted arylalkyl, - (alkylene or substituted alkylene) -ON (R') 2 (alkylene or substituted alkylene) -C (O) SR ', - (alkylene or substituted alkylene) -S-S- (aryl or substituted aryl), -C (O) R', -C (O) 2 R 'or-C (O) N (R') 2 Wherein each R "is independently hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, alkylaryl, substituted alkylaryl, arylalkyl, or substituted arylalkyl;
or R is 5 Is L-X, wherein
X is selected from the group consisting of: marking; a dye; a polymer; a water-soluble polymer; derivatives of polyethylene glycol; a photocrosslinking agent; a cytotoxic compound; a drug; affinity labeling; a photoaffinity label; a reactive compound; a resin; a second protein or polypeptide analog; an antibody or antibody fragment; a metal chelator; a cofactor; a fatty acid; a carbohydrate; a polynucleotide; DNA; RNA; an antisense polynucleotide; saccharides, water-soluble dendrimers, cyclodextrins, biomaterials; a nanoparticle; spin labeling; a fluorophore-containing moiety; a radioactive portion; novel functional groups; groups that interact covalently or non-covalently with other molecules; a light-shielding portion; a photoisomerizable moiety; biotin; biotin analogues; a moiety incorporating a heavy atom; a chemically cleavable group; a photocleavable group; an extended side chain; a carbon-bonded sugar; a redox active agent; an aminothiopropionic acid; a toxic moiety; isotopically labeled moieties; a biophysical probe; phosphorescent groups; a chemiluminescent group; an electron dense group; a magnetic group; an insertion group; a chromophore; an energy transfer agent; a bioactive agent; a detectable label; and any combination thereof; and L is optional and when present is a linker selected from the group consisting of: alkylene, substituted alkylene, alkenylene, substituted alkenylene, -O-) (alkylene or substituted alkylene) -, -S- (alkylene or substituted alkylene) -, -S (O) k - (wherein k is 1, 2 or 3), -S (O) k (alkylene or substituted alkylene) -, -C (O) - (alkylene or substituted alkylene) -, -C (S) - (alkylene or substituted alkylene) -, -N (R ') -, NR' - (alkylene or substituted alkylene) -, -C (O) N (R ') -, CON (R') - (alkylene or substituted alkylene) -, -CSN (R ') -, CSN (R') - (alkylene or substituted alkylene) -, -N (R ') CO- (alkylene or substituted alkylene) -, -N (R') C (O) O-, alkylene or substituted alkylene) -O-n=cr '-, - (alkylene or substituted alkylene) -C (O) NR' - (alkylene or substituted alkylene) -, - (alkylene or substituted alkylene) -S (O) k - (alkylene or substituted alkylene) -S-, - (alkylene or substituted alkylene) -S-S-, -S (O) k N(R')-、-N(R')C(O)N(R')-、-N(R')C(S)N(R')-、-N(R')S(O) k N(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 ') -, wherein each R' is independently H, alkyl or substituted alkyl.
These unnatural amino acids can be in the form of salts, or can be incorporated into unnatural amino acid polypeptides, polymers, polysaccharides, or polynucleotides and optionally post-translationally modified.
Other non-limiting examples of these amino acids include amino acids having the following structure:
Figure BDA0001546710350000891
These unnatural amino acids can be in the form of salts, or can be incorporated into unnatural amino acid polypeptides, polymers, polysaccharides, or polynucleotides and optionally post-translationally modified.
In addition, these amino acids include amino acids having the structure of formula (XIV):
Figure BDA0001546710350000892
wherein:
a is optional and when present is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkyl, substituted lower heterocycloalkyl, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene;
b is optional and when present is a linker selected from the group consisting of: lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O- (alkylene or substituted alkylene) -, -S- (alkylene or substituted alkylene) -, -S (O) k - (wherein k is 1, 2 or 3), -S (O) k (alkylene or substituted alkylene) -, -C (O) -, -NS (O) 2 -、-OS(O) 2 -C (O) - (alkylene or substituted alkylene) -, -C (S) - (alkylene or substituted alkylene) -, -N (R '), -NR ' - (alkylene or substituted alkylene) -, -C (O) N (R '), -CON (R ') - (alkylene or substituted alkylene) -, -CSN (R ') - (alkylene or substituted alkylene) -, -N (R ') CO- (alkylene or substituted alkylene) -, -N (R ') C (O) O-, -S (O) k N(R')-、-N(R')C(O)N(R')-、-N(R')C(S)N(R')-、-N(R')S(O) k N(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 ') -, wherein each R' is independently H, alkyl or substituted alkyl;
k is-NR 6 R 7 Or-n=cr 6 R 7 ；
R 1 Is H, an amino protecting group, a resin, an amino acid, a polypeptide or a polynucleotide; and is also provided with
R 2 Is OH, an ester protecting group, a resin, an amino acid, a polypeptide or a polynucleotide;
R 3 and R is R 4 Each independently is H, halogen, lowerAlkyl or substituted lower alkyl, or R 3 And R is R 4 Or two R 3 The radicals optionally form cycloalkyl or heterocycloalkyl;
R 6 and R is R 7 Each independently selected from the group consisting of: H. alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, polyoxyalkylene, substituted polyoxyalkylene, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkylaryl, arylalkyl, and substituted arylalkyl, -C (O) R', -C (O) 2 R"、-C(O)N(R") 2 Wherein each R "is independently hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, alkylaryl, substituted alkylaryl, arylalkyl, or substituted arylalkyl; or R is 6 Or R is 7 Is L-X, wherein
X is selected from the group consisting of: marking; a dye; a polymer; a water-soluble polymer; derivatives of polyethylene glycol; a photocrosslinking agent; a cytotoxic compound; a drug; affinity labeling; a photoaffinity label; a reactive compound; a resin; a second protein or polypeptide analog; an antibody or antibody fragment; a metal chelator; a cofactor; a fatty acid; a carbohydrate; a polynucleotide; DNA; RNA; an antisense polynucleotide; saccharides, water-soluble dendrimers, cyclodextrins, biomaterials; a nanoparticle; spin labeling; a fluorophore-containing moiety; a radioactive portion; novel functional groups; groups that interact covalently or non-covalently with other molecules; a light-shielding portion; a photoisomerizable moiety; biotin; biotin analogues; a moiety incorporating a heavy atom; a chemically cleavable group; a photocleavable group; an extended side chain; a carbon-bonded sugar; a redox active agent; an aminothiopropionic acid; a toxic moiety; isotopically labeled moieties; a biophysical probe; phosphorescent groups; a chemiluminescent group; an electron dense group; a magnetic group; an insertion group; a chromophore; an energy transfer agent; a bioactive agent; a detectable label; and any combination thereof; and L is optional and when present is A linker selected from the group consisting of: alkylene, substituted alkylene, alkenylene, substituted alkenylene, -O- (alkylene or substituted alkylene) -, -S- (alkylene or substituted alkylene) -, -S (O) k - (wherein k is 1, 2 or 3), -S (O) k (alkylene or substituted alkylene) -, -C (O) - (alkylene or substituted alkylene) -, -C (S) - (alkylene or substituted alkylene) -, -N (R ') -, -NR ' - (alkylene or substituted alkylene) -, -C (O) N (R ') -, CON (R ') - (alkylene or substituted alkylene) -, -CSN (R ') - (alkylene or substituted alkylene) -, -N (R ') CO- (alkylene or substituted alkylene) -, -N (R ') C (O) O-, -S (O) k N(R')-、-N(R')C(O)N(R')-、-N(R')C(S)N(R')-、-N(R')S(O) k N(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 ') -, wherein each R' is independently H, alkyl or substituted alkyl.
These unnatural amino acids can be in the form of salts, or can be incorporated into unnatural amino acid polypeptides, polymers, polysaccharides, or polynucleotides and optionally post-translationally modified.
These amino acids further comprise amino acids having the structure of formula (XVI):
Figure BDA0001546710350000911
wherein:
a is optional and when present is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkyl, substituted lower heterocycloalkyl, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene;
B is optional and when present is a linker selected from the group consisting of: lower levelAlkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O- (alkylene or substituted alkylene) -, -S- (alkylene or substituted alkylene) -, -S (O) k - (wherein k is 1, 2 or 3), -S (O) k (alkylene or substituted alkylene) -, -C (O) -, -NS (O) 2 -、-OS(O) 2 -C (O) - (alkylene or substituted alkylene) -, -C (S) - (alkylene or substituted alkylene) -, -N (R '), -NR ' - (alkylene or substituted alkylene) -, -C (O) N (R '), -CON (R ') - (alkylene or substituted alkylene) -, -CSN (R ') - (alkylene or substituted alkylene) -, -N (R ') CO- (alkylene or substituted alkylene) -, -N (R ') C (O) O-, -S (O) k N(R')-、-N(R')C(O)N(R')-、-N(R')C(S)N(R')-、-N(R')S(O) k N(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 ') -, wherein each R' is independently H, alkyl or substituted alkyl;
R 1 is H, an amino protecting group, a resin, an amino acid, a polypeptide or a polynucleotide; and is also provided with
R 2 Is OH, an ester protecting group, a resin, an amino acid, a polypeptide or a polynucleotide;
R 3 and R is R 4 Each independently is H, halogen, lower alkyl or substituted lower alkyl, or R 3 And R is R 4 Or two R 3 The radicals optionally form cycloalkyl or heterocycloalkyl;
R 6 and R is R 7 Each independently selected from the group consisting of: H. alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, polyoxyalkylene, substituted polyoxyalkylene, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkylaryl, arylalkyl, and substituted arylalkyl, -C (O) R', -C (O) 2 R"、-C(O)N(R") 2 Wherein R' are each independently hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, aryl, substituted arylHeteroaryl, alkylaryl, substituted alkylaryl, arylalkyl, or substituted arylalkyl; or R is 6 Or R is 7 Is L-X, wherein
X is selected from the group consisting of: marking; a dye; a polymer; a water-soluble polymer; derivatives of polyethylene glycol; a photocrosslinking agent; a cytotoxic compound; a drug; affinity labeling; a photoaffinity label; a reactive compound; a resin; a second protein or polypeptide analog; an antibody or antibody fragment; a metal chelator; a cofactor; a fatty acid; a carbohydrate; a polynucleotide; DNA; RNA; an antisense polynucleotide; saccharides, water-soluble dendrimers, cyclodextrins, biomaterials; a nanoparticle; spin labeling; a fluorophore-containing moiety; a radioactive portion; novel functional groups; groups that interact covalently or non-covalently with other molecules; a light-shielding portion; a photoisomerizable moiety; biotin; biotin analogues; a moiety incorporating a heavy atom; a chemically cleavable group; a photocleavable group; an extended side chain; a carbon-bonded sugar; a redox active agent; an aminothiopropionic acid; a toxic moiety; isotopically labeled moieties; a biophysical probe; phosphorescent groups; a chemiluminescent group; an electron dense group; a magnetic group; an insertion group; a chromophore; an energy transfer agent; a bioactive agent; a detectable label; and any combination thereof; and L is optional and when present is a linker selected from the group consisting of: alkylene, substituted alkylene, alkenylene, substituted alkenylene, -O- (alkylene or substituted alkylene) -, -S- (alkylene or substituted alkylene) -, -S (O) k - (wherein k is 1, 2 or 3), -S (O) k (alkylene or substituted alkylene) -, -C (O) - (alkylene or substituted alkylene) -, -C (S) - (alkylene or substituted alkylene) -, -N (R ') -, -NR ' - (alkylene or substituted alkylene) -, -C (O) N (R ') -, CON (R ') - (alkylene or substituted alkylene) -, -CSN (R ') - (alkylene or substituted alkylene) -, -N (R ') CO- (alkylene or substituted alkylene) -, -N (R ') C (O) O-, -S (O) k N(R')-、-N(R')C(O)N(R')-、-N(R')C(S)N(R')-、-N(R')S(O) k N(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 ') -, wherein each R' is independently H, alkyl or substituted alkyl.
These unnatural amino acids can be in the form of salts, or can be incorporated into unnatural amino acid polypeptides, polymers, polysaccharides, or polynucleotides and optionally post-translationally modified.
Furthermore, these amino acids comprise amino acids having the structure of formula (XVII):
Figure BDA0001546710350000931
wherein:
b is optional and when present is a linker selected from the group consisting of: lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O- (alkylene or substituted alkylene) -, -S- (alkylene or substituted alkylene) -, -S (O) k - (wherein k is 1, 2 or 3), -S (O) k (alkylene or substituted alkylene) -, -C (O) -, -NS (O) 2 -、-OS(O) 2 -C (O) - (alkylene or substituted alkylene) -, -C (S) - (alkylene or substituted alkylene) -, -N (R '), -NR ' - (alkylene or substituted alkylene) -, -C (O) N (R '), -CON (R ') - (alkylene or substituted alkylene) -, -CSN (R ') - (alkylene or substituted alkylene) -, -N (R ') CO- (alkylene or substituted alkylene) -, -N (R ') C (O) O-, -S (O) k N(R')-、-N(R')C(O)N(R')-、-N(R')C(S)N(R')-、-N(R')S(O) k N(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 ') -, wherein each R' is independently H, alkyl or substituted alkyl;
R 1 is H, an amino protecting group, a resin, an amino acid, a polypeptide or a polynucleotide; and is also provided with
R 2 Is OH, an ester protecting group, a resin, an amino acid, a polypeptide or a polynuclearA glycoside acid;
R 6 and R is R 7 Each independently selected from the group consisting of: H. alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, polyoxyalkylene, substituted polyoxyalkylene, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkylaryl, arylalkyl, and substituted arylalkyl, -C (O) R', -C (O) 2 R"、-C(O)N(R") 2 Wherein each R "is independently hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, alkylaryl, substituted alkylaryl, arylalkyl, or substituted arylalkyl; or R is 6 Or R is 7 Is L-X, wherein
X is selected from the group consisting of: marking; a dye; a polymer; a water-soluble polymer; derivatives of polyethylene glycol; a photocrosslinking agent; a cytotoxic compound; a drug; affinity labeling; a photoaffinity label; a reactive compound; a resin; a second protein or polypeptide analog; an antibody or antibody fragment; a metal chelator; a cofactor; a fatty acid; a carbohydrate; a polynucleotide; DNA; RNA; an antisense polynucleotide; saccharides, water-soluble dendrimers, cyclodextrins, biomaterials; a nanoparticle; spin labeling; a fluorophore-containing moiety; a radioactive portion; novel functional groups; groups that interact covalently or non-covalently with other molecules; a light-shielding portion; a photoisomerizable moiety; biotin; biotin analogues; a moiety incorporating a heavy atom; a chemically cleavable group; a photocleavable group; an extended side chain; a carbon-bonded sugar; a redox active agent; an aminothiopropionic acid; a toxic moiety; isotopically labeled moieties; a biophysical probe; phosphorescent groups; a chemiluminescent group; an electron dense group; a magnetic group; an insertion group; a chromophore; an energy transfer agent; a bioactive agent; a detectable label; and any combination thereof; and L is optional and when present is a linker selected from the group consisting of: alkylene, substituted alkylene, alkenylene, substituted alkenylene, -O-, -O- (alkylene or substituted alkylene) -, -S- (alkylene or substituted alkylene) -, -S (O) k - (wherein k is 1, 2 or 3), -S (O) k (alkylene or substituted alkylene) -, -C (O) - (alkylene or substituted alkylene) -, -C (S) - (alkylene or substituted alkylene) -, -N (R ') -, -NR ' - (alkylene or substituted alkylene) -, -C (O) N (R ') -, CON (R ') - (alkylene or substituted alkylene) -, -CSN (R ') - (alkylene or substituted alkylene) -, -N (R ') CO- (alkylene or substituted alkylene) -, -N (R ') C (O) O-, -S (O) k N(R')、-N(R')C(O)N(R')-、-N(R')C(S)N(R')-、-N(R')S(O) k N(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 ') -, wherein each R' is independently H, alkyl or substituted alkyl.
Non-limiting examples of these amino acids include amino acids having the following structure:
Figure BDA0001546710350000941
these unnatural amino acids can be in the form of salts, or can be incorporated into unnatural amino acid polypeptides, polymers, polysaccharides, or polynucleotides and optionally post-translationally modified.
In addition, these amino acids include amino acids having the structure of formula (XVIII):
Figure BDA0001546710350000951
wherein:
b is optional and when present is a linker selected from the group consisting of: lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O- (alkylene or substituted alkylene) -, -S- (alkylene or substituted alkylene) -, -S (O) k - (wherein k is 1, 2 or 3), -S (O) k (alkylene orSubstituted alkylene) -, -C (O) -, -NS (O) 2 -、-OS(O) 2 -C (O) - (alkylene or substituted alkylene) -, -C (S) - (alkylene or substituted alkylene) -, -N (R '), -NR ' - (alkylene or substituted alkylene) -, -C (O) N (R '), -CON (R ') - (alkylene or substituted alkylene) -, -CSN (R ') - (alkylene or substituted alkylene) -, -N (R ') CO- (alkylene or substituted alkylene) -, -N (R ') C (O) O-, -S (O) k N(R')-、-N(R')C(O)N(R')-、-N(R')C(S)N(R')-、-N(R')S(O) k N(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 ') -, wherein each R' is independently H, alkyl or substituted alkyl;
R 1 is H, an amino protecting group, a resin, an amino acid, a polypeptide or a polynucleotide; and is also provided with
R 2 Is OH, an ester protecting group, a resin, an amino acid, a polypeptide or a polynucleotide;
R 6 and R is R 7 Each independently selected from the group consisting of: H. alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, polyoxyalkylene, substituted polyoxyalkylene, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkylaryl, arylalkyl, and substituted arylalkyl, -C (O) R', -C (O) 2 R"、-C(O)N(R") 2 Wherein each R "is independently hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, alkylaryl, substituted alkylaryl, arylalkyl, or substituted arylalkyl; or R is 6 Or R is 7 Is L-X, wherein
X is selected from the group consisting of: marking; a dye; a polymer; a water-soluble polymer; derivatives of polyethylene glycol; a photocrosslinking agent; a cytotoxic compound; a drug; affinity labeling; a photoaffinity label; a reactive compound; a resin; a second protein or polypeptide analog; an antibody or antibody fragment; a metal chelator; a cofactor; a fatty acid; a carbohydrate; core aggregationA glycoside acid; DNA; RNA; an antisense polynucleotide; saccharides, water-soluble dendrimers, cyclodextrins, biomaterials; a nanoparticle; spin labeling; a fluorophore-containing moiety; a radioactive portion; novel functional groups; groups that interact covalently or non-covalently with other molecules; a light-shielding portion; a photoisomerizable moiety; biotin; biotin analogues; a moiety incorporating a heavy atom; a chemically cleavable group; a photocleavable group; an extended side chain; a carbon-bonded sugar; a redox active agent; an aminothiopropionic acid; a toxic moiety; isotopically labeled moieties; a biophysical probe; phosphorescent groups; a chemiluminescent group; an electron dense group; a magnetic group; an insertion group; a chromophore; an energy transfer agent; a bioactive agent; a detectable label; and any combination thereof; and L is optional and when present is a linker selected from the group consisting of: alkylene, substituted alkylene, alkenylene, substituted alkenylene, -O- (alkylene or substituted alkylene) -, -S- (alkylene or substituted alkylene) -, -S (O) k - (wherein k is 1, 2 or 3), -S (O) k (alkylene or substituted alkylene) -, -C (O) - (alkylene or substituted alkylene) -, -C (S) - (alkylene or substituted alkylene) -, -N (R ') -, -NR ' - (alkylene or substituted alkylene) -, -C (O) N (R ') -, CON (R ') - (alkylene or substituted alkylene) -, -CSN (R ') - (alkylene or substituted alkylene) -, -N (R ') CO- (alkylene or substituted alkylene) -, -N (R ') C (O) O-, -S (O) k N(R')-、-N(R')C(O)N(R')-、-N(R')C(S)N(R')-、-N(R')S(O) k N(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 ') -, wherein each R' is independently H, alkyl or substituted alkyl; and is also provided with
R a Each independently selected from the group consisting of: H. halogen, alkyl, substituted alkyl, -N (R') 2 、-C(O) k R '(wherein k is 1, 2 or 3), -C (O) N (R') 2 -OR' and-S (O) k R'; wherein each R' is independently H, alkyl or substituted alkyl and n is 0 to 8.
These unnatural amino acids can be in the form of salts, or can be incorporated into unnatural amino acid polypeptides, polymers, polysaccharides, or polynucleotides and optionally post-translationally modified.
Non-limiting examples of these amino acids include amino acids having the following structure:
Figure BDA0001546710350000961
these unnatural amino acids can be in the form of salts, or can be incorporated into unnatural amino acid polypeptides, polymers, polysaccharides, or polynucleotides and optionally post-translationally modified.
Oxime-based unnatural amino acids can be synthesized by methods already described in the art or by methods described herein, comprising: (a) Reacting a hydroxylamine-containing unnatural amino acid with a carbonyl-or dicarbonyl-containing reagent; (b) Reacting a carbonyl-or dicarbonyl-containing unnatural amino acid with a hydroxylamine-containing reagent; or (c) reacting the oxime-containing unnatural amino acid with certain reagents that contain a carbonyl or dicarbonyl group, including, for example, a ketone-containing reagent or an aldehyde-containing reagent. Representative, non-limiting examples of these synthetic methods are presented in fig. 5 and 6.
D. Cellular uptake of unnatural amino acids
Unnatural amino acid absorption by eukaryotic cells is a common concern when designing and selecting unnatural amino acids, including but not limited to for incorporation into polypeptides. For example, the high charge density of α -amino acids suggests that these compounds are unlikely to penetrate cells. Natural amino acids are taken up in eukaryotic cells by a collection of protein-based transport systems. A rapid screen to assess which unnatural amino acids, if any, are taken up by cells can be performed (examples 15 and 16 herein illustrate non-limiting examples of tests that can be performed on unnatural amino acids). See, e.g., U.S. patent publication No. 2004/198637, entitled "Protein Arrays," which is incorporated herein by reference in its entirety, and Liu, D.R., and Schultz, P.G. (1999) Progress toward the evolution of an orga nism with an expanded genetic code.PNAS United States96:4780-4785. Although absorption is amenable to analysis by a variety of assays, an alternative to designing unnatural amino acids that conform to cellular absorption pathways is to provide biosynthetic pathways for amino acid production in vivo.
Typically, unnatural amino acids produced by cellular uptake as described herein are produced at concentrations (including, but not limited to, natural cell amounts) sufficient for efficient protein biosynthesis, but not to such an extent as to affect the concentration of other amino acids or to deplete cellular resources. Typical concentrations produced in this way are about 10mM to about 0.05mM.
E. Biosynthesis of unnatural amino acids
Many biosynthetic pathways already exist in cells for the production of amino acids and other compounds. Although the biosynthetic methods for a particular unnatural amino acid may not exist in nature, including but not limited to cells, the methods and compositions described herein provide these methods. For example, biosynthetic pathways for unnatural amino acids can be produced in a host cell by adding new enzymes or modifying existing host cell pathways. Other novel enzymes include naturally occurring enzymes or artificially evolved enzymes. For example, the biosynthesis of para-aminophenylalanine (as presented by the example in WO 2002/085923 entitled "In vivo incorporation of unnatural amino acids") relies on the addition of a combination of known enzymes from other organisms. Genes for these enzymes can be introduced into eukaryotic cells by transforming the cells with plasmids that include these genes. When expressed in cells, these genes provide an enzymatic pathway for the synthesis of the desired compounds. Examples of the types of enzymes optionally added are provided herein. Other enzyme sequences are found, for example, in the Genbank (Genbank). Artificially evolved enzymes can be added to cells in the same way. In this way, the cellular machinery and source of the cell is manipulated to produce unnatural amino acids.
A variety of methods are available for preparing novel enzymes for use in biosynthetic pathways or evolving existing pathways. For example, include, but are not limited to, those described by Maxygen, inc. (available on the world Wide Web)www.maxygen.comObtained) the developed recursive recombination can be used to develop novel enzymes and pathways. See, e.g., stemmer (1994), rapid evolution of a protein in vitro by DNA shuffling,Nature370 (4) 389-391; and Stemmer, (1994), DNA shuffling by random fragmentation and reassembly: in vitro recombination for molecular evolution,Proc.Natl.Acad.Sci.USA.,91:10747-10751. By Genencor (available on the world Wide Webgenencor.comObtained) developed Similarly DesignPath TM Optionally used in metabolic pathway engineering, which includes, but is not limited to, pathways used to engineer the production of unnatural amino acids in cells. This technique uses a combination of new genes, including but not limited to those identified by functional genomics, molecular evolution, and design, to engineer existing pathways in host organisms. Diversa Corporation (available on the world Wide Web)diversa.comObtained) also provides techniques for rapid screening of libraries of genes and gene pathways, including, but not limited to, the generation of new pathways for the production of unnatural amino acids by biosynthesis.
Typically, unnatural amino acids produced in engineered biosynthetic pathways as described herein are produced at concentrations (including, but not limited to, natural cell amounts) sufficient for efficient protein biosynthesis, but not to such an extent as to affect other amino acid concentrations or deplete cellular resources. Typical concentrations produced in vivo in this way are about 10mM to about 0.05mM. Once the cells are transformed with a plasmid comprising genes for the enzymes required for the production of a particular pathway and unnatural amino acids are produced, in vivo selection is optionally used to further optimize the production of unnatural amino acids for ribosomal protein synthesis and cell growth.
F. Other synthetic methods
The unnatural amino acids described herein can be synthesized using methods described in the art or using techniques described herein or a combination thereof. As an aid, the following table provides various starting electrophiles and nucleophiles that can be combined to produce the desired functional groups. The information provided is intended to be illustrative and not limiting of the synthetic techniques described herein.
Table 1: examples of covalent bonds and precursors thereof
Figure BDA0001546710350000981
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Figure BDA0001546710350000991
In general, carbon electrophiles are susceptible to attack by a complementary nucleophile (including carbon nucleophiles), wherein the attacking nucleophile provides an electron pair to the carbon electrophile to form a new bond between the nucleophile and the carbon electrophile.
Non-limiting examples of carbon nucleophiles include, but are not limited to, alkyl Grignard, alkenyl Grignard, aryl Grignard, and alkynyl Grignard, organolithium, organozinc, alkyl tin, alkenyl tin, aryl tin, and alkynyl tin (organotin), alkyl borane, alkenyl borane, aryl borane, and alkynyl borane (organoborane and organoborates); these carbon nucleophiles have the advantage of being kinetically stable in water or polar organic solvents. Other non-limiting examples of carbon nucleophiles include phosphonium salts, enols, and enolate reagents; these carbon nucleophiles have the advantage of being relatively easy to produce from precursors well known to those skilled in the art of synthetic organic chemistry. When used with a carbon electrophile, the carbon nucleophile forms a new carbon-carbon bond between the carbon nucleophile and the carbon electrophile.
Non-limiting examples of non-carbon nucleophiles suitable for coupling with the carbon electrophiles include, but are not limited to, primary and secondary amines, thiols, thiolates, and sulfides, alcohols, alkoxides, azides, hemi-carbazide, and the like. When used with carbon electrophiles, these non-carbon nucleophiles typically produce heteroatom bonds (C-X-C), where X is a heteroatom, including but not limited to oxygen, sulfur, or nitrogen.
VI polypeptide with unnatural amino acid
For convenience, the forms, properties, and other characteristics of the compounds described in this section have been described generally and/or with specific examples. However, the forms, properties and other features described in this section should not be limited to the general description or specific examples provided in this section, but are equally well applicable to all compounds within the scope of formulas I-XVIII, XXX-XXXIV (a and B) and XXXX-xxxiii, including any sub-or specific compounds within the scope of formulas I-XVIII, XXX-XXXIV (a and B) and XXXX-xxxiii described in the specification, claims and drawings herein.
The compositions and methods described herein provide for the incorporation of at least one unnatural amino acid into a polypeptide. Unnatural amino acids can be present at any position on a polypeptide, including any terminal position or any internal position of the polypeptide. In contrast to homologous naturally occurring amino acid polypeptides, it is preferred that the unnatural amino acid does not disrupt the activity and/or tertiary structure of the polypeptide unless such disruption of activity and/or tertiary structure is for one purpose of incorporating the unnatural amino acid into the polypeptide. Furthermore, the incorporation of unnatural amino acids into polypeptides can modify the activity (e.g., manipulate the therapeutic efficacy of the polypeptide; improve the safety profile of the polypeptide; modulate the pharmacokinetics, pharmacology, and/or potency (e.g., increase water solubility, bioavailability; increase serum half-life; increase therapeutic half-life; modulate immunogenicity; modulate biological activity; or extend circulation time) of the polypeptide to some extent relative to homologous naturally occurring amino acid polypeptides; provide additional functional groups to the polypeptide; incorporate tags, labels, or detectable signals into the polypeptide; facilitate the separation properties of the polypeptide; and any combination of the foregoing modifications) and/or tertiary structure without causing disruption of activity and/or tertiary structure at all. Such modifications to the activity and/or tertiary structure are typically one goal of achieving such incorporation, although incorporation of unnatural amino acids into polypeptides can also have no effect on the activity and/or tertiary structure of the polypeptide relative to homologous naturally occurring amino acid polypeptides. Accordingly, non-natural amino acid polypeptides, compositions comprising non-natural amino acid polypeptides, methods for making such polypeptides and polypeptide compositions, methods for purifying, isolating, and characterizing such polypeptides and polypeptide compositions, and methods of using such polypeptides and polypeptide compositions are considered within the scope of the present disclosure. Furthermore, the non-natural amino acid polypeptides described herein can also be linked to another polypeptide (including, for example, a non-natural amino acid polypeptide or a naturally occurring amino acid polypeptide).
The unnatural amino acid polypeptides described herein can be prepared by biosynthesis or non-biosynthesis. Biosynthesis means any method that utilizes a translation system (cellular or non-cellular) that includes the use of at least one of the following components: a polynucleotide, a codon, a tRNA, and a ribosome. Non-biosynthesis means any method that does not utilize a translation system: the method can be further classified into a method using a solid-state peptide synthesis method and a solid-state peptide synthesis method; a method utilizing at least one enzyme; and methods that do not utilize at least one enzyme; in addition, any of this subdivision may overlap and many methods may utilize a combination of these subdivision.
The methods, compositions, strategies, and techniques described herein are not limited to a particular type, kind, or family of polypeptides or proteins. Indeed, virtually any polypeptide may comprise at least one unnatural amino acid described herein. By way of example only, the polypeptide may be homologous to a therapeutic protein selected from the group consisting of: alpha-1 antitrypsin, angiostatin, anti-hemolytic factor, antibody, apolipoprotein, apoprotein, atrial natriuretic factor, atrial natriuretic polypeptide, atrial peptide, C-X-C chemokine, T39765, NAP-2, ENA-78, gro-a, gro-b, gro-C, IP-10, GCP-2, NAP-4, SDF-1, PF4, MIG, calcitonin, C-kit ligand, cytokine, CC chemokine, monocyte chemokine-1, monocyte chemokine-2, monocyte chemokine-3, monocyte inflammatory protein-1 alpha, monocyte inflammatory protein-iβ, RANTES, 1309, R83915, R91733, HCC1, T58847, D31065, T64262, CD40 ligand, C-kit ligand, collagen, and pharmaceutical compositions containing the same Colony Stimulating Factor (CSF), complement factor 5a, complement inhibitor, complement receptor 1, cytokine, epithelial neutrophil activating peptide-78, MIP-16, MCP-1, epidermal Growth Factor (EGF), epithelial neutrophil activating peptide, erythropoietin (EPO), epidermal exfoliative toxin, factor IX, factor VII, factor VIII, factor X, fibroblast Growth Factor (FGF), fibrinogen, fibronectin, tetrahelical bundle protein, G-CSF, glp-1, GM-CSF, glucocerebrosidase, gonadotropin, growth factor receptor, grf, hedgehog protein, hemoglobin, hepatocyte growth factor (hGF), hirudin, human growth hormone (hGH), human serum albumin, ICAM-1, ICAM-1 receptor, LFA-1 receptor, insulin-like growth factor (IGF), IGF-I, IGF-II, interferon (IFN), IFN-alpha, IFN-beta, IFN-gamma, interleukin (IL), IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, keratinocyte Growth Factor (KGF), lactoferrin, leukemia inhibitory factor, luciferase, neurogenin, neutrophil Inhibitory Factor (NIF), oncostatin M, osteogenic protein, oncogene products, paracitin, parathyroid hormone, PD-ECSF, PDGF, peptide hormones, pleiotrophin, protein A, protein G, pth, pyrogenic exotoxin A, neutropy the therapeutic agents include, but are not limited to, heat exotoxin B, heat exotoxin C, pyy, relaxin, renin, SCF, small biosynthetic proteins, soluble complement receptor I, soluble I-CAM 1, soluble interleukin receptor, soluble TNF receptor, somatostatin, growth hormone, streptokinase, superantigen, staphylococcal enterotoxin, SEA, SEB, SEC1, SEC2, SEC3, SED, SEE, steroid hormone receptor, superoxide dismutase, toxic shock syndrome toxin, thymosin alpha 1, tissue type plasminogen activator, tumor Growth Factor (TGF), tumor necrosis factor alpha, tumor necrosis factor beta, tumor Necrosis Factor Receptor (TNFR), VLA-4 protein, VCAM-1 protein, vascular Endothelial Growth Factor (VEGF), urokinase, mos, ras, raf, met, p, tat, fos, myc, jun, myb, rel, estrogen receptor, progesterone receptor, testosterone receptor, aldosterone receptor, LDL receptor, and corticosterone. In related or other embodiments, the unnatural amino acid polypeptide can also be homologous to any polypeptide member of the growth hormone supergene family.
The unnatural amino acid polypeptide can be further modified as described elsewhere in this disclosure, or the unnatural amino acid polypeptide can be used without further modification. Unnatural amino acids can be incorporated into polypeptides for a variety of purposes, including, but not limited to, tailoring changes in protein structure and/or function, changing the size of protease target sites, acidity, nucleophilicity, hydrogen bonding, hydrophobicity, accessibility, targeting moieties (including, but not limited to, for polypeptide arrays), and the like. Polypeptides comprising unnatural amino acids can have enhanced or even entirely new catalytic or biophysical properties. By way of example only, the following properties may be engineered by including unnatural amino acids in polypeptides: toxicity, biodistribution, structural properties, spectroscopic properties, chemical and/or photochemical properties, catalytic capabilities, half-life (including but not limited to serum half-life), ability to react with other molecules (including but not limited to covalent or non-covalent), and the like. Compositions having polypeptides comprising at least one unnatural amino acid are useful for, including, but not limited to, novel therapies, diagnostics, catalytic enzymes, industrial enzymes, binding proteins, including, but not limited to, antibodies, and research, including, but not limited to, research on protein structure and function. See, e.g., dougherty, (2000) Unnatural Amino Acids as Probes of Protein Structure and Function, Current Opinion in Chemical Biology,4:645-652。
In addition, the side chains of the unnatural amino acid component of polypeptides can provide the polypeptide with a variety of other functional groups; by way of example only, and not limitation, the side chain of the unnatural amino acid portion of a polypeptide can comprise any of the following: marking; a dye; a polymer; a water-soluble polymer; derivatives of polyethylene glycol; a photocrosslinking agent; a cytotoxic compound; a drug; affinity labeling; a photoaffinity label; a reactive compound; a resin; a second protein or polypeptide analog; an antibody or antibody fragment; a metal chelator; a cofactor; a fatty acid; a carbohydrate; a polynucleotide; DNA; RNA; an antisense polynucleotide; saccharides, water-soluble dendrimers, cyclodextrins, biomaterials; a nanoparticle; spin labeling; a fluorophore-containing moiety; a radioactive portion; novel functional groups; groups that interact covalently or non-covalently with other molecules; a light-shielding portion; an actinic radiation excitable moiety; a ligand; a photoisomerizable moiety; biotin; biotin analogues; a moiety incorporating a heavy atom; a chemically cleavable group; a photocleavable group; an extended side chain; a carbon-bonded sugar; a redox active agent; an aminothiopropionic acid; a toxic moiety; isotopically labeled moieties; a biophysical probe; phosphorescent groups; a chemiluminescent group; an electron dense group; a magnetic group; an insertion group; a chromophore; an energy transfer agent; a bioactive agent; a detectable label; a small molecule; inhibitory ribonucleic acid, radionucleotide; a neutron capture agent; derivatives of biotin; a quantum dot; nano transitorin; a radiotransferrin; abzymes, activating complex activators, viruses, adjuvants, aglycones, allergens, angiostatin, anti-hormones, antioxidants, aptamers, guide RNAs, saponins, shuttle vectors, macromolecules, pseudoepitopes, receptors, reverse micelles, and any combination thereof.
In one aspect, the composition comprises at least one polypeptide having at least one (including but not limited to at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten or more) unnatural amino acids. These unnatural amino acids can be the same or different. In addition, there may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more than 20 different or identical unnatural amino acid in a polypeptide that includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more than 20 different sites. In another aspect, the composition comprises a polypeptide in which at least one (but less than all) of the specific amino acids present in the polypeptide have been replaced with unnatural amino acids. For a given polypeptide having more than one unnatural amino acid, these unnatural amino acids can be the same or different (such as, for example only, the polypeptide can comprise two or more different types of unnatural amino acids, or can comprise two identical unnatural amino acids). For a given polypeptide having more than two unnatural amino acids, the unnatural amino acids can be the same, different, or a combination of multiple numbers of unnatural amino acids of the same species with at least one different unnatural amino acid.
Although embodiments of the unnatural amino acid polypeptides described herein can be synthesized chemically by solid phase peptide synthesis methods (such as, for example only, on solid resins), solution phase peptide synthesis methods, and/or without the aid of enzymes, other embodiments of the unnatural amino acid polypeptides described herein are susceptible to synthesis by cell membranes, cellular extracts, or lysate systems, or by in vivo systems (such as, for example only, cellular mechanisms using prokaryotic or eukaryotic cells). In other or additional embodiments, one key feature of the unnatural amino acid polypeptides described herein is that they can be synthesized using ribosomes. In other or additional embodiments of the unnatural amino acid polypeptides described herein, the unnatural amino acid polypeptides can be synthesized by combinations of methods, including, but not limited to, solid resins, with no assistance from enzymes, with assistance from ribosomes, and/or by combinations of in vivo systems.
The synthesis of unnatural amino acid polypeptides by ribosomal and/or in vivo systems has advantages and features that differ from unnatural amino acid polypeptides synthesized on solid resins or without the aid of enzymes. These advantages and features include different impurity profiles: systems that utilize ribosomes and/or in vivo systems should have impurities derived from the biological system utilized, which include host cell proteins, membrane moieties, and lipids, while the impurity profile from systems that utilize solid resins and/or do not aid in enzymes may include organic solvents, protecting groups, resin materials, coupling reagents, and other chemicals used in the synthesis procedure. In addition, the isotopic pattern of the unnatural amino acid polypeptide synthesized by using ribosomes and/or in vivo systems can reflect the isotopic pattern of raw materials utilized by the cell; on the other hand, the isotopic pattern of an unnatural amino acid polypeptide synthesized on a solid resin and/or without the aid of enzymes can reflect the isotopic pattern of an amino acid utilized in the synthesis. Furthermore, unnatural amino acids synthesized through the use of ribosomes and/or in vivo systems can be substantially free of the D-isomer of the amino acid and/or can be readily incorporated into the structure of the polypeptide, and/or can provide little or no internal amino acid deletion polypeptide. On the other hand, non-natural amino acid polypeptides synthesized by solid resins and/or without enzymes may have a higher content of D-isomers of amino acids and/or a lower content of internal cysteine amino acids and/or a higher percentage of internal amino acid deletion polypeptides. Furthermore, one skilled in the art should be able to distinguish between non-natural amino acid polypeptides synthesized by using ribosomes and/or in vivo systems and non-natural amino acid polypeptides synthesized by solid resins and/or without enzymes.
Compositions and methods comprising nucleic acids and oligonucleotides
A. Universal recombinant nucleic acid methods for use herein
In many embodiments of the methods and compositions described herein, nucleic acids encoding polypeptides of interest (including, for example, GH polypeptides) are isolated, cloned, and engineered, typically using recombinant methods. These embodiments are useful for (including, but not limited to) protein expression or in the production of variants, derivatives, expression cassettes or other sequences derived from polypeptides. In some embodiments, the sequence encoding the polypeptide is operably linked to a heterologous promoter.
The nucleotide sequence encoding the polypeptide comprising the unnatural amino acid can be synthesized from the amino acid sequence of the parent polypeptide, and then the nucleotide sequence can be altered to effect the introduction (i.e., incorporation or substitution) or removal (i.e., deletion or substitution) of the relevant amino acid residue. The nucleotide sequence may be conveniently modified by site-directed mutagenesis in accordance with conventional methods. Alternatively, the nucleotide sequence may be prepared by chemical synthesis, including, but not limited to, by use of an oligonucleotide synthesizer, wherein the oligonucleotide is designed according to the amino acid sequence of the desired polypeptide, and preferably selects those codons favored by the host cell in which the recombinant polypeptide will be produced. For example, several small oligonucleotides encoding portions of a desired polypeptide may be synthesized and assembled by PCR, ligation, or ligation chain reaction. See, e.g., barany et al, proc. Natl. Acad. Sci.88:189-193 (1991); U.S.6,521,427, which is incorporated herein by reference.
The unnatural amino acid methods and compositions described herein utilize conventional techniques in the field of recombinant genetics. The basic literature disclosing general methods for the unnatural amino acid methods and compositions described herein includes Sambrook et al, molecular Cloning, A Laboratory Manual (3 rd edition, 2001); kriegler, gene Transfer and Expression: A Laboratory Manual (1990); and Current Protocols in Molecular Biology (Ausubel et al, 1994).
General works describing molecular biology techniques include Berger and Kimmel, guide to Molecular Cloning Techniques, methods in Enzymology, volume 152, academic Press, inc., san Diego, CA (Berger); sambrook et al, molecular Cloning-A Laboratory Manual (2 nd edition), volumes 1-3, cold Spring Harbor Laboratory, cold Spring Harbor, new York,1989 ("Sambrook"), current Protocols in Molecular Biology, f.m. Ausubel et al, current Protocols, greene Publishing Associates, inc. And John Wiley & Sons, inc. Co-pending (1999 supplement year-round ") (" Ausubel "). These works describe the formation of mutations, the use of vectors, promoters, and many other related topics, which involve the production of genes or polynucleotides including, but not limited to, selector codons, orthogonal tRNA's, orthogonal synthetases, and pairs thereof, that contain proteins that contain unnatural amino acids.
Various types of mutations are formed for use in the unnatural amino acid methods and compositions described herein for a variety of purposes, including, but not limited to, for producing novel synthetases or tRNA's, for mutating tRNA molecules, for mutating polynucleotides encoding synthetases, libraries of tRNA's, for producing libraries of synthetases, for producing selector codons, for inserting selector codons encoding unnatural amino acids in a protein or polypeptide of interest. Including, but not limited to, site-directed mutagenesis, random point mutagenesis, homologous recombination, DNA shuffling or other recursive mutagenesis methods, chimeric construction, mutagenesis using uracil containing templates, oligonucleotide directed mutagenesis, phosphorothioate modified DNA mutagenesis, mutagenesis using gapped double-stranded DNA or analogues thereof, or any combination thereof. Other suitable methods include point mismatch repair, mutant formation using host strains lacking repair, restriction-selection and restriction-purification, deletion mutant formation, mutagenesis induced by total gene synthesis, double strand break repair, and the like. Mutant formations, including but not limited to chimeric constructs, are also encompassed by the unnatural amino acid methods and compositions described herein. In one embodiment, the mutant formation may be guided by known information of the naturally occurring molecule or of the mutated or mutated naturally occurring molecule, including but not limited to sequence comparisons, physical properties, crystal structure or the like.
These and other related procedures are described by the literature and examples presented herein. Other information is found in the following publications and references cited therein: ling et al Approaches to DNA mutagenesis: an oversview, anal biochem.254 (2): 157-178 (1997); dale et al, oligonucleotides-directed random mutagenesis using the phosphorothioate method, methods mol. Biol.57:369-374 (1996); smith, in vitro mutagenesis, ann.Rev.Genet.19:423-462 (1985); botstein and Shortle, strategies and applications of in vitro mutagenesis, science 229:1193-1201 (1985); carter, site-directed mutagenesis, biochem. J.237:1-7 (1986); kunkel, the efficiency of oligonucleotide directed mutagenesis, in Nucleic Acids & Molecular Biology (Eckstein, f. And liley, d.m. j., spring Verlag, berlin)) (1987); kunkel, rapid and efficient site-specific mutagenesis without phenotypic selection, proc. Natl. Acad. Sci. USA 82:488-492 (1985); kunkel et al Rapid and efficient site-specific mutagenesis without phenotypic selection, methods in enzymol.154,367-382 (1987); bass et al, mutant Trp repressors with new DNA-binding specificities, science 242:240-245 (1988); methods in enzymol.100:468-500 (1983); methods in enzymol.154:329-350 (1987); zoller and Smith, oligonucleotides-directed mutagenesis using Ml 3-modified vectors: an efficient and general procedure for the production of point mutations in any DNA fragment, nucleic Acids Res.10:6487-6500 (1982); zoller and Smith, oligonucleotides-directed mutagenesis of DNA fragments cloned into M vectors, methods in enzymol.100:468-500 (1983); zoller and Smith, oligonucleotides-directed mutagenesis: a simple method using two Oligonucleotide primers and a single-stranded DNA template, methods in enzymol.154:329-350 (1987); taylor et al, the use of phosphorothioate-modified DNA in restriction enzyme reactions to prepare nicked DNA, nucleic acids Res.13:8749-8764 (1985); taylor et al The rapid generation of oligonucleotide-directed mutations at high frequency using phosphorothioate-modified DNA, nucl. Acids Res.13:8765-8785 (1985); nakamaye and Eckstein, inhibition of restriction endonuclease Nci I cleavage by phosphorothioate groups and its application to oligonucleotide-directed mutagenesis, nucl. Acids Res.14:9679-9698 (1986); sayers et al, 5'-3'Exonucleases in phosphorothioate-based oligonucleotide-directed mutagenesis, nucl. Acids Res.16:791-802 (1988); sayers et al Strand specific cleavage of phosphorothioate-containing DNA by reaction with restriction endonucleases in the presence ofethidium bromide, (1988) Nucl. Acids Res.16:803-814; kramer et al The gapped duplex DNA approach to oligonucleotide-directed mutation construction, nucleic acids Res.12:9441-9456 (1984); kramer and Fritz, oligonucleotides-directed construction of mutations via gapped duplex DNA, methods in enzymol.154:350-367 (1987); kramer et al Improved enzymatic in vitro reactions in the gapped duplex DNA approach to oligonucleotide-directed construction of mutations, nucleic acids Res.16:7207 (1988); fritz et al, oligonucleotides-directed construction of mutations: a gapped duplex DNA procedure without enzymatic reactions in vitro, nucl. Acids Res.16:6987-6999 (1988); kramer et al, point Mismatch Repair, cell 38:879-887 (1984); carter et al Improved oligonucleotide site-directed mutagenesis using M vectors, nucl. Acids Res.13:4431-4443 (1985); carter, improved oligonucleotide-directed mutagenesis using Ml 3.3 vectors, methods in enzymol.154:382-403 (1987); eghtedarzadeh and Henikoff, use of oligonucleotides to generate large deletions, nucl. Acids Res.14:5115 (1986); wells et al Importance of hydrogen-bond formation in stabilizing the transition state of subtilisin, phil.Trans.R.Soc.Lond.A 317:415-423 (1986); nambiar et al, total synthesis and cloning of a gene coding for the ribonuclease S protein, science 223:1299-1301 (1984); sakmar and Khorana, total synthesis and expression of a gene for the a-subunit of bovine rod outer segment guanine nucleotide-binding protein (transducin), nucl. Acids Res.14:6361-6372 (1988); wells et al Cassette mutagenesis: an efficient method for generation of multiple mutations at defined sites, gene 34:315-323 (1985); grundstrom et al, oligonucleotides-directed mutagenesis by microscale 'shot-gun' gene synthesis, nucleic acids Res.13:3305-3316 (1985); mandecki, oligonucleotides-directed double-strand break repair in plasmids of Escherichia coli: a method 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 et al Nature Biotechnology,19:456-460 (2001), W.P.C.stemmer, nature 370,389-91 (1994); and I.A.Lorimer, I.Pastan, nucleic Acids Res.23,3067-8 (1995). Additional details regarding many of these approaches can be found in volume Methods in Enzymology, volume 154, which also describes effective control of the problem of failure in various mutation forming methods.
Methods and compositions described herein also include the use of eukaryotic host cells, non-eukaryotic host cells, and organisms to incorporate unnatural amino acids into a living organism through orthogonal tRNA/RS pairs. The host cell is genetically engineered (including, but not limited to) with a polynucleotide corresponding to a polypeptide described herein and a construct comprising a polynucleotide corresponding to a polypeptide described herein, including, but not limited to, a vector corresponding to a polypeptide described herein, which may be, for example, a cloning vector and an expression vector, including, but not limited to, transformation, transduction, or transfection. For example, the coding region of the orthogonal tRNA, the orthogonal tRNA synthetase, and the protein to be derived are operably linked to a gene expression control element that is functional in the desired host cell. Vectors may be in the form of, for example, plasmids, cosmids, phages, bacteria, viruses, naked and conjugated polynucleotides. The vector is introduced into cells and/or microorganisms using standard methods, including electroporation (from m et al, proc. Natl. Acad. Sci. USA 82,5824 (1985)), infection by viral vectors, high-speed ballistic penetration by small particles with nucleic acids within the matrix of the beads or particles, or onto surfaces (Klein et al, nature 327,70-73 (1987)), and/or the like.
Engineered host cells can be cultured in conventional nutrient media modified for such activities as, for example, screening steps, activating promoters, or selecting transformants. These cells may optionally be cultured as transgenic organisms. Other suitable references including, but not limited to, those concerning cell isolation and culture (e.g., concerning subsequent nucleic acid isolation) include fresnel (1994) Culture of Animal Cells, a Manual of Basic Technique, 3 rd edition, wiley-list, new York, and references cited therein; payne et al (1992) Plant Cell and Tissue Culture in Liquid Systems John Wiley & Sons, inc. New York, N.Y.; gamborg and Phillips (ed) (1995) Plant Cell, tissue and Organ Culture; fundamental Methods Springer Lab Manual Springer-Verlag (Berlin Heidelberg New York) and Atlas and Parks (ed) The Handbook of Microbiological Media (1993) CRC Press, boca Raton, FL.
Several well-known methods of introducing a target nucleic acid into a cell are available, any of which can be used in the methods and compositions described herein. It comprises: fusion of recipient cells with DNA-containing bacterial protoplasts, electroporation, ballistic bombardment, infection with viral vectors (discussed further herein), and the like. Bacterial cells can be used to amplify the number of plasmids containing a DNA construct corresponding to a polypeptide described herein. Bacteria are grown to log stage and plasmids within the bacteria can be isolated by a variety of methods known in the art (see, e.g., sambrook). In addition, numerous kits are commercially available for purification of plasmids from bacteria (see, e.g., easyPrep) TM 、FlexiPrep TM Both from Pharmacia Biotech; strataClean TM From Stratagene; QIAprep TM From Qiagen). The isolated and purified plasmid is then further processed to produce other plasmids for use in transfecting cells or for incorporation into related vectors for infecting organisms. Typical vectors contain transcription and translation terminators, transcription and translation initiation sequences, and promoters suitable for use in regulating expression of a particular nucleic acid of interest. Vectors optionally include a general expression cassette containing at least one independent terminator sequence, sequences allowing replication of the cassette in eukaryotes or prokaryotes or both, including but not limited to shuttle vectors, and selectable markers for both prokaryotic and eukaryotic systems. The vector is suitable for replication and integration in prokaryotes, eukaryotes, or preferably both. See, gillim and Smith, gene 8:81 (1979); roberts et al, nature,328:731 (1987); schneider, E.et al, protein expr. Purif.6 (1): 10-14 (1995); ausubel, sambrook, berger (all as above). Catalogues of bacteria and phages suitable for cloning are provided by, for example, ATCC, e.g., the ATCC Catalogue of bacteria and bacteriophage (1992) southern a et al, code published by ATCC. Other basic procedures and potential theoretical considerations for sequencing, cloning, and other aspects of molecular biology are also found in Watson et al (1992) Recombinant DNA Second Edition Scientific American Books, NY. In addition, substantially any nucleic acid (and substantially any labeled nucleic acid, whether standard or not) may be customized or ordered standard from any of a variety of commercial sources, such as the Midland Certified Reagent Company (Midland, TX) mcrc.com) The Great American Gene Company (Ramona, CA, on the world Wide Web)genco.comObtained), expressGen inc (Chicago, IL, on the world wide webexpressgen.comObtained), operon Technologies inc (Alameda, CA) and many other sources.
B. Selecting codons
The selector codons encompassed in the methods and compositions described herein detail the genetic codon framework of the protein biosynthesis machinery. For example, the selector codon includes, but is not limited to, a unique three base codon, a nonsense codon, such as a stop codon, including, but not limited to, an amber codon (UAG) or an opal codon (UGA), an unnatural codon, a four or more base codon, a rare codon, or the like. There is a broad range of numbers of selector codons that can be introduced into a desired gene or polynucleotide, including, but not limited to, one or more, two or more, three or more, 4, 5, 6, 7, 8, 9, 10, or more in a single polynucleotide encoding at least a portion of a polypeptide of interest.
In one embodiment, the method comprises using a selector codon as a stop codon for in vivo incorporation of one or more unnatural amino acids. For example, an O-tRNA that recognizes a stop codon (including, but not limited to, UAG) is produced and aminoacylated with a desired unnatural amino acid by an O-RS. The O-tRNA is not recognized by the aminoacyl-tRNA synthetase of the naturally occurring host. Conventional site-directed mutagenesis may be used to create a stop codon (including, but not limited to, UAG) introduced at a site of interest in a polypeptide of interest. See, e.g., sayers, J.R. et al (1988), 5',3'Exonuclease in phosphorothioate-based oligomicleotide-directed mutagenesis, nucleic Acids Res,16 (3): 791-802. When the O-RS, the O-tRNA and the nucleic acid that encodes the polypeptide of interest are combined in vivo, an unnatural amino acid is incorporated in response to the UAG codon to yield a polypeptide that contains the unnatural amino acid at the indicated position.
The incorporation of unnatural amino acids can be performed in vivo without significant confusion in eukaryotic host cells. For example, because the suppression efficiency of a UAG codon depends on competition between an O-tRNA (including, but not limited to, an amber suppressor tRNA) and a eukaryotic release factor (including, but not limited to, eRF), which binds to a stop codon and causes release of a growing peptide from the ribosome, the suppression efficiency can be modulated by, including, but not limited to, increasing the expression level of the O-tRNA and/or the suppressor tRNA.
Selector codons also include extended codons that include, but are not limited to, four or more base codons (such as four, five, six or more base codons). Examples of four base codons include, but are not limited to AGGA, CUAG, UAGA, CCCU and analogs thereof. Examples of five base codons include, but are not limited to AGGAC, CCCCU, CCCUC, CUAGA, CUACU, UAGGC and analogs thereof. Features of the methods and compositions described herein include the use of codons that are extended according to frameshift suppression. Codons of four or more bases may insert one or more unnatural amino acids into the same protein, including but not limited to. For example, codons of four or more bases are read as a single amino acid in the presence of a mutant O-tRNA (including, but not limited to, a specific frameshift suppressor tRNA) that has an anticodon loop (e.g., has at least 8-10nt anticodon loops). In other embodiments, the anticodon loop is decodable, including, but not limited to, at least one four base codon, at least one five base codon, or at least one six base codon or more. Because there are 256 possible four base codons, multiple unnatural amino acids in the same cell can be encoded using codons of four or more bases. See Anderson et al, (2002) Exploring the Limits of Codon and Anticodon Size, chemistry and Biology,9:237-244; magliry, (2001) Expanding the Genetic Code: selection of Efficient Suppressors of Four-base Codons and Identification of "Shift" Four-base Codons with a Library Approach in Escherichia coli, J.mol.biol.307:755-769.
For example, four base codons have been used to incorporate unnatural amino acids into proteins using in vitro biosynthetic methods. See, for example, ma et al, (1993) Biochemistry,32:7939-7945; and Hohsaka et al, (1999) J.am.chem.Soc,121:34-40. The NBU derivatives of 2-naphthylalanine and lysine were simultaneously incorporated into streptavidin in vitro using CGGG and AGGU with two chemical acylated frameshift suppressor tRNA's. See, e.g., hohsaka et al, (1999) J.am.chem.Soc,121:12194-12195. In an in vivo study, moore et al studied the ability of tRNALeu derivatives with NCUA anti-codons to inhibit UAGN codons (N may be U, A, G or C), and found that quad UAGA could be decoded with 13% to 26% efficiency from tRNALeu with UCUA anti-codons, with only a few decoding in the 0 or-1 frames. See Moore et al, (2000) J.mol.biol.,298:195-205. In one embodiment, extended codons based on rare codons or nonsense codons can be used in the methods and compositions described herein, which can reduce missense read-through and frameshift suppression at other unwanted sites.
For a given system, a selector codon can also comprise one of the natural three base codons, with no (or little) use of the natural base codon by the endogenous system. For example, it includes a system that lacks a tRNA that recognizes a natural three base codon, and/or a system in which the three base codon is a rare codon.
The selector codon optionally comprises unnatural base pairs. These unnatural base pairs further expand the existing genetic symbology. One extra base pair increases the number of triplet codons from 64 to 125. The nature of the third base pair includes stable and selective base pairing, efficient enzymatic incorporation into DNA with high fidelity by a polymerase, and efficient continuous primer extension after primary unnatural base pair synthesis. Descriptions of unnatural base pairs that may be suitable for methods and compositions include, for example, hirao et al, (2002) An unnatural base pair for incorporating amino acid analogues into protein, nature Biotechnology,20:177-182, and also see Wu, Y.et al (2002) J.am.chem.Soc.124:14626-14630. Other related publications are listed herein.
For in vivo use, the unnatural nucleoside is membrane permeable and phosphorylated to form the corresponding triphosphate. In addition, the increased genetic information is stable and not disrupted by cellular enzymes. Previous efforts by Benner and others have utilized hydrogen bond types that are different from those in typical Watson-Crick base pairs, with the most notable example being the iso-C: iso-G pair. See, e.g., switzer et al, (1989) J.am.chem.Soc,111:8322-8322; and Picccirilli et al, (1990) Nature,343:33-37; kool, (2000) Curr.Opin.chem.biol.,4:602-608. These bases are often mismatched to some extent with the natural base and cannot be enzymatically replicated. Kool and co-workers demonstrated that hydrophobic stacking interactions between bases can replace hydrogen bonds to drive base pair formation. See, kool, (2000) curr. Opin. Chem. Biol.,4:602-608; and Guckian and Kool, (1998) Angew.chem.int.ed.Engl.,36 (24): 2825-2828). In an effort to develop unnatural base pairs that meet all of the above requirements, schultz, romesberg and co-workers have systematically synthesized and studied a range of unnatural hydrophobic bases. PICS self-pairing was found to be more stable than natural base pairing and it can be efficiently incorporated into DNA by the Klenow Fragment (KF) of E.coli DNA polymerase I. See, for example, mcMinn et al, (1999) J.am.chem.Soc,121:11585-11586; and Ogawa et al, (2000) J.am.chem.Soc,122:3274-3278. The 3MN-3MN self pairing can be synthesized by KF with sufficient efficiency and selectivity for biological functions. See, for example, ogawa et al, (2000) J.am.chem.Soc,122:8803-8804. However, both bases act as chain terminators for further replication. More recently, mutant DNA polymerases have been developed that can be used to replicate PICS self-pairing. In addition, the 7AI self-pairing can be replicated. See, e.g., tae et al, (2001) J.am.chem.Soc,123:7439-7440. Novel metal base pairs Dipic: py have also been developed that form stable pairs upon binding to Cu (II). See, meggers et al, (2000) J.am.chem.Soc,122:10714-10715. Because the extended codons and unnatural codons are inherently orthogonal to the natural codons, the unnatural amino acid methods described herein can take advantage of this property to generate orthogonal tRNA's with respect thereto.
Translation bypass systems can also be used to incorporate unnatural amino acids into desired polypeptides. In the translational bypass system, large sequences are incorporated into genes, but are not translated into proteins. The sequence contains a structure that acts as a signal that induces ribosomes to cross the sequence and continue translation downstream of the insertion.
In certain embodiments, the protein or polypeptide of interest (or portion thereof) in the methods and/or compositions described herein is encoded by a nucleic acid. Typically, the nucleic acid comprises at least one selector codon, at least two selector codons, at least three selector codons, at least four selector codons, at least five selector codons, at least six selector codons, at least seven selector codons, at least eight selector codons, at least nine selector codons, ten or more selector codons.
Genes encoding proteins or polypeptides of interest may be mutagenized under the direction of "Mutagenesis and Other Molecular Biology Techniques" using methods well known to those skilled in the art and described herein to include, for example, one or more selector codons for incorporation of unnatural amino acids. For example, nucleic acids of the protein of interest are mutagenized to include one or more selector codons, providing for the incorporation of one or more unnatural amino acids. The methods and compositions described herein include any such variant, including, but not limited to, mutant forms of any protein (e.g., comprising at least one unnatural amino acid). Similarly, the methods and compositions described herein comprise the corresponding nucleic acids, i.e., any nucleic acid having one or more selector codons that encode or permit in vivo incorporation of one or more unnatural amino acids.
Nucleic acid molecules encoding polypeptides of interest, including by way of example only GH polypeptides, can be susceptible to mutation to introduce cysteines at any desired position in the polypeptide. Cysteine is widely used to introduce reactive molecules, water-soluble polymers, proteins, or a variety of other molecules onto a protein of interest. Methods suitable for incorporating cysteines into desired positions of polypeptides are well known in the art, such as those described in U.S. patent No. 6,608,183 (which is incorporated herein by reference in its entirety), as well as standard mutation forming techniques. The use of such techniques for introducing and utilizing cysteine may be combined with the techniques for introducing and utilizing unnatural amino acids described herein.
In vivo production of polypeptides comprising unnatural amino acids
For convenience, the in vivo production of polypeptides comprising unnatural amino acids described in this section is described generally and/or with specific examples. However, in vivo production of polypeptides comprising unnatural amino acids described in this section should not be limited to the general description or specific examples provided in this section, but rather the in vivo production of polypeptides comprising unnatural amino acids described in this section is equally well suited for all compounds within the categories of formulas I-XVIII, XXX-XXXIV (A and B) and XXXXX-XXXXIII, including any or specific compounds within the categories of formulas I-XVIII, XXX-XXXIV (A and B) and XXXXXXX-XXXXIII described in the specification, claims and drawings herein.
The polypeptides described herein can be produced in vivo using tRNA's and tRNA synthetases that are modified to add or replace amino acids that are not encoded in a naturally occurring system.
Methods of producing tRNA's and tRNA synthetases that use amino acids that are not encoded in a naturally occurring system are described, for example, in U.S. patent application publication No. 2003/0082575 (Ser. No. 10/126,927) and 2003/0108885 (Ser. No. 10/126,931), which are incorporated by reference in their entireties. These methods include the generation of translation mechanisms that function independently of synthetases and tRNA's that are endogenous to the translation system (and thus sometimes referred to as "orthogonal"). In one embodiment, the translation system comprises a polynucleotide encoding a polypeptide; the polynucleotide may be an mRNA transcribed from the corresponding DNA, or the mRNA may be produced from an RNA viral vector; in addition, the polynucleotide includes a selector codon that corresponds to a pre-specified site for incorporation of an unnatural amino acid. The translation system further includes a tRNA for and (where appropriate) also an unnatural amino acid, where the tRNA has specificity/recognition for/a selector codon; in other embodiments, the unnatural amino acid is aminoacylated. Unnatural amino acids include those unnatural amino acids having the structure of any of formulas I-XVIII, XXX-XXXIV (A and B), and XXXX-XXXXIII described herein. In other or additional embodiments, the translation system includes an aminoacyl-synthetase that is specific for the tRNA, and in other or additional embodiments, the translation system includes an orthogonal tRNA and an orthogonal aminoacyl-tRNA synthetase. In other or additional embodiments, the translation system includes at least one of: a plasmid comprising the aforementioned polynucleotide (such as, for example, in the form of DNA only), a genomic DNA comprising the aforementioned polynucleotide (such as, for example, in the form of DNA only), or a genomic DNA into which the aforementioned polynucleotide is integrated (in other embodiments, the integration is stable integration). In other or additional embodiments of the translation system, the selector codon is selected from the group consisting of an amber codon, an ocher codon, a opal codon, a unique codon, a rare codon, a non-natural codon, a five base codon, and a four base codon. In other or additional embodiments of the translation system, the tRNA is a suppressor tRNA. In other or additional embodiments, the unnatural amino acid polypeptide is synthesized from a ribosome.
In other or additional embodiments, the translation system includes an orthogonal tRNA (O-tRNA) and an orthogonal aminoacyl tRNA synthetase (O-RS). Typically, in a translation system, an O-RS preferentially aminoacylates an O-tRNA with at least one unnatural amino acid and the O-tRNA recognizes at least one selector codon that is not recognized by other tRNAs in the system. The translation system thus inserts unnatural amino acids into the polypeptides produced in the system in response to the encoded selector codon, thereby "substituting" the unnatural amino acids into the position in the encoded polypeptide.
A variety of orthogonal tRNA's and aminoacyl tRNA synthetases for inserting specific synthetic amino acids into polypeptides have been described in the art and are generally suitable for use in the methods described herein to produce the unnatural amino acid polypeptides described herein. For example, ketone-specific O-tRNA/aminoacyl-tRNA synthetases are described in Wang, L. Et al, proc. Natl. Acad. Sci. USA 100 (1): 56-61 (2003), zhang, Z. Et al, biochem.42 (22): 6735-6746 (2003). Exemplary O-RSs, or portions thereof, are encoded by polynucleotide sequences and include the amino acid sequences disclosed in U.S. patent application publications 2003/0082575 and 2003/0108885, each of which is incorporated herein by reference in its entirety. Corresponding O-tRNA molecules for use with O-RSs are also described in U.S. patent application publication No. 2003/0082575 (Ser. No. 10/126,927) and 2003/0108885 (Ser. No. 10/126,931), which are incorporated herein by reference in their entirety. In addition, mehl et al, J.am.chem.Soc.2003;125:935-939, santoro et al Nature Biotechnology, month 10 2002; 20:1044-1048, which is incorporated herein by reference in its entirety, discusses screening methods and aminoacyl tRNA synthetases and tRNA molecules that are used to incorporate para-aminophenylalanine into polypeptides.
Exemplary O-tRNA sequences suitable for use in the methods described herein include, but are not limited to, the nucleotide sequences SEQ ID NOS: 1-3 as disclosed in U.S. patent application publication No. 2003/0108885 (Ser. No. 10/126,931), which is incorporated herein by reference. Other examples of O-tRNA/aminoacyl-tRNA synthetase pairs that are specific for a particular unnatural amino acid are described in U.S. patent application publication No. 2003/0082575 (Ser. No. 10/126,927), which is incorporated by reference herein in its entirety. Combining O-RS and O-tRNA's containing both ketone and azide amino acids in Saccharomyces cerevisiae is described in Chin, J.W. et al, science 301:964-967 (2003).
Uses for the O-tRNA/aminoacyl-tRNA synthetases include selecting specific codons that encode an unnatural amino acid. Although any codon can be used, it is generally desirable to select codons that are rarely or never used in the cell in which the O-tRNA/aminoacyl-tRNA synthetase is expressed. By way of example only, exemplary codons include nonsense codons, such as stop codons (amber, ocher, and opal), four or more base codons, and other natural three base codons that are rarely or not used.
Specific selector codons can be introduced into appropriate positions in the polynucleotide coding sequence using mutation-forming methods known in the art, including, but not limited to, site-directed mutagenesis, cassette mutagenesis, restriction-selection mutagenesis, and the like.
Methods for producing components useful in protein biosynthesis mechanisms that incorporate unnatural amino acids, such as O-RS, O-tRNA and orthogonal O-tRNA/O-RS pairs, are described in Wang, L. et al, science 292:498-500 (2001); chin, J.W. et al, J.Am.chem.Soc.124:9026-9027 (2002); zhang, Z.et al, biochemistry 42:6735-6746 (2003). Methods and compositions for in vivo incorporation of unnatural amino acids are described in U.S. patent application publication 2003/0082575 (Ser. No. 10/126,927), which is incorporated by reference herein in its entirety. Methods for selecting orthogonal tRNA-tRNA synthetase pairs for use in an in vivo translation system of an organism are also described in U.S. patent application publication Nos. 2003/0082575 (Ser. No. 10/126,927) and 2003/0108885 (Ser. No. 10/126,931), which are incorporated herein by reference in their entirety. In addition, PCT publication No. WO 04/035743 entitled "Site Specific Incorporation of Keto Amino Acids into proteins," which is incorporated by reference in its entirety, describes orthogonal RS and tRNA pairs for incorporating keto amino acids. PCT publication No. WO 04/094593, entitled "Expanding the Eukaryotic Genetic Code," which is incorporated herein by reference in its entirety, describes orthogonal RS and tRNA pairs for incorporating non-naturally encoded amino acids into eukaryotic host cells.
The method for producing at least one recombinant orthogonal aminoacyl-tRNA synthetase (O-RS) comprises: (a) Generating a pool of (optionally mutant) aminoacyl-tRNA synthetases (RSs) derived from a first organism, where the first organism comprises (but is not limited to) a prokaryotic organism, such as, by way of example only, methanococcus jannaschii, methanobacterium thermoautotrophicum, salmonella, escherichia coli, archaeoglobus fulgidus, thermococcus furiosus, thermococcus renavium, thermus thermophilus, or an analog thereof, or a eukaryotic organism; (b) Selecting (and/or screening) members of a library of RSs (optionally mutant RSs) that aminoacylate an orthogonal tRNA (O-tRNA) in the presence of an unnatural amino acid and a natural amino acid, thereby providing a pool of active (optionally mutant) RSs; and/or, (c) selecting (optionally by negative selection) active RSs (including but not limited to mutant RSs) in the pool that preferentially aminoacylate the O-tRNA in the absence of an unnatural amino acid, thereby providing at least one recombinant O-RS; wherein at least one of the recombinant O-RSs preferentially aminoacylates the O-tRNA with the unnatural amino acid.
In one embodiment, the RS is an inactive RS. Inactive RSs may be generated by mutating active RSs. Merely by way of example, an inactive RS can be generated by mutating 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 at least about 10 or more amino acids to different amino acids, including but not limited to alanine.
The mutant RS libraries can be generated using a variety of techniques known in the art, including, but not limited to, rational design based on the three-dimensional RS structure of the protein, or mutant formation of RS nucleotides in random or rational design techniques. By way of example only, mutant RSs may be generated by site-specific mutations, random mutations, recombinant mutations that create diversity, chimeric constructs, rational design, and other methods described herein or known in the art.
In one embodiment, active members of the library of RSs (optionally mutant RSs) are selected (and/or screened), including but not limited to those members that aminoacylate an orthogonal tRNA (O-tRNA) in the presence of an unnatural amino acid and a natural amino acid, including but not limited to: introducing a positive selection or screening marker (including but not limited to an antibiotic resistance gene or analog thereof) and (optionally a mutant) RS library into a plurality of cells, wherein the positive selection and/or screening marker comprises at least one selector codon including but not limited to an amber codon, an ocher codon, a opal codon, a unique codon, a rare codon, an unnatural codon, a five base codon, and a four base codon; growing the plurality of cells in the presence of a selection agent; identifying cells that survive (or exhibit a specific response) in the presence of the selection and/or screening agent by suppressing at least one selector codon in the positive selection or screening marker, thereby providing a subset of positive selection cells of the pool containing active (optionally mutant) RSs. Optionally, the selection and/or screening agent concentration may vary.
In one aspect, the positive selection marker is a Chloramphenicol Acetyl Transferase (CAT) gene and the selector codon is an amber stop codon in the CAT gene. Optionally, the positive selection marker is a beta-lactamase gene and the selector codon is an amber stop codon in the beta-lactamase gene. In another aspect, positive screening markers include fluorescent or luminescent screening markers or affinity-based screening markers (including but not limited to cell surface markers).
In one embodiment, active RSs (optionally mutants) in the negative selection or screening pool, including but not limited to those active RSs that preferentially aminoacylate the O-tRNA in the absence of an unnatural amino acid, include but are not limited to: introducing a negative selection or screening marker into a plurality of cells of a second organism in a pool of active (optionally mutant) RSs from a positive selection or screening, wherein the negative selection or screening marker comprises at least one selector codon including, but not limited to, an antibiotic resistance gene including, but not limited to, a Chloramphenicol Acetyl Transferase (CAT) gene; and identifying cells that survive or exhibit a specific screening response in a first medium supplemented with an unnatural amino acid and a screening or selecting agent, but that do not survive or exhibit a specific response in a second medium not supplemented with an unnatural amino acid and a selecting or screening agent, thereby providing surviving cells or screened cells with at least one recombinant O-RS. Merely by way of example, the CAT identification protocol optionally serves as a positive selection and/or negative screening for determining the appropriate O-RS recombinants. For example, the clone pool is replicated on a growth plate containing CAT (which includes at least one selector codon), optionally with or without one or more unnatural amino acids. The only colonies grown on plates containing unnatural amino acids are therefore considered to contain recombinant O-RS. In one aspect, the concentration of the selective (and/or screening) agent is varied. In some aspects, the first organism is different from the second organism. Thus, the first organism and/or the second organism optionally comprises: prokaryotes, eukaryotes, mammals, escherichia coli, fungi, yeasts, archaebacteria, eubacteria, plants, insects, protozoa, and the like. In other embodiments, the screening label comprises a fluorescent or luminescent screening label or an affinity based screening label.
In another embodiment, screening or selecting (including but not limited to) active (optionally mutant) RSs in the pool includes (but is not limited to): isolating the pool of active mutant RS from positive selection step (b); introducing a pool of negative selection or screening markers, wherein the negative selection or screening markers comprise at least one selector codon (including, but not limited to, a toxic marker gene comprising, but not limited to, a ribonuclease barnase gene comprising at least one selector codon) and an active (optionally mutant) RS into a plurality of cells of a second organism; and identifying cells that survive or exhibit a specific screening response in a first medium that is not supplemented with an unnatural amino acid, but that do not survive or exhibit a specific screening response in a second medium that is supplemented with an unnatural amino acid, thereby providing surviving cells or screened cells with at least one recombinant O-RS, where the at least one recombinant O-RS is specific for the unnatural amino acid. In one aspect, the at least one selector codon comprises about two or more selector codons. These embodiments optionally can include wherein at least one selector codon includes two or more selector codons, and wherein the first organism is different from the second organism (including, but not limited to), each organism optionally including, but not limited to, a prokaryote, eukaryote, mammal, escherichia coli, fungus, yeast, archaebacteria, eubacteria, plants, insects, protozoa, and the like. Also, some aspects comprise wherein the negative selection marker comprises a ribonuclease barnase gene (which comprises at least one selector codon). Other aspects include wherein the screening label optionally comprises a fluorescent or luminescent screening label or an affinity-based screening label. In embodiments herein, screening and/or selecting optionally includes a change in screening and/or selection stringency.
In another embodiment, the method of producing at least one recombinant orthogonal aminoacyl-tRNA synthetase (O-RS) can further comprise: (d) isolating at least one recombinant O-RS; (e) Generating a second set of O-RSs (optionally mutated) derived from at least one recombinant O-RS; and, (f) repeating steps (b) and (c) until a mutant O-RS is obtained that includes the ability to preferentially aminoacylate the O-tRNA. Optionally, repeating steps (d) - (f) (including, but not limited to) at least about twice. In one aspect, the second set of mutant O-RSs derived from the at least one recombinant O-RS may be produced by mutagenesis, including but not limited to random mutagenesis, site-specific mutagenesis, recombination, or a combination thereof.
The stringency of the selection/screening step includes, but is not limited to, positive selection/screening step (b), negative selection/screening step (c) or both positive and negative selection/screening steps (b) and (c), in which method the selection/screening stringency is optionally altered. In another embodiment, the positive selection/screening step (b), the negative selection/screening step (c), or both the positive and negative selection/screening steps (b) and (c) comprise the use of a reporter gene, wherein the reporter gene is detected by Fluorescence Activated Cell Sorting (FACS) or wherein the reporter gene is detected by luminescence. Optionally, the reporter gene is displayed on the cell surface, on a phage display or the like and is selected according to affinity or catalytic activity involving the unnatural amino acid or the like. In one embodiment, the mutant synthetases are displayed on the cell surface, on a phage display, or the like.
Methods of producing a recombinant orthogonal tRNA (O-tRNA) include, but are not limited to: (a) Generating a pool of mutant tRNA's derived from at least one tRNA, including but not limited to, suppressor tRNA's from a first organism; (b) Selecting (including but not limited to) a negative selection or screening a pool of (optionally mutant) tRNA's that were aminoacylated by an aminoacyl-tRNA synthetase (RS) from a second organism in the absence of RS from the first organism, thereby providing a pool of tRNA's (optionally mutants); and, (c) selecting or screening for members of the pool of tRNA's (optionally mutants) aminoacylated by the introduced orthogonal RS (O-RS), thereby providing at least one recombinant O-tRNA; wherein at least one of the recombinant O-tRNA recognizes a selector codon and is not efficiently recognized by an RS from the second organism and is preferentially aminoacylated by the O-RS. In some embodiments, the at least one tRNA is a suppressor tRNA and/or a unique three base codon that includes natural and/or unnatural bases, or is a nonsense codon, rare codon, unnatural codon, a codon that includes at least 4 bases, an amber codon, an ocher codon, or an opal stop codon. In one embodiment, the recombinant O-tRNA has an increase in orthogonality. It will be appreciated that in some embodiments, the O-tRNA is optionally introduced into the first organism from the second organism without modification. In various embodiments, the first organism is the same as or different from the second organism and is optionally selected from (including, but not limited to) prokaryotes (including, but not limited to, methanococcus jannaschii, methanobacterium thermoautotrophicum, escherichia coli, halophilus, etc.), eukaryotes, mammals, fungi, yeast, archaebacteria, eubacteria, plants, insects, protists, and the like. In addition, the recombinant tRNA is optionally aminoacylated with an unnatural amino acid, where the unnatural amino acid is biosynthesized in vivo, either naturally or by genetic manipulation. Optionally, the unnatural amino acid is added to the growth medium of at least the first organism or the second organism, where the unnatural amino acid is capable of achieving an appropriate intracellular concentration to allow incorporation into the unnatural amino acid polypeptide.
In one aspect, selecting (including but not limited to) a negative selection or screening pool for (optionally mutant) tRNA aminoacylated by an aminoacyl-tRNA synthetase (step (b)) comprises: introducing a pool of a toxic marker gene (wherein the toxic marker gene comprises at least one selector codon (or a gene necessary for a gene or organism that results in the production of a toxic or static agent, wherein such marker gene comprises at least one selector codon)) and (optionally a mutant) tRNA into a plurality of cells from a second organism; and selecting a surviving cell, wherein the surviving cell comprises a pool of (optionally mutant) tRNA's comprising at least one orthogonal tRNA or nonfunctional tRNA. For example, surviving cells can be selected by using a comparative ratio cell density assay.
Alternatively, the toxic marker gene may comprise two or more selector codons. In another embodiment of the methods described herein, the toxic marker gene is a ribonuclease barnase gene, wherein the ribonuclease barnase gene comprises at least one amber codon. Optionally, the ribonuclease barnase gene may comprise two or more amber codons.
In one embodiment, selecting or screening (optionally mutant) a member of the pool of tRNA's aminoacylated by the introduced orthogonal RS (O-RS) can comprise: introducing a pool of positive selection or screening marker genes (wherein the positive marker genes include a drug resistance gene (including but not limited to a beta-lactamase gene including at least one selector codon, such as at least one amber stop codon) or genes necessary for the organism, or genes that detoxify toxic agents) and an O-RS and (optionally mutant) tRNA into a plurality of cells from a second organism; and identifying surviving cells or screened cells grown in the presence of a selection or screening agent, including but not limited to an antibiotic, thereby providing a cell pool having at least one recombinant tRNA, wherein at least one recombinant tRNA is aminoacylated by an O-RS and an amino acid is inserted into a translation product encoded by a positive marker gene in response to the at least one selector codon. In another embodiment, the concentration of the selection and/or screening agent is varied.
Methods of producing specific O-tRNA/O-RS pairs are provided. Methods include (but are not limited to): (a) Generating a pool of mutant tRNA's derived from at least one tRNA from a first organism; (b) A pool of (optionally mutant) trnas in the negative selection or screening pool that were aminoacylated by an aminoacyl-tRNA synthetase (RS) from a second organism in the absence of an RS from the first organism, thereby providing a pool of (optionally mutant) trnas; (c) The members aminoacylated by the introduced orthogonal RS (O-RS) in the pool of (optionally mutant) tRNA are selected or screened to provide at least one recombinant O-tRNA. The at least one recombinant O-tRNA recognizes a selector codon and is not efficiently recognized by an RS from the second organism and is preferentially aminoacylated by the O-RS. The method also comprises (d) generating a pool of (optionally mutant) aminoacyl-tRNA synthetases (RSs) derived from at least one RS from a third organism; (e) Selecting or screening a pool of mutant RSs for preferential aminoacylation of a member of the at least one recombinant O-tRNA in the presence of an unnatural amino acid and a natural amino acid, thereby providing a pool of active (optionally mutant) RSs; and (f) preferentially aminoacylating the active (optionally mutant) RS of the at least one recombinant O-tRNA in the absence of the unnatural amino acid in the negative selection or screening pool, thereby providing at least one specific O-tRNA/O-RS pair, where the at least one specific O-tRNA/O-RS pair comprises at least one recombinant O-RS that is specific for the unnatural amino acid and at least one recombinant O-tRNA. Specific O-tRNA/O-RS pairs produced by the methods described herein are included in the scope and methods described herein. For example, the specific O-tRNA/O-RS pair can include (including, but not limited to) a mutRNATyr-mutTyrRS pair (such as a mutRNATyr-SS12TyrRS pair), a mutRNALeu-mutLeuRS pair, a mutRNAThr-mutThrRS pair, a mutRNAGlu-mutGluRS pair, or the like. In addition, these methods comprise wherein the first organism and the third organism are the same (including, but not limited to, methanococcus jannaschii).
Methods for selecting orthogonal tRNA-tRNA synthetase pairs for use in an in vivo translation system of a second organism are also included in the methods described herein. Methods include (but are not limited to): introducing a marker gene, a tRNA, and an aminoacyl-tRNA synthetase (RS) that is isolated from or derived from a first organism into a first set of cells from a second organism; introducing the marker gene and the tRNA into a duplicate cell set from a second organism; and selecting surviving cells in a first group that are not viable in the duplicate cell group or screening for cells in the duplicate cell group that are not capable of producing the specific screening response, wherein the first group and the duplicate cell group are grown in the presence of a selection or screening agent, wherein the surviving cells or the screened cells comprise orthogonal tRNA-tRNA synthetase pairs for use in an in vivo translation system of a second organism. In one embodiment, the comparing and selecting or screening comprises in vivo complementation assays. The concentration of the selection or screening agent may vary.
Organisms described herein include a variety of organisms and a variety of combinations. In one embodiment, the organism is optionally a prokaryotic organism, including, but not limited to, methanococcus jannaschii, methanobacterium thermoautotrophicum, salmonella, escherichia coli, archaeoglobus fulgidus, pyrococcus furiosus, thermococcus celer, thermus thermophilus, or an analog thereof. Alternatively, the organism is a eukaryotic organism that includes, but is not limited to, plants (including, but not limited to, complex plants such as monocots or dicots), algae, protozoa, fungi (including, but not limited to, yeast, etc.), animals (including, but not limited to, mammals, insects, arthropods, etc.), or the like.
A. Expression in non-eukaryotic and eukaryotic organisms
The techniques disclosed in this section are applicable to the expression of the unnatural amino acid polypeptides described herein in non-eukaryotic and eukaryotic organisms.
To obtain high levels of expression of the cloned polynucleotide, the polynucleotide encoding the desired polypeptide is typically subcloned into an expression vector containing a strong promoter to direct transcription, a transcription/translation terminator, and if a nucleic acid encoding a protein, a ribosome binding site for translation initiation is typically subcloned. Suitable bacterial promoters are described, for example, in Sambrook et al, and Ausubel et al.
Bacterial expression systems for expressing polypeptides can be used in (including, but not limited to) E.coli, bacillus sp, pseudomonas fluorescens, pseudomonas aeruginosa, pseudomonas putida, and Salmonella (Palva et al, gene 22:229-235 (1983); mosbach et al, nature 302:543-545 (1983)). Kits for these expression systems are commercially available. Eukaryotic expression systems for mammalian cells, yeast and insect cells are commercially available. In the case where an orthogonal tRNA and aminoacyl tRNA synthetase (described elsewhere herein) are used to express the polypeptide, the host cell used for expression is selected for its ability to use the orthogonal component. Exemplary host cells include Gram-positive bacteria (Gram-positive bacteria) (including, but not limited to, brevibacterium (B.brevis) or Bacillus subtilis (B.subtilis) or Streptomyces (Streptomyces)), and Gram-negative bacteria (Gram-negative bacteria) (E.coli or Pseudomonas fluorescens, pseudomonas aeruginosa, pseudomonas putida), as well as yeast and other eukaryotic cells. Cells comprising O-tRNA/O-RS pairs can be used as described herein.
Eukaryotic or non-eukaryotic host cells as described herein provide the ability to synthesize polypeptides comprising large, suitable amounts of unnatural amino acids. In one aspect, the composition optionally comprises (but is not limited to) at least 10 micrograms, at least 50 micrograms, at least 75 micrograms, at least 100 micrograms, at least 200 micrograms, at least 250 micrograms, at least 500 micrograms, at least 1 milligram, at least 10 milligrams, at least 100 milligrams, at least 1 gram, or 1 gram of the polypeptide comprising the unnatural amino acid or more, or an amount obtainable by an in vivo polypeptide production process (details regarding recombinant protein production and purification are provided herein). In another aspect, the polypeptide is optionally present in the composition at a concentration of at least 10 micrograms of polypeptide per liter, at least 50 micrograms of polypeptide per liter, at least 75 micrograms of polypeptide per liter, at least 100 micrograms of polypeptide per liter, at least 200 micrograms of polypeptide per liter, at least 250 micrograms of polypeptide per liter, at least 500 micrograms of polypeptide per liter, at least 1 milligram of polypeptide per liter, or at least 10 milligrams of polypeptide per liter or more in (including, but not limited to) a cell lysate, buffer, pharmaceutical buffer, or other liquid suspension (including, but not limited to) a volume of between about 1nl to about 100 liters. The production of large amounts (including, but not limited to, greater than would normally be obtained by other methods (including, but not limited to, in vitro translation)) of proteins in eukaryotic cells comprising at least one unnatural amino acid is a feature of the methods, techniques, and compositions described herein.
Eukaryotic or non-eukaryotic host cells as described herein provide the ability to biosynthesize proteins including large, suitable amounts of unnatural amino acids. For example, a polypeptide comprising an unnatural amino acid can be produced at a concentration of at least 10 micrograms/liter, at least 50 micrograms/liter, at least 75 micrograms/liter, at least 100 micrograms/liter, at least 200 micrograms/liter, at least 250 micrograms/liter, or at least 500 micrograms/liter, at least 1 milligram/liter, at least 2 milligram/liter, at least 3 milligram/liter, at least 4 milligram/liter, at least 5 milligram/liter, at least 6 milligram/liter, at least 7 milligram/liter, at least 8 milligram/liter, at least 9 milligram/liter, at least 10 milligram/liter, at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900 milligram/liter, 1 gram/liter, 5 gram/liter, 10 gram/liter, or more polypeptide in a cell extract, cell lysate, culture medium, buffer, and/or the like.
1.Expression system, culture and isolation
The techniques disclosed in this section can be applied to expression systems, cultures, and separations of the unnatural amino acid polypeptides described herein. The unnatural amino acid polypeptide can be expressed in any number of suitable expression systems, including, but not limited to, yeast, insect cells, mammalian cells, and bacteria. A description of an exemplary expression system is provided herein.
YeastAs used herein, the term "yeast" includes any of a variety of yeasts capable of expressing a gene encoding a non-natural amino acid polypeptide. These yeasts include, but are not limited to, ascomycetes (Endomycetes), basidiomycetes, and yeasts belonging to the phylum semi-known (Fungium) and (Saccharomyces) division into two families, the genus Spyomyces (Spyomyceae) and the family Saccharomyces (Saccharomyces) the latter includes four subfamilies, the subfamilies (Schizosaccharomyces) (e.g., schizosaccharomyces (genus Schizosaccharomyces)), the subfamilies (Nadsonoideae), the subfamilies (Lipomycideae) (Pichia pastoris) (e.g., saccharomyces), the genus Kluyveromyces (genus Kluyveromyces) and the genus Saccharomyces (genus Saccharomyces)) and the basidiomyces (Saccharomyces) include the subfamilies (genus Leucosporidium), the rhodotorula (rhodosporidium), the genus Phaffia (rhodosporum) (e.g., saccharomyces (26), the subfamilies (Saccharomyces) and the subfamilies (e.g., saccharomyces (35), the genus Saccharomyces (Saccharomyces) and the genus Saccharomyces (e.g., saccharomyces (37), the subfamily (Saccharomyces) and the subfamilies (Saccharomyces) of the genus Saccharomyces (Saccharomyces).
In certain embodiments, pichia, kluyveromyces, saccharomyces, schizosaccharomyces, hansenula (Hansenula), candida (tonoplastis), and species within candida (including, but not limited to, pichia pastoris (p. Pastoris), p. Gullerimondii, saccharomyces cerevisiae (s. Cerevisiae), brewer's yeast (s. Carlsbergensis), saccharifying yeast (s. Dialecticus), douglas yeast (s. Douglas), s. Khiyveri, s. Norbensis, oval yeast (s. Oviformis), kluyveromyces lactis (k. Lacti), kluyveromyces fragilis (k. Fragis), candida albicans (c. Albicans), candida maltosa (c. Maculosa), and han.
The selection of suitable yeasts for the expression of the unnatural amino acid polypeptide is within the skill of those skilled in the art. In selecting yeast hosts for expression, suitable hosts may include, but are not limited to, those that exhibit good secretion capacity, low proteolytic activity, and overall activity, for example. Yeasts are commonly available from a variety of sources including, but not limited to, the yeast genetic reserve center of the university of california (Berkeley, CA) biophysical and medical physical lines, (Yeast Genetic Stock Center, department of Biophysics and Medical Physics, university of California (Berkeley, CA)) and the american type culture collection (American Type Culture Collection) ("ATCC") (Manassas, VA).
The term "yeast host" or "yeast host cell" encompasses yeasts that can or have been used as recipients of recombinant vectors or other transfer DNA. The term encompasses the progeny of the original yeast host cell that has received the recombinant vector or other transfer DNA. It will be appreciated that due to sporadic or deliberate mutation, the progeny of a single parent cell are completely identical in morphology or genome or total DNA complementary to the original parent. Offspring of a parent cell that are substantially similar to a parent to be characterized by a relevant property, such as the presence of a nucleotide sequence encoding an unnatural amino acid polypeptide, are included in the offspring that are meant by this definition.
Expression and transformation vectors (including extrachromosomal replicons or integrating vectors) have been developed for transformation into a number of yeast hosts. For example, expression vectors have been developed for Saccharomyces cerevisiae (Sikorski et al, GENETICS (1998) 112:19; ito et al, J.BACTERIIOL (1983) 153:163; hinnen et al, PROC.NATL. ACAD. SCI. USA (1978) 75:1929); candida albicans (Kurtz et al, M) OL .C ELL .B IOL (1986) 6:142); candida maltosa (Kunze et al, J.BASIC MICrobiol. (1985) 25:141); hansenula polymorpha (Gleeson et al, J.GEN.MICROBIOL. (1986) 132:3459; roggenkamp et al, M) OL Gen.genet. (1986) 202:302); kluyveromyces fragilis (Das et al, J.BACTERIIOL (1984) 158:1165); kluyveromyces lactis (De Louvencourt et al J.BACTERIOL. (1983) 154:737;Van den Berg et al)BIO/TECHNOLOGY (1990) 8:135); guilerimondii (Kunze et al, J.BASIC MICROBIOL. (1985) 25:141); pichia pastoris (U.S. Pat. No. 5,324,639; no. 4,929,555; and No. 4,837,148; cragg et al, mol. Cell. BIOL (1985) 5:3376); schizosaccharomyces pombe (Schizosaccharomyces pombe) (beacon and Nurse, NATURE (1981) 300:706); yarrowia lipolytica (Y.lipolytica) (Davidow et al, CURR.GENET. (1985) 10:380 (1985); gaillindin et al, CURR.G.) ENET (1985) 10:49); aspergillus nidulans (A. Nidulans) (Ballance et al, BIOCHEM. BIOPHYS. RES. COMMUN. (1983) 112:284-89; tilburn et al, G) ENE (1983) 26:205-221; and Yelton et al, PROC NATL ACAD. SCI. USA (1984) 81:1470-74); aspergillus niger (A. Niger) (Kelly and Hynes, EMBO J. (1985) 4:475-479); reesia (EP 0 244 234); and filamentous fungi such as Neurospora (Neurospora), penicillium (Penicillium), curvularia (Tolypocladium) (WO 91/00357), each of which is incorporated herein by reference in its entirety.
Control sequences for yeast vectors include, but are not limited to, those derived from, for example, alcohol Dehydrogenase (ADH) (EP 0 284 044); enolase; glucokinase; glucose-6-phosphate isomerase; glyceraldehyde-3-phosphate dehydrogenase (GAP or GAPDH); hexokinase; phosphofructokinase; 3-phosphoglycerate mutase; and the promoter region of the gene for pyruvate kinase (PyK) (EP 0 329 203). Yeast PH05 gene encoding acid phosphatase may also provide a suitable promoter sequence (Miyanohara et al, PROC. NATL. A CAD SCI.USA (1983) 80:1). Other suitable promoter sequences for yeast hosts may include 3-phosphoglycerate kinase (Hitzeman et al, J. BIOL. CHEM. (1980) 255 (4): 12073-12080); and other glycolytic enzymes such as pyruvate decarboxylase, triose phosphate isomerase, and glucose phosphate isomerase (Holland et al, BIOCHEMISTRY (1978) 17 (23): 4900-4907; hess et al, J.ADV.ENZYME REG. (1969) 7:149-167). Inducible yeast promoters having the additional advantage of transcription controlled by growth conditions can comprise alcohol dehydrogenase 2; heterocytochrome C; an acid phosphatase; metallothionein; glyceraldehyde-3-phosphate dehydrogenase; degrading enzymes associated with nitrogen metabolism; the start of enzymes responsible for maltose and galactose utilization A mover region. Suitable vectors and promoters for use in yeast expression are further described in EP 0073 657.
Yeast enhancers can also be used with yeast promoters. In addition, synthetic promoters may also act as yeast promoters. For example, an Upstream Activating Sequence (UAS) of a yeast promoter can be ligated to a transcription activating region of another yeast promoter to produce a synthetic hybrid promoter. Examples of such hybrid promoters include ADH regulatory sequences linked to GAP transcriptional activation regions. See U.S. patent nos. 4,880,734 and 4,876,197, which are incorporated by reference in their entirety. Other examples of hybrid promoters include promoters consisting of the regulatory sequences of the ADH2, GAL4, GAL10 or PHO5 genes in combination with the transcriptional activation region of a glycolytic enzyme gene, such as GAP or PyK. See EP 0 164 556. In addition, the yeast promoter may comprise a naturally occurring promoter of non-yeast origin that has the ability to bind to yeast RNA polymerase and initiate transcription.
Other control elements that may form part of a yeast expression vector include, for example, terminators from GAPDH or enolase genes (Holland et al, J.BlOL. C) HEM . (1981) 256:1385). In addition, the origin of replication from the 2. Mu. Plasmid origin is suitable for use in yeast. Suitable selection genes for use in yeast are the trpl genes present in yeast plasmids. See Tschumper et al, GENE (1980) 10:157; kingsman et al, GENE (1979) 7:141. the trpl gene provides a selectable marker for mutant strains of yeast lacking the ability to grow in tryptophan. Similarly, leu 2-deficient yeast strains (ATCC 20,622 or 38,626) are complemented by known plasmids having the Leu2 gene.
Methods of introducing exogenous DNA into a yeast host include, but are not limited to, transformation of spheroplasts or intact yeast host cells treated with alkali metal cations. For example, transformation of yeast may be according to Hsiao et al, P ROC .N ATL .A CAD .S CI USA (1979) 76:3829, van Solingen et al, J.BACT. (1977) 130:946. However, other methods of introducing DNA into cells (such as by nuclear injection, electroporation, or protoplast fusion) may also be used according to S AMBROOK Et al, M OLECULAR C LONING :A L AB .M ANUAL (2001) Is generally used as described in the specification. The yeast host cells can then be cultured using standard techniques known to those skilled in the art.
Other methods of expressing heterologous proteins in yeast host cells are described in U.S. patent publication No. 20020055169, U.S. patent No. 6,361,969; 6,312,923; 6,183,985; 6,083,723; 6,017,731; 5,674,706; 5,629,203; 5,602,034; and 5,089,398; U.S. recheck patent nos. RE37,343 and RE35,749; PCT published patent application WO 99/07862; WO 98/37208; and WO 98/26080; european patent application EP 0 946 736; EP 0 732 403; EP 0 480 480; WO 90/10277; EP 0 460 071; EP 0 340 986; EP 0 329 203; EP 0 324 274; and EP 0 164 556. See also Gellissen et al ANTONIE VAN LEEUWENHOEK (1992) 62 (l-2): 79-93; romanos et al, YEAST (1992) 8 (6): 423-488; goeddel, M ETHODS IN ENZYMOLOGY (1990) 185:3-7, each of which is incorporated by reference herein IN its entirety.
During the amplification phase using standard fed-batch fermentation methods, yeast host strains can be grown in the fermenter. The fermentation process may be regulated due to differences in the carbon utilization pathways or expression control patterns of the particular yeast host. By way of example only, fermentation of a Saccharomyces host may require a single glucose feed, a complex nitrogen source (e.g., casein hydrolysate), and supplementation with multiple vitamins, while for optimal growth and expression, the methylotrophic yeast Pichia pastoris may require glycerol, methanol, and trace mineral feeds, but only simple ammonium (nitrogen) salts. See, for example, U.S. Pat. nos. 5,324,639; eniott et al, J.PROTEIN CHEM (1990) 9:95; and Fieschko et al, BIOTECH.BlOENG. (1987) 29:1113, each of which is incorporated herein by reference in its entirety.
However, these fermentation processes may have certain common characteristics independent of the yeast host strain used. For example, growth limiting nutrients (typically carbon) may be added to the fermenter during the amplification stage to allow maximum growth. In addition, fermentation processes typically use fermentation media designed to contain sufficient amounts of carbon, nitrogen, basic salts, phosphorus, and other secondary nutrients (vitamins, trace minerals, salts, etc.). Examples of fermentation media suitable for use with pichia are described in U.S. Pat. nos. 5,324,639 and 5,231,178, each of which is incorporated herein by reference in its entirety.
Baculovirus-infected insect cellsThe term "insect host" or "insect host cell" refers to an insect that can or has been used as a recipient for a recombinant vector or other transfer DNA. The term encompasses the offspring of the original insect host cell that has been transfected. It will be appreciated that due to sporadic or deliberate mutation, the progeny of a single parent cell need not be completely identical in morphology or genome or total DNA complementary to the original parent. Offspring of a parent cell that are substantially similar to a parent to be characterized by a relevant property, such as the presence of a nucleotide sequence encoding an unnatural amino acid polypeptide, are included in the offspring that are meant by this definition.
The selection of suitable insect cells for expression of the polypeptides is well known to those skilled in the art. Several insect species are well described in the art and are commercially available, including, but not limited to, aedes aegypti (Aedes aegypti), silkworm (Bombyx mori), drosophila (Drosophila melanogaster), spodoptera frugiperda (Spodoptera frugiperda), and Trichoplusia ni (Trichoplusia ni). In selecting insect hosts for expression, suitable hosts may include, but are not limited to, those that exhibit, inter alia, good secretion capacity, low proteolytic activity, and overall activity. Insects are commonly available from a variety of sources including, but not limited to, the insect genetic reserve center (the Insect Genetic Stock Center, department of Biophysics and Medical Physics, university of California (Berkeley, CA)) of the university of california (Berkeley, CA) biophysical and medical physical lines, and the american type culture collection (American Type Culture Collection) ("ATCC") (Manassas, VA).
Typically, components of baculovirus-infected insect expression systems comprise a transfer vector, typically a bacterial plasmid, containing a fragment of the baculovirus genome and convenient restriction sites for insertion of a heterologous gene to be expressed; wild-type baculovirus having sequences homologous to baculovirus-specific fragments in the transfer vector (which allows homologous recombination of heterologous genes into the baculovirus genome); and suitable insect host cells and growth media. Materials, methods and techniques used in constructing vectors, transfecting cells, selecting plaques, growing cells in culture, and the like are known in the art and manuals describing these techniques are available.
After insertion of the heterologous gene into the transfer vector, the vector and the wild-type viral genome are transfected into insect host cells, where the vector and viral genome recombine. The packaged recombinant virus is expressed and the recombinant plaques identified and purified. Materials and methods for baculovirus/insect cell expression systems are available in kit form, for example, from Invitrogen corp (Carlsbad, CA). An illustrative technique is described in SUMMERS AND SMITH, TEXAS AGRICULTURAL EXPERIMENT STATION BULLETIN, 1555 (1987), which is incorporated herein by reference. See also RICHARDSON,39METHODS IN MOLECULAR BIOLOGY:BACULOVIRUS EXPRESSION PROTOCOLS (1995); AUSUBEL et al CURRENT PROTOCOLS IN MOLECULAR B IOLOGY 16.9-16.11 (1994); KING and POSSEE, THE BACULOVIRUS SYSTEM: A LABORATORY GUIDE (1992); and O' REILLY et al BACULOVIRUS EXPRESSION VECTORS: A LABORATORY MANUAL (1992).
The use of baculovirus/insect cell expression systems to produce a variety of heterologous proteins is described in the following references and these techniques may be suitable for preparing the unnatural amino acid polypeptides described herein. See, for example, U.S. patent No. 6,368,825; 6,342,216; 6,338,846; 6,261,805; 6,245,528,6,225,060; 6,183,987; 6,168,932; 6,126,944; 6,096,304; 6,013,433; 5,965,393; 5,939,285; 5,891,676; 5,871,986; 5,861,279; 5,858,368; 5,843,733; 5,762,939; 5,753,220; 5,605,827; 5,583,023; 5,571,709; 5,516,657; 5,290,686; WO 02/06305; WO 01/90390; WO 01/27301; WO 01/05956; WO 00/55345; WO 00/20032WO 99/51721; WO 99/45130; WO 99/31257; WO 99/10515; WO 99/09193; WO 97/26332; WO 96/29400; WO 96/25496; WO 96/06161; WO 95/20672; WO 93/03173; WO 92/16619; WO 92/03628; WO 92/01801; WO 90/14428; WO 90/10078; WO 90/02566; WO 90/02186; WO 90/01556; WO 89/01038; WO 89/01037; WO 88/07082, each of which is incorporated herein by reference in its entirety.
Vectors suitable for use in baculovirus/insect cell expression systems include, but are not limited to, insect expression and transfer vectors derived from the baculovirus, spodoptera frugiperda (Autographa californica) nuclear polyhedrosis virus (AcNPV), which are viral expression vectors independent of helper virus. Viral expression vectors derived from this system typically use a strong viral polyhedrin gene promoter to drive expression of heterologous genes. See generally Reilly et al BACULOVIRUS EXPRESSION VECTORS: A LABORATORY MANUAL (1992).
The above components, including the promoter, leader region (if necessary), coding sequence of interest, and transcription termination sequence, are typically assembled into an intermediate translocation construct (transfer vector) prior to insertion of the foreign gene into the baculovirus genome. The intermediate translocation constructs are typically maintained in replicons, such as extrachromosomal elements (e.g., plasmids) that are capable of stable maintenance in a host, such as a bacterium. Replicons should have a replication system, thus allowing them to be maintained in a suitable host for cloning and amplification. More specifically, the plasmid may contain a polyhedrin polyadenylation signal (Miller et al, ANN. REV. MICROBIOL. (1988) 42:177) and a prokaryotic ampicillin resistance (amp) gene, as well as an origin of replication for selection and propagation in E.coli.
One common transfer vector used to introduce foreign genes into AcNPV is pAc373. Many other vectors known to those of skill in the art have also been designed to include, for example, pVL985, which changes the polyhedrin start codon from ATG to ATT, and which introduces a BamHI cloning site 32 base pairs downstream of ATT. See Luckow and Summers, VIROLOGY 170:31-39 (1989). Other commercially available vectors include, for example, PBlueBac4.5/V5-His; pBlueBacHis2; pMelBac; pBlueBac4.5 (Invitrogen Corp., carlsbad, calif.).
After insertion of the heterologous gene, the transfer vector and the wild-type baculovirus genome are co-transfected into an insect cell host. Illustrative methods for introducing heterologous DNA into desired sites in baculoviruses are described in sum ers and SMITH, TEXAS AGRICULTURAL EXPERIMENT STATION BULLETIN, 1555 (1987); smith et al, MOL.CELL.BIOL (1983) 3:2156; luckow and Summers, VIROLOGY (1989) 170:31-39. For example, insertion into a gene such as a polyhedrin gene may be performed by homologous double crossover recombination; it is also possible to insert restriction enzyme sites engineered into the desired baculovirus gene. See Miller et al, blOESSAYS (1989) 4:91.
Transfection may be performed using TROTTER and W OOD 39, METHODS IN MOLECULAR BIOLOGY (1995); mann and King, J.G EN .V IROL The method described in (1989) 70:3501 is carried out by electroporation. Alternatively, liposomes can be used to transfect insect cells with recombinant expression vectors and baculoviruses. See, e.g., liebman et al, blOTECHNlQUES (1999) 26 (1): 36; graves et al, BIOCHEMISTRY (1998) 37:6050; nomura et al, J.BIOL.CHEM (1998) 273 (22): 13570; schmidt et al, PROTEIN EXPRESSION AND PURIFICATION (1998) 12:323; siffert et al, NATURE GENETICS (1998) 18:45; t (T) ILKINS CELL BIOLOGY, A LABORATORY HANDBOOK 145-154 (1998); cai et al PROTEIN EXPRESSION AND PURIFICATION (1997) 10:263; dolphin et al, NATURE GENETICS (1997) 17:491; kost et al, GENE (1997) 190:139; jakobsson et al J.B IOL CHEM (1996) 271:22203; rowles et al J.BIOL.CHEM (1996) 271 (37): 22376; reversey et al J.B IOL CHEM (1996) 271 (39): 23607-10; stanley et al, J.B IOL CHEM (1995) 270:4121; sisk et al, J.VIROL. (1994) 68 (2): 766; and Peng et al, BIOTECHNIQUES (1993) 14.2:274. Commercially available liposomes comprise (for example)
Figure BDA0001546710350001261
And->
Figure BDA0001546710350001262
(Invitrogen, corp., carlsbad, calif.). In addition, calcium phosphate transfection may be used. See T ROTTER And W is OOD 39, METHODS IN MOLECULAR BIOLOGY (1995); kitts, NAR (1990) 18 (19): 5667; and Mann and King, J.GEN.VIROL. (1989) 70:3501.
Baculovirus expression vectors typically contain a baculovirus promoter. A baculovirus promoter is any DNA sequence capable of binding to a baculovirus RNA polymerase and initiating transcription of the downstream (3') of the coding sequence (e.g., structural gene) into mRNA. The promoter should have a transcription initiation region that is typically located near the 5' end of the coding sequence. This transcription initiation region typically comprises an RNA polymerase binding site and a transcription initiation site. Baculovirus promoters may also have a second domain, called an enhancer, which (if present) is typically remote from the structural gene. Furthermore, expression may be regulatory or constitutive.
Structural genes transcribed in large quantities late in the infectious cycle provide particularly useful promoter sequences. Examples include sequences derived from the gene encoding the viral polyhedrin (FRIESEN et al, the Regulation of Baculovirus Gene Expression in THE MOLECULAR BIOLOGY OF BACULOVIRUSES (1986); EP 0 127 839 and 0 155 476) and the gene encoding the p10 protein (Vlak et al, J.GEN.VlROL. (1988) 69:765).
The newly formed baculovirus expression vector is enveloped in an infectious recombinant baculovirus and the plaque can then be grown, for example, by Miller et al, BIOESSAYS (1989) 4:91; SUMMERS and SMITH, TEXAS AGRICULTURAL EXPERIMENT STATION BULLETIN NO.1555 (1987).
Recombinant baculovirus expression vectors have been developed for infection into several insect cells. For example, recombinant baculoviruses have been developed, particularly for use in the following insects: aedes aegypti (ATCC No. CCL-125), silkworm (ATCC No. CRL-8910), drosophila (ATCC No. 1963), spodoptera frugiperda, and Spodoptera frugiperda. See WO 89/046,699; wright, NATURE (1986) 321:718; carbonell et al, J.VlROL. (1985) 56:153; smith et al, mol.cell.BIOL (1983) 3:2156. See generally Fraser et al, IN VITRO cell.dev.BIOL (1989) 25:225. More specifically, cell lines for baculovirus expression vector systems typically include, but are not limited to, sf9 (spodoptera frugiperda) (ATCC No. CRL-1711), sf21 (spodoptera frugiperda) (Invitrogen corp., catalog No. 11497-013 (Carlsbad, CA)), tri-368 (spodoptera frugiperda), and High-Five TM BTI-TN-5B1-4 (Spodoptera frugiperda).
Cells and media for direct expression and fusion expression of heterologous polypeptides in baculovirus/expression are commercially available.
Bacteria and method for producing sameBacterial expression techniques are well known in the art. A variety of vectors are available for use in bacterial hosts. The vector may be a single copy or low or high multiple copy vector. Vectors may be used for cloning and/or expression. Because of the rich literature on vectors, the commercial availability of many vectors, and even manuals describing the vectors and their restriction maps and features, need not be widely discussed herein. As is well known, vectors typically include markers that permit selection, which can provide resistance, prototrophy, or immunity to cytotoxic agents. Typically, there are multiple markers that provide different features.
A bacterial promoter is any DNA sequence capable of binding to a bacterial RNA polymerase and initiating transcription of the downstream (3 ") coding sequence (e.g., structural gene) into mRNA. The promoter should have a transcription initiation region that is typically located near the 5' end of the coding sequence. This transcription initiation region typically comprises an RNA polymerase binding site and a transcription initiation site. The bacterial promoter may also have a second domain, called an operator, which may overlap with an adjacent RNA polymerase binding site where RNA synthesis begins. The operator allows for down-regulated (inducible) transcription, as the gene repressor protein can bind to the operator and thereby inhibit transcription of a particular gene. Constitutive expression can occur in the absence of a negative regulatory element, such as an operator. Alternatively, upregulation may be achieved by gene activation of the protein binding sequence (which, if present, is usually closest to (5') of the RNA polymerase binding sequence). An example of a gene-activated protein is the metabolite-activated protein (CAP) which helps initiate transcription of the lac operon in E.coli [ Raibaud et al, ANNU. REV. GENET. (1984) 18:173]. Regulated expression may thus be positive or negative, thereby enhancing or attenuating transcription.
Sequences encoding metabolic pathway enzymes provide particularly useful promoter sequences. Examples include promoter sequences derived from sugar-metabolizing enzymes such as galactose, lactose (lac) [ Chang et al, NATURE (1977) 198:1056] and maltose. Other examples include promoter sequences derived from biosynthetic enzymes such as tryptophan (trp) (Goeddel et al, NUC. ACIDS RES (1980) 8:4057; yeverton et al, NUCL. ACIDS RES (1981) 9:731; U.S. Pat. No. 4,738,921; IFNPub. 036 776 and No. 121 775), each of which is incorporated by reference in its entirety. Beta-galactosidase (bla) promoter system [ Weissmann (1981) "The cloning of Interferon and other migtakes." In Interferon 3 (I.Gress et al) ], phage lambda PL [ Shimatake et al, NATURE (1981) 292:128], and T5[ U.S. Pat. No. 4,689,406 ], each of which is incorporated herein by reference In its entirety, the promoter systems also provide suitable promoter sequences. Preferred methods contemplated herein utilize a strong promoter (such as a T7 promoter) to induce high level production of the polypeptide. Examples of such vectors include, but are not limited to, the pET29 series from Novagen, and the pPOP vector described in WO99/05297 (which is incorporated herein by reference in its entirety). These expression systems produce high levels of polypeptides in the host without compromising host cell viability or growth parameters.
In addition, synthetic promoters, which are not found in nature, also act as bacterial promoters. For example, the transcriptional activation sequence of one bacterial or phage promoter may be linked to the operator sequence of another bacterial or phage promoter to produce a synthetic hybrid promoter [ U.S. Pat. No. 4,551,433 ]]. For example, the tac promoter is a hybrid trp-lac promoter consisting of both the trp promoter and the lac operator sequence regulated by the lac repressor [ Amann et al, GENE (1983) 25:167; de Boer et al, P ROC .NATL.ACAD.SCI.(1983)80:21]. In addition, the bacterial promoter may comprise a naturally occurring promoter of non-bacterial origin having the ability to bind to bacterial RNA polymerase and initiate transcription. Naturally occurring promoters of non-bacterial origin may also be compatible with RNA polymeraseCoupled to produce high levels of expression of some genes in prokaryotes. Phage T7RNA polymerase/promoter System is an example of a coupled promoter System [ Studier et al J.M OL .B IOL (1986) 189:113; tabor et al Proc Natl. Acad. Sci. (1985) 82:1074]. In addition, the hybrid promoter may also include a phage promoter and an E.coli operator region (IFNPub. 267 851).
In addition to functional promoter sequences, effective ribosome binding sites are also suitable for expression of foreign genes in prokaryotes. In E.coli, the ribosome binding site is called the Shine-Dalgarno (SD) sequence and comprises the initiation codon (ATG) and a sequence of 3-9 nucleotides in length located 3-11 nucleotides upstream of the initiation codon [ Shine et al, NATURE (1975) 254:34]. The SD sequence is believed to promote mRNA binding to ribosomes by base pairing between the SD sequence and the 3' end of E.coli 16S rRNA [ Steitz et al, "Genetic signals and nucleotide sequences in messenger RNA", in Biological Regulation and Development: gene Expression (R.F.Goldberger, 1979) ]. For expression of eukaryotic and prokaryotic genes with weak ribosome binding sites [ Sambrook et al, "Expression of cloned genes in Escherichia coli", molecular Cloning: A Laboratory Manual,1989].
The term "bacterial host" or "bacterial host cell" refers to a bacterium that can or has been used as a recipient for a recombinant vector or other transfer DNA. The term encompasses the progeny of an original bacterial host cell that has been transfected. It will be appreciated that the progeny of a single parent cell need not be completely identical in morphology or in genome or in total DNA complementary to the original parent, due to accidental or deliberate mutation. Progeny of a parent cell that is substantially similar to the parent to be characterized by the relevant property, such as the presence of a nucleotide sequence encoding the desired polypeptide, are included in the progeny that are meant by this definition.
The selection of suitable host bacteria for expression of the desired polypeptide is well known to those skilled in the art. In selecting bacterial hosts for expression, suitable hosts may include, but are not limited to, those that display, inter alia, at least one of, and preferably at least two of, the following characteristics: good inclusion body formation ability, low proteolytic activity, good secretion ability, good soluble protein production ability, and overall activity. Bacterial hosts are commonly available from a variety of sources including, but not limited to, the bacterial genetic reserve centers (the Bacterial Genetic Stock Center, department of Biophysics and Medical Physics, university of California (Berkeley, CA)) of the university of california (Berkeley, CA) biophysical and medical physical lines; american type culture Collection (American Type Culture Collection) ("ATCC") (Manassas, va.). Industrial/medical fermentation typically uses bacteria derived from strain K (e.g. W3110) or bacteria derived from strain B (e.g. BL 21). These strains are particularly suitable because their growth parameters are extremely well known and active. In addition, these strains are nonpathogenic, which is commercially important for safety and environmental reasons. In one embodiment of the methods described and contemplated herein, the E.coli host includes, but is not limited to, a strain of BL21, DH10B, or derivatives thereof. In another embodiment of the methods described and contemplated herein, the E.coli host is a deproteinized strain, including, but not limited to OMP-and LON-. In another embodiment, the bacterial host is a species of Pseudomonas, such as Pseudomonas fluorescens, pseudomonas aeruginosa, and Pseudomonas putida. An example of a Pseudomonas expressing strain is Pseudomonas fluorescens biotype I, strain MB101 (Dow Chemical).
Once a recombinant host cell strain has been established (i.e., an expression construct has been introduced into a host cell and the host cell with the appropriate expression construct isolated), the recombinant host cell strain is cultured under conditions suitable for the production of the polypeptide. The method of cultivation of the recombinant host cell strain will depend on the nature of the expression construct employed and the host cell itself. Recombinant host strains are typically cultured using methods well known in the art. Recombinant host cells are typically cultured in liquid media containing absorbable sources of carbon, nitrogen, and inorganic salts, and optionally vitamins, amino acids, growth factors, and other protein culture supplements well known in the art. The liquid medium used to culture the host cells may optionally contain antibiotics or antifungal agents to prevent the growth of unwanted microorganisms and/or compounds including, but not limited to, antibiotics used to select host cells containing the expression vector.
Recombinant host cells can be cultured in batch or continuous form, where the cells are collected in batch or continuous form (in the case where the desired polypeptide accumulates within the cell) or the culture supernatant is collected. For preparation in prokaryotic host cells, batch culture and cell harvesting are preferred.
In one embodiment, the unnatural amino acid polypeptides described herein are purified after expression in a recombinant system. The polypeptide may be purified from the host cell or culture medium by a variety of methods known in the art. In general, many polypeptides produced in bacterial host cells are poorly soluble or insoluble (in the form of inclusion bodies). In one embodiment, amino acid substitutions can be readily made in a selected polypeptide for the purpose of increasing the solubility of a recombinantly produced polypeptide using the methods disclosed herein as well as those known in the art. In the case of insoluble polypeptides, the polypeptides may be collected from the host cell lysate by centrifugation or filtration and may be further followed by homogenization of the cells. In the case of poorly soluble polypeptides, compounds including, but not limited to, polyethylenimine (PEI) may be added to induce precipitation of partially soluble polypeptides. The precipitated polypeptide may then be conveniently collected by centrifugation or filtration. The recombinant host cells can be disrupted or homogenized to release inclusion bodies from the cells using a variety of methods well known to those of skill in the art. Host cell disruption or homogenization may be performed using well known techniques including, but not limited to, enzymatic cell disruption, sonication, dunus homogenization (dounce homogenization), or high pressure release disruption. In one embodiment of the methods described and contemplated herein, the E.coli host cells are disrupted to release inclusion bodies of the polypeptide using high pressure release techniques. It has been found that the yield of insoluble polypeptide in the form of inclusion bodies can be increased by using only one channel of the E.coli host cell through the homogenizer. When processing inclusion bodies of polypeptides, it is advantageous to minimize the repeated homogenization time in order to maximize the yield of inclusion bodies without loss due to factors such as solubilization, mechanical shearing, or proteolysis.
The insoluble or precipitated polypeptide may then be solubilized using any of a number of suitable solubilizing agents known in the art. For example, the polypeptide is solubilized with urea or guanidine hydrochloride. The volume of dissolved polypeptide should be minimized so that large volume preparations can be made using readily disposable batch sizes. This factor can be important in large scale commercial plants where recombinant hosts can grow in volumes of thousands of liters. In addition, when manufacturing polypeptides in large scale commercial settings, especially for human medical use, caustic chemicals that can damage machinery and containers, or the polypeptide product itself, should be avoided, if possible. It has been shown in the methods described and contemplated herein that the more caustic denaturant guanidine hydrochloride can be replaced with the milder denaturant urea to solubilize polypeptide inclusion bodies. The use of urea significantly reduces the risk of damage to stainless steel equipment used in the manufacture and purification of polypeptides while effectively dissolving the polypeptide inclusion bodies.
In the case of soluble polypeptides, the peptide may be secreted into the periplasmic space or into the culture medium. In addition, the soluble peptide may be present in the cytoplasm of the host cell. The soluble peptide may be concentrated prior to the purification step. The soluble peptides can be concentrated from, for example, cell lysates or culture media using standard techniques, including but not limited to those described herein. In addition, the host cells can be disrupted and the soluble peptide released from the cytoplasmic or periplasmic space of the host cells using standard techniques, including but not limited to those described herein.
When the polypeptide is prepared as a fusion protein, it is preferred to remove the fusion sequence. Removal of the fusion sequence may be accomplished by methods including, but not limited to, enzymatic cleavage or chemical cleavage, with enzymatic cleavage being preferred. Enzymatic removal of the fusion sequence can be accomplished using methods well known to those skilled in the art. The choice of enzyme used to remove the fusion sequence is determined by the nature of the fusion, and the reaction conditions are dictated by the choice of enzyme. Chemical cleavage can be accomplished using reagents including, but not limited to, cyanogen bromide, TEV protease, and other reagents. The cleaved polypeptide is optionally purified from the cleaved fusion sequence by well known methods. These methods will be determined by the nature and properties of the fusion sequences and polypeptides. Purification methods may include, but are not limited to, size exclusion chromatography, hydrophobic interaction chromatography, ion exchange chromatography, or dialysis, or any combination thereof.
The polypeptide is also optionally purified to remove DNA from the protein solution. DNA may be removed by any suitable method known in the art, including but not limited to precipitation or ion exchange chromatography. In one embodiment, the DNA is removed by precipitation with a nucleic acid precipitating agent such as, but not limited to, protamine sulfate. The polypeptide may be separated from the precipitated DNA using standard well known methods, including but not limited to centrifugation or filtration. In the context of using polypeptides to treat humans, removal of host nucleic acid molecules is an important factor, and the methods described herein reduce host cell DNA to a pharmaceutically acceptable level.
Methods of small-scale or large-scale fermentation may also be used in protein expression, including but not limited to fermentors, shake flasks, fluidized bed bioreactors, hollow fiber bioreactors, roller bottle culture systems, and stirred tank bioreactor systems. Each of these processes may be performed in a batch, fed-batch, or continuous mode process.
The human form of the unnatural amino acid polypeptides described herein can generally be recovered using methods standard in the art. For example, the culture medium or cell lysate may be centrifuged or filtered to remove cell debris. The supernatant may be concentrated or diluted to the desired volume or diafiltered into a suitable buffer to condition the formulation for further purification. Further purification of the unnatural amino acid polypeptides described herein includes, but is not limited to, separating deamidated and truncated forms of the polypeptide variants from the corresponding intact forms.
Any of the following exemplary procedures can be used to purify the unnatural amino acid polypeptides described herein: affinity chromatography; anion or cation exchange chromatography (using (including but not limited to) DEAE SEPHAROSE); silica chromatography; reversed phase HPLC; gel filtration (using (including but not limited to) SEPHADEX G-75); hydrophobic interaction chromatography; size exclusion chromatography; metal chelate chromatography; ultrafiltration/diafiltration; precipitating with ethanol; precipitating ammonium sulfate; focusing the chromatogram; displacement chromatography; electrophoresis procedures (including but not limited to preparative isoelectric focusing), differential solubility (including but not limited to ammonium sulfate precipitation), SDS-PAGE, extraction, or any combination thereof.
The polypeptides encompassed in the methods and compositions described herein, including but not limited to polypeptides comprising unnatural amino acids, antibodies to polypeptides comprising unnatural amino acids, binding partners for polypeptides comprising unnatural amino acids, can be partially purified or substantially purified to homogeneity according to standard procedures known and used by those of skill in the art. Thus, the polypeptides described herein may be recovered and purified by any of a number of methods well known in the art, including, but not limited to, ammonium sulfate or ethanol precipitation, acid or base extraction, column chromatography, affinity column chromatography, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, hydroxyapatite chromatography, lectin chromatography, gel electrophoresis, and any combination thereof. In preparing properly folded mature proteins, a protein refolding step can be used if necessary. High Performance Liquid Chromatography (HPLC), affinity chromatography or other suitable methods may be used in the final purification step where high purity is desired. In one embodiment, antibodies raised against (or polypeptides comprising) unnatural amino acids are used as purification reagents for affinity-based purification of polypeptides comprising one or more unnatural amino acids, including but not limited to. After partial purification or homogeneity as desired, the polypeptides may optionally be used for a variety of utilities, including, but not limited to, as an assay component, therapeutic agent, prophylactic agent, diagnostic agent, research agent, and/or as an immunogen for antibody production.
In addition to other references noted herein, a variety of purification/protein folding methods are well known in the art, including, but not limited to, those set forth in the following: the R.scope(s) is (are) used,Protein Purification,Springer-Verlag,N.Y.(1982)；Deutscher,methods in Enzymologv volume 182 Guide to Protein Purification,Academic Press,Inc.N.Y.(1990)；Sandana(1997)Bioseparation of ProteinsAcademic Press, inc.; bollag et al (1996)Protein Methods.Wiley-Lists, 2 nd edition, NY; walker (1996)The Protein Protocols HandbookHumana Press, NJ; harris and Angal (1990)Protein Purification Applications:A Practical ApproachIRL Press at Oxford, oxford, england; harris and AngalProtein Purification Methods:A Practical Approach IRL Press at Oxford,Oxford,England；Scopes(1993)Protein Purification Principles and Practice 3 rd editionSpringer Verlag, NY; janson and Ryden (1998)Protein Purification:Principles.High Resolution Methods and Application, 2 nd editionWiley-VCH, NY; walker (1998)Protein Protocols on CD-ROMHumana Press, NJ; and references cited therein.
One advantage of producing a polypeptide comprising at least one unnatural amino acid in a eukaryotic host cell or a non-eukaryotic host cell is that the polypeptide will typically fold in its native conformation. However, in certain embodiments of the methods and compositions described herein, after synthesis, expression, and/or purification, the polypeptide can have a conformation that differs from the desired conformation of the relevant polypeptide. In one aspect of the methods and compositions described herein, the expressed protein is optionally denatured and subsequently renatured. This optional denaturation and renaturation is accomplished using methods known in the art, wherein these methods include, but are not limited to, adding chaperonin (chaperonin) to the polypeptide of interest, and dissolving the polypeptide in chaotropes, including, but not limited to, guanidine hydrochloride, and using protein disulfide isomerase.
In general, it is sometimes desirable to denature and reduce the expressed polypeptides and then refold the polypeptides into a preferred conformation. For example, the refolding can be achieved by adding guanidine, urea, DTT, DTE, and/or chaperonin to the translation product of interest. Methods for reducing, denaturing and renaturating proteins are well known to those skilled in the art (see, above references, and Debinski et al (1993)J.Biol.Chem.268:14065-14070; kreitman and Pastan (1993)Bioconjug.Chem..4:581-585; and Buchner et al, (1992)Anal.Biochem..205:263-270). Debinski et al, for example, describe denaturation and reduction of inclusion body proteins in guanidine-DTE. Proteins may be refolded in a redox buffer containing (including, but not limited to) oxidized glutathione and L-arginine. Refolding reagents can flow or otherwise move into contact with one or more polypeptides or other expression products, or vice versa.
In the case of prokaryotic production of non-natural amino acid polypeptides, the polypeptides thus produced may be misfolded and thus lack or have reduced biological activity. The biological activity of the protein can be restored by "refolding". In one embodiment, the misfolded polypeptide is refolded by dissolving (wherein the polypeptide is also insoluble), unfolding, and reducing the polypeptide chain using, for example, one or more chaotropic agents including, but not limited to, urea and/or guanidine, and a reducing agent capable of reducing disulfide bonds, including, but not limited to, dithiothreitol, DTT, or 2-mercaptoethanol, 2-ME. At moderate concentrations of chaotropic agents, an oxidizing agent (including but not limited to oxygen, cystine, or cystamine) is then added, which allows the reformation of disulfide bonds. The unfolded or misfolded polypeptide may be refolded using standard methods known in the art, such as those described in U.S. Pat. nos. 4,511,502, 4,511,503, and 4,512,922, each of which is incorporated herein by reference in its entirety. Polypeptides may also be co-folded with other proteins to form heterodimers or heteromultimers. After refolding or co-folding, the polypeptide is optionally further purified.
Purification of the unnatural amino acid polypeptide can also be accomplished using a variety of techniques, including, but not limited to, those described herein, e.g., hydrophobic interaction chromatography, size exclusion chromatography, ion exchange chromatography, reverse phase high performance liquid chromatography, affinity chromatography, and the like, or any combination thereof. Other purifications may also include the step of drying or precipitating the purified protein.
After purification, the unnatural amino acid polypeptide can be exchanged into different buffers and/or concentrated by any of a variety of methods known in the art, including, but not limited to, diafiltration and dialysis. hGH provided as a single purified protein may be subjected to aggregation and precipitation. In certain embodiments, the purified unnatural amino acid polypeptide can be at least 90% pure (as measured by reverse phase high performance liquid chromatography (RP-HPLC) or sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)). In certain other embodiments, the purified unnatural amino acid polypeptide can be at least 95% pure, or at least 98% pure, or at least 99% or more pure. Regardless of the exact number of purities of the unnatural amino acid polypeptides, the unnatural amino acid polypeptides are sufficiently pure for use as a pharmaceutical product or for further processing, including, but not limited to, conjugation with water-soluble polymers (such as PEG).
In certain embodiments, the non-natural amino acid polypeptide molecule can be used as a therapeutic agent in the absence of other active ingredients or proteins (other than excipients, carriers, and stabilizers, serum albumin, and the like), and in certain embodiments, the non-natural amino acid polypeptide molecule can be complexed with another polypeptide or polymer.
2.Purification of unnatural amino acid polypeptides
General purification methodsThe techniques disclosed in this section are applicable to the general purification of the unnatural amino acid polypeptides described herein.
Any of a variety of separation steps may be performed on the cell lysate extract, the culture medium, the inclusion bodies, the periplasmic space of the host cells, the cytoplasm of the host cells, or other materials including the desired polypeptide, or any mixture of polypeptides produced by any separation step including, but not limited to, affinity chromatography, ion exchange chromatography, hydrophobic interaction chromatography, gel filtration chromatography, high performance liquid chromatography ("HPLC"), reverse phase HPLC ("RP-HPLC"), expanded bed adsorption, or any combination and/or repetition thereof, and in any suitable order.
The equipment and other necessary materials for performing the techniques described herein are commercially available. Pumps, fraction collectors, monitors, recorders and the whole system are available from, for example, applied Biosystems (Foster City, CA), bio-Rad Laboratories, inc. (Hercules, CA), amersham Biosciences, inc. (Piscataway, NJ). Chromatographic materials including, but not limited to, exchange matrix materials, media, and buffers are also available from these companies.
Equilibration and other steps in the column chromatography methods described herein, such as washing and elution, can be accomplished more quickly using specialized equipment such as pumps. Commercially available pumps include (but are not limited to)
Figure BDA0001546710350001341
Pump P-50, peristaltic pump P-1, pump P-901, and pump P-903 (Amersham Biosciences, piscataway, N.J.).
Examples of fraction collectors include Redifrac fraction collector, FRAC-100 and FRAC-200 fraction collector
Figure BDA0001546710350001342
Fraction collector (Amersham Biosciences, piscataway, NJ). Mixers can also be used to create pH and linear concentration gradients. Commercially available mixers include gradient mixer GM-1 and inline mixers (Amersham Biosciences, piscataway, NJ).
Any commercially available monitor may be used to monitor the chromatographic process. These monitors can be used to collect information such as UV, fluorescence, pH and conductivity. Examples of detectors include monitor UV-1,
Figure BDA0001546710350001343
S II, monitor UV-M II, monitor UV-900, monitor UPC-900, monitor pH/C-900, and conductivity monitor (Amersham Biosciences, piscataway, N.J.). In fact, various +.>
Figure BDA0001546710350001344
The entire system of the system (Piscataway, NJ) is commercially available.
In one embodiment of the methods and compositions described herein, for example, a polypeptide can be reduced and denatured by first denaturing the resulting purified polypeptide in urea, followed by dilution in TRIS buffer containing a reducing agent (such as DTT) at a suitable pH. In another embodiment, the polypeptide is denatured in urea at a concentration ranging between about 2M to about 9M, followed by dilution in TRIS buffer at a pH ranging from about 5.0 to about 8.0. The refolded mixture of this example can then be incubated. In one embodiment, the refolded mixture is incubated at room temperature for 4 to 24 hours. The reduced and denatured polypeptide mixture may then be further isolated or purified.
As set forth herein, the pH of the first polypeptide mixture may be first adjusted, followed by any subsequent separation steps. In addition, the first polypeptide mixture, or any subsequent mixture thereof, may be concentrated using techniques known in the art. Furthermore, the elution buffer comprising the first polypeptide mixture or any subsequent mixture thereof may be exchanged for a buffer suitable for the next separation step using techniques well known to those skilled in the art.
Ion exchange chromatographyThe techniques disclosed in this section are applicable to ion chromatography of the unnatural amino acid polypeptides described herein.
In one embodiment, and as an optional additional step, the first polypeptide mixture may be subjected to ion exchange chromatography. See generally ION EXCHANGE CHROMATOGRAPHY: PRINCIPLES AND METHODS (catalog No. 18-1114-21,Amersham Biosciences (Piscataway, NJ)). The commercially available ion exchange column comprises
Figure BDA0001546710350001351
And +.>
Figure BDA0001546710350001352
Column (Amersham Biosciences, piscataway, NJ). These columns use strong anion exchangers, such as Q +.>
Figure BDA0001546710350001353
Fast Flow、Q/>
Figure BDA0001546710350001354
High Performance, and Q
Figure BDA0001546710350001355
XL; strong cation exchangers, such as SP +.>
Figure BDA0001546710350001356
High Performance、SP
Figure BDA0001546710350001357
Fast Flow, and SP->
Figure BDA0001546710350001358
XL; weak anion exchangers, e.g. DEAE
Figure BDA0001546710350001359
Fast Flow; and weak cation exchangers, such as CM +. >
Figure BDA00015467103500013510
Figure BDA00015467103500013511
Fast Flow (Amersham Biosciences, piscataway, NJ). The polypeptides at any stage of the purification process may be subjected to anion or cation exchange column chromatography to isolate the substantially purified polypeptides. The cation exchange chromatography step may be performed using any suitable cation exchange matrix. The cation exchange matrix includes, but is not limited to, fibrous, porous, nonporous, particulate, beaded, or crosslinked cation exchange matrix materials. These cation exchange matrix materials include, but are not limited to, cellulose, agarose, dextran, polyacrylate, polyethylene, polystyrene, silica, polyether, or a complex of any of the foregoing. After adsorption of the polypeptide onto the cation exchanger matrix, the substantially purified polypeptide may be eluted by contacting the matrix with a buffer having a sufficiently high pH or ionic strength to displace the polypeptide from the matrix. Suitable buffers for use in high pH elution of the substantially purified polypeptide include, but are not limited to, citrate, phosphate, formate, acetate, HEPES, and MES buffers at concentrations ranging from at least about 5mM to at least about 100mMAnd (3) liquid.
Reversed phase chromatographyThe techniques disclosed in this section are applicable to reverse phase chromatography of the unnatural amino acid polypeptides described herein.
RP-HPLC can be performed according to suitable protocols known to those skilled in the art to purify proteins. See, e.g., pearson et al, ANAL biochem (1982) 124:217-230 (1982); rivier et al, J.CHROM. (1983) 268:112-119; kunitani et al, J.CHROM. (1986) 359:391-402. The polypeptide may be subjected to RP-HPLC to isolate the substantially purified polypeptide. In this regard, a variety of lengths may be used, including but not limited to at least about C 3 To at least about C 30 At least about C 3 To at least about C 20 Or at least about C 3 To at least about C 18 ) Silica-derived resins having alkyl functional groups of the resin. Alternatively, a polymeric resin may be used. For example, tosoHaas Amberchrome CG sd resin, which is a styrene polymer resin, can be used. Cyano or polymeric resins having a variety of alkyl chain lengths may also be used. In addition, the RP-HPLC column can be washed with a solvent such as ethanol. Suitable elution buffers containing ion-pair reagents and organic modifiers such as methanol, isopropanol, tetrahydrofuran, acetonitrile or ethanol can be used to wash the polypeptide from the RP-HPLC column. The most common ion pair reagents include, but are not limited to, acetic acid, formic acid, perchloric acid, phosphoric acid, trifluoroacetic acid, heptafluorobutyric acid, triethylamine, tetramethylammonium, tetrabutylammonium, triethylammonium acetate. Elution may be performed using one or more gradients or isocratic conditions, where gradient conditions preferably reduce separation time and reduce peak width. Another approach involves using two gradients with different solvent concentration ranges. Examples of suitable elution buffers for use herein may include, but are not limited to, ammonium acetate and acetonitrile solutions.
Hydrophobic interaction chromatography purification technologyThe techniques disclosed in this section are applicable to hydrophobic interaction chromatographic purification of the unnatural amino acid polypeptides described herein.
Hydrophobic Interaction Chromatography (HIC) may be performed on the polypeptide. See generally HYDROPHOBIC INTERACTION CHROMATOGRAPHY HANDBOOK:PRINCIPLES AND METHODS (catalog number 18-1020-90,Amersham Biosciences (Piscataway, N.J.), which is incorporated herein by reference). Suitable HIC matrices may include, but are not limited to, alkyl or aryl substituted matrices, such as butyl, hexyl, octyl, or phenyl substituted matrices, including agarose, cross-linked agarose, sepharose, cellulose, silica, dextran, polystyrene, polymethacrylate matrices, and mixed forms of resins including, but not limited to, polyvinylamine resins or butyl or phenyl substituted polymethacrylate matrices. Commercial sources of hydrophobic interaction column chromatography include, but are not limited to
Figure BDA0001546710350001361
And +.>
Figure BDA0001546710350001362
Column (Amersham Biosciences, piscataway, NJ). Briefly, prior to loading, the HIC column may be equilibrated using standard buffers known to those of ordinary skill in the art, such as acetic acid/sodium chloride solution or HEPES containing ammonium sulfate. Ammonium sulfate may be used as a buffer for loading the HIC column. After loading the polypeptide, the column may then be washed using standard buffers and conditions to remove unwanted material, but the polypeptide is retained on the HIC column. The polypeptide may be eluted with about 3 to about 10 column volumes of standard buffer (such as HEPES buffer containing EDTA and lower ammonium sulfate concentration than the equilibration buffer, or acetic acid/sodium chloride buffer). A gradual decreasing linear salt gradient using, for example, a potassium phosphate gradient, may also be used to elute the polypeptide molecule. The eluate may then be concentrated, for example, by filtration, such as diafiltration or ultrafiltration. Diafiltration may be used to remove salts used to elute the polypeptide.
Other purification techniquesThe techniques disclosed in this section can be applied to other purification techniques for the unnatural amino acid polypeptides described herein.
The first polypeptide mixture or any subsequent mixture thereof may be subjected to, for example, GEL FILTRATION (GEL FILTRATION: PRINCIPLES AND METHODS (catalog number 18-1022-18, amer)sham Biosciences, piscataway, NJ), which is incorporated herein by reference in its entirety, hydroxyapatite chromatography (another separation step suitable for matrices including, but not limited to, HA-ultragel, high Resolution (Calbiochem), CHT ceramic hydroxyapatite (BioRad), bio-Gel HTP hydroxyapatite (BioRad)), HPLC, expanded bed adsorption, ultrafiltration, diafiltration, lyophilization, and the like to remove any excess salts and replace the buffer with a buffer suitable for the next separation step or even formulation of the final pharmaceutical product. Various techniques, including but not limited to those described herein, can be used to monitor the yield of a polypeptide comprising a substantially purified polypeptide at each step described herein. These techniques can also be used to assess the yield of a substantially purified polypeptide after the final separation step. For example, any of a number of reversed-phase high pressure liquid chromatography columns having a variety of alkyl chain lengths may be used (such as cyano RP-HPLC, C 18 RP-HPLC) and cation exchange HPLC and gel filtration HPLC to monitor the yield of polypeptide.
The purity can be determined using standard techniques such as SDS-PAGE or by measuring the polypeptide using Western blot methods (Western blot) as well as ELISA assays. For example, polyclonal antibodies can be raised against proteins isolated from negative control yeast fermentation and cation exchange recovery. These antibodies can also be used to detect the presence of contaminating host cell proteins.
In certain embodiments, the polypeptide yield after each purification step may be 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% of the polypeptide in the starting material of each purification step.
RP-HPLC material Vydac C4 (Vydac) consists of silica gel particles with C on the surface 4 -an alkyl chain. The separation of polypeptide from protein impurities is based on hydrophobic phases Differences in the strength of interaction. Elution was performed with a gradient of acetonitrile in dilute trifluoroacetic acid. Preparative HPLC was performed using a stainless steel tube column (packed with 2.8 to 3.2 liters of Vydac C4 silica gel). The hydroxyapatite ultragel eluate was acidified by addition of trifluoroacetic acid and loaded onto a Vydac C4 column. For washing and elution, a gradient of acetonitrile in dilute trifluoroacetic acid was used. Fractions were collected and immediately neutralized with phosphate buffer. Polypeptide fractions within the limits of IPC are pooled.
The DEAE Sepharose (Pharmacia) material consists of Diethylaminoethyl (DEAE) groups covalently bound to the surface of agarose gel beads. Binding of the polypeptide to the DEAE group is mediated by ionic interactions. Acetonitrile and trifluoroacetic acid were passed through the column without residence. After these materials are washed out, trace impurities are removed by washing the column with low pH acetate buffer. The column is then washed with neutral phosphate buffer and the polypeptide eluted with a buffer having increased ionic strength. The column was packed with DEAE Sepharose fast flow. The column volume was adjusted to ensure polypeptide loading in the range of 3-10mg of polypeptide per ml of gel. The column was washed with water and equilibration buffer (sodium phosphate/potassium phosphate). Pooled fractions of HPLC eluate were loaded and the column was washed with equilibration buffer. The column was then washed with wash buffer (sodium acetate buffer), followed by equilibration buffer. The polypeptides were then eluted from the column with elution buffers (sodium chloride, sodium phosphate/potassium phosphate) and collected as a single fraction according to the main elution profile. The eluate of the DEAE Sepharose column was adjusted to the indicated conductivity. The resulting drug substance was sterile filtered into Teflon vials and stored at-70 ℃.
The yield and purity of a polypeptide comprising one or more unnatural amino acids can be assessed using a variety of methods and procedures, including, but not limited to, bradford assay, SDS-PAGE, silver stained SDS-PAGE, coomassie blue stained SDS-PAGE (coomassie stained SDS-PAGE), mass spectrometry, including, but not limited to MALDI-TOF, and other methods known to those of skill in the art for characterizing proteins.
Other methods include, but are not limited to, the step of removing endotoxin. Endotoxins are Lipopolysaccharides (LPS) located on the outer membrane of gram-negative host cells, such as e.coli. Methods of reducing endotoxin content include, but are not limited to, purification techniques using silica carriers, glass powders, or hydroxyapatite, reverse phase chromatography, affinity chromatography, size exclusion chromatography, anion exchange chromatography, hydrophobic interaction chromatography, combinations of these methods, and the like. Modification or other methods may be required to remove contaminants such as co-migratory proteins from the polypeptide of interest. Methods of measuring endotoxin content are known to those of ordinary skill in the art and include, but are not limited to, limulus amoebocyte lysate (Limulus Amebocyte Lysate, LAL) assays.
Other methods and procedures include, but are not limited to, SDS-PAGE coupled with protein staining methods, immunoblotting, matrix assisted laser desorption/ionization-mass spectrometry (MALDI-MS), liquid chromatography/mass spectrometry, isoelectric focusing, analytical anion exchange, chromatofocusing, and circular dichroism.
In certain embodiments, amino acids of formulas I-XVIII, XXX-XXXIV (A and B), and XXXXX-XXXXIII, comprising any sub-or specific compound within the scope of formulas I-XVIII, XXX-XXXIV (A and B), and XXXXX-XXXXIII, can be incorporated into polypeptides by biosynthesis, thereby producing unnatural amino acid polypeptides. In other embodiments, these amino acids are incorporated at specific sites within the polypeptide. In other embodiments, these amino acids are incorporated into polypeptides using a translation system. In other embodiments, these translation systems include: (i) A polynucleotide encoding a polypeptide, wherein the polynucleotide comprises a selector codon that corresponds to a predetermined site that incorporates the amino acid, and (ii) a tRNA that comprises an amino acid, wherein the tRNA is specific for the selector codon. In other embodiments of these translation systems, the polynucleotide is an mRNA produced in the translation system. In other embodiments of these translation systems, the translation system comprises a plasmid or phage comprising a polynucleotide. In other embodiments of these translation systems, the translation system comprises genomic DNA comprising a polynucleotide. In other embodiments of these translation systems, the polynucleotide is stably integrated into the genomic DNA. In other embodiments of these translation systems, the translation systems include a tRNA that is specific for a selector codon, where the selector codon is selected from the group consisting of an amber codon, an ocher codon, a opal codon, a unique codon, a rare codon, an unnatural codon, a five-base codon, and a four-base codon. In other embodiments of these translation systems, the tRNA is an suppressor tRNA. In other embodiments of these translation systems, the translation system includes a tRNA that is to be aminoacylated to the amino acid described above. In other embodiments of these translation systems, the translation system includes an aminoacyl-synthetase that is specific for the tRNA. In other embodiments of these translation systems, the translation systems include an orthogonal tRNA and an orthogonal aminoacyl tRNA synthetase. In other embodiments of these translation systems, the polypeptide is synthesized from ribosomes, and in other embodiments, the translation system is an in vivo translation system comprising cells selected from the group consisting of bacterial cells, archaeal cells, and eukaryotic cells. In other embodiments, the cell is an E.coli cell, a yeast cell, a cell from a Pseudomonas species, a mammalian cell, a plant cell, or an insect cell. In other embodiments of these translation systems, the translation system is an in vitro translation system comprising a cell extract from a bacterial cell, an archaeal cell, or a eukaryotic cell. In other embodiments, the cell extract is a cell from an escherichia coli cell, a species from pseudomonas, a yeast cell, a mammalian cell, a plant cell, or an insect cell. In other embodiments, at least a portion of the polypeptide is synthesized by solid phase or solution phase peptide synthesis methods or combinations thereof, and in other embodiments, further comprising ligating the polypeptide to another polypeptide. In other embodiments, amino acids of formulas I-XVIII, XXX-XXXIV (a and B) and XXXX-xxxiii comprising any of the sub-or specific compounds within the formulas I-XVIII, XXX-XXXIV (a and B) and XXXX-xxxiii may be biosynthetically incorporated into a polypeptide, wherein the polypeptide is a protein homologous to a therapeutic protein selected from the group consisting of: alpha-1 antitrypsin, angiostatin, anti-hemolytic factor, antibody, apolipoprotein, apoprotein, atrial natriuretic factor, atrial natriuretic polypeptide, atrial peptide, C-X-C chemokine, T39765, NAP-2, ENA-78, gro-a, gro-b, gro-C, IP-10, GCP-2, NAP-4, SDF-1, PF4, MIG, calcitonin, C-kit ligand, cytokine, CC chemokine, monocyte chemokine-1, monocyte chemokine-2, monocyte chemokine-3, monocyte inflammatory protein-1 alpha, monocyte inflammatory protein-iβ, RANTES, 1309, R83915, R91733, HCC1, T58847, D31065, T64262, CD40 ligand, C-kit ligand, collagen, and pharmaceutical compositions containing the same Colony Stimulating Factor (CSF), complement factor 5a, complement inhibitor, complement receptor 1, cytokine, epithelial neutrophil activating peptide-78, MIP-16, MCP-1, epidermal Growth Factor (EGF), epithelial neutrophil activating peptide, erythropoietin (EPO), epidermal exfoliative toxin, factor IX, factor VII, factor VIII, factor X, fibroblast Growth Factor (FGF), fibrinogen, fibronectin, tetrahelical bundle protein, G-CSF, glp-1, GM-CSF, glucocerebrosidase, gonadotropin, growth factor receptor, grf, hedgehog protein, hemoglobin, hepatocyte growth factor (hGF), hirudin, human growth hormone (hGH), human serum albumin, ICAM-1, ICAM-1 receptor, LFA-1 receptor, insulin-like growth factor (IGF), IGF-I, IGF-II, interferon (IFN), IFN-alpha, IFN-beta, IFN-gamma, interleukin (IL), IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, keratinocyte Growth Factor (KGF), lactoferrin, leukemia inhibitory factor, luciferase, neurogenin, neutrophil Inhibitory Factor (NIF), oncostatin M, osteogenic protein, oncogene products, paracitin, parathyroid hormone, PD-ECSF, PDGF, peptide hormones, pleiotrophin, protein A, protein G, pth, pyrogenic exotoxin A, neutropy the therapeutic agents include, but are not limited to, heat exotoxin B, heat exotoxin C, pyy, relaxin, renin, SCF, small biosynthetic proteins, soluble complement receptor I, soluble I-CAM 1, soluble interleukin receptor, soluble TNF receptor, somatostatin, growth hormone, streptokinase, superantigen, staphylococcal enterotoxin, SEA, SEB, SEC1, SEC2, SEC3, SED, SEE, steroid hormone receptor, superoxide dismutase, toxic shock syndrome toxin, thymosin alpha 1, tissue type plasminogen activator, tumor Growth Factor (TGF), tumor necrosis factor alpha, tumor necrosis factor beta, tumor Necrosis Factor Receptor (TNFR), VLA-4 protein, VCAM-1 protein, vascular Endothelial Growth Factor (VEGF), urokinase, mos, ras, raf, met, p, tat, fos, myc, jun, myb, rel, estrogen receptor, progesterone receptor, testosterone receptor, aldosterone receptor, LDL receptor, and corticosterone.
B. Post-translational modification in vivo
By producing polypeptides of interest having at least one unnatural amino acid in eukaryotic cells, these polypeptides may comprise eukaryotic post-translational modifications. In certain embodiments, the protein comprises at least one unnatural amino acid and at least one post-translational modification by a eukaryotic cell in vivo, where the post-translational modification is not by a prokaryotic cell. For example, post-translational modifications include, but are not limited to, acetylation, acylation, lipid-modification, palmitoylation, palmitate addition, phosphorylation, glycolipid-bond modification, glycosylation, and the like. In one aspect, post-translational modification comprises binding an oligosaccharide (including, but not limited to) (GlcNAc-Man) via a GlcNAc-asparagine linkage 2 -Man-GlcNAc)) to asparagine. See table 1, which lists some examples of N-linked oligosaccharides of eukaryotic proteins (other residues may also be present, which are not shown). In another aspect, post-translational modification comprises attachment of an oligosaccharide (including, but not limited to, gal-GalNAc, gal-GlcNAc, etc.) to serine or threonine by GalNAc-serine or GalNAc-threonine linkages, or GlcNAc-serine or GlcNAc-threonine linkages.
Table 1: examples of oligosaccharides linked by GlcNAc linkage
Figure BDA0001546710350001401
In another aspect, the post-translational modification comprises proteolytic processing of a precursor (including, but not limited to, a calcitonin precursor, a calcitonin gene related peptide precursor, preproparathyroid hormone, preproinsulin, proinsulin, pro-opiomelanocortin, and the like), assembly into a multi-subunit protein or macromolecular assembly, translation to another site in the cell (including, but not limited to, an organelle such as the endoplasmic reticulum, golgi apparatus, nucleus, lysosomes, peroxisomes, mitochondria, chloroplasts, vacuoles, and the like, or via the secretory pathway). In certain embodiments, the protein comprises a secretion or localization sequence, an epitope tag, a FLAG tag, a polyhistidine tag, a GST fusion, or an analog thereof.
One advantage of unnatural amino acids is that they exist with other chemical moieties that can be used to add other molecules. These modifications may be made in vivo or in vitro in eukaryotic or non-eukaryotic cells. Thus, in certain embodiments, the post-translational modification is by an unnatural amino acid. For example, post-translational modification may be performed by nucleophilic-electrophilic reactions. Most translations currently used for selective modification of proteins involve the formation of covalent bonds between nucleophilic and electrophilic reaction partners, including, but not limited to, reactions of alpha-haloketones with histidine or cysteine side chains. The selectivity in these cases is determined by the number and accessibility of nucleophilic residues in the protein. Other more selective reactions may be used in the polypeptides described herein or prepared using the methods described herein, including, but not limited to, in vitro and in vivo reactions of unnatural keto-amino acids with hydrazides or aminooxy compounds. See, for example, cornish et al, (1996) Am.Chem.Soc.118:8150-8151; mahal et al, (1997)Science,276:1125-1128; wang et al, (2001)Science292:498-500; chin et al, (2002)Am.Chem.Soc.124:9026-9027; chin et al, (2002)Proc.Natl.Acad.Sci..99:11020-11024; wang et al, (2003)Proc.Natl.Acad.Set100:56-61; zhang et al, (2003)Biochemistry.42:6735-6746; chin et al, (2003)Science300:964-967. This allows the selective labelling of almost any protein with a number of reagents, including fluorophores, cross-linking agents, sugar derivatives, and cytotoxic molecules. See also 1 month and 16 days 2003U.S. patent application Ser. No. 10/686,944, entitled "Glycoprotein synthesis," which is incorporated by reference herein. Post-translational modifications, including but not limited to through azido amino acids, can also be made through staudinger ligation (Staudinger ligation), including but not limited to with triarylphosphine reagents. See, for example, kiick et al, (2002) Incorporation of azides into recombinant proteins for chemoselective modification by the Staudinger ligtation,PNAS 99(1):19-24。
IX. alternative systems for the preparation of unnatural amino acid polypeptides
Several strategies have been used to introduce unnatural amino acids into proteins in non-recombinant host cells, mutagenized host cells, or cell-free systems. The alternative systems disclosed in this section can be applied to the preparation of the unnatural amino acid polypeptides described herein. For example, derivatizing amino acids with reactive side chains (such as Lys, cys, and Tyr) converts lysine to N 2 -acetyl-lysine. Chemical synthesis also provides a simple method of incorporating unnatural amino acids. With the recent development of enzymatic and natural chemical ligation of peptide fragments, it is possible to make larger proteins. See e.g. p.e.dawson and s.b.h.kent,Annu.Rev.Biochem,69:923 (2000). Chemical peptide ligation and native chemical ligation are described in U.S. patent No. 6,184,344, U.S. patent publication No. 2004/01388412, U.S. patent publication No. 2003/0208046, WO 02/098902, and WO 03/042235, which are all incorporated herein by reference. General in vitro biosynthesis methods in which suppressor tRNA chemically acylated with a desired unnatural amino acid is added to an in vitro extract capable of supporting protein biosynthesis have been used to site-specifically incorporate over 100 unnatural amino acids into a wide variety of proteins of virtually any size. See for example V.W.Cornish, D.Mendel and p.g.schultz,Angew.Chem.Int.Ed.Engl.1995,34:621-633(1995)；C.J.Noren,S.J.Anthony-Cahill,M.C.Griffith,P.G.Schultz,A general method for site-specific incorporation of unnatural amino acids into proteins,Science244182-188 (1989); J.D.Bain, C.G.Glabe, T.A.Dix, A.R.Chamberlin,E.S.Diala,Biosynthetic site-specific incorporation of a unnatural amino acid into a polypeptide,J.Am.Chem.Soc.1118013-8014 (1989). Various functional groups have been introduced into proteins to study protein stability, protein folding, enzymatic mechanisms, and signal transduction.
An in vivo approach called selective pressure incorporation was developed to develop hybridization of wild-type synthetases. See for example N.Budisa, C.Minks, S.Alefelder, W.Wenger, F.M.Dong, L.Moroder and r.huber, FASEB J..13:41-51 (1999). An auxotrophic strain in which the relevant metabolic pathway that supplies a particular natural amino acid to a cell is cut off is grown in minimal medium containing a limited concentration of the natural amino acid while transcription of the target gene is inhibited. At the beginning of the stationary growth phase, the natural amino acids are depleted and replaced with unnatural amino acid analogs. Recombinant protein expression is induced such that proteins containing non-native analogs accumulate. For example, using this strategy, o-fluorophenylalanine, m-fluorophenylalanine, and p-fluorophenylalanine have been incorporated into proteins, and exhibit two characteristic shoulders in the UV spectrum that can be readily identified. See for example C.Minks, R.Huber, L.Moroder and n.budisk,Anal.Biochem.29-34 (2000); trifluoromethionine has been used to replace methionine in phage T4 lysozyme by 19 F NMR was used to study its interaction with chitosan oligosaccharide ligands, see e.g., H.Duewel, E.Daub, V.Robinson and J.F.Honek,Biochemistry,36:3404-3416 (1997); and the incorporation of trifluoroleucine in place of leucine results in an increase in the thermal and chemical stability of the leucine zipper protein. See, e.g., Y.Tang, G.Ghirlanda, W.A.Petka, T.Nakajima, W.F.DeGrado and d.a. tirrell, angelw. Chem.Int.Ed.Engl..40 (8):1494-1496 (2001). In addition, selenomethionine and telluromethionine are incorporated into various recombinant proteins to facilitate resolution of phases in X-ray crystallography. See for example W.A.Hendrickson, J.R.Horton and d.m. lemaster,EMBO J.1665-1672 (1990); J.O.Boles, K.Lewinski, M.Kunkle, J.D.Odom, B.Dunlap, L.Lebioda and m.hatada,Nat.Struct.Biol283-284 (1994); N.Budisa, B.Steipe, P.Demange, C.Eckerskorn, J.Kellermann and R.huber,Eur.J.Biochem.230:788-796 (1995); and N.Budisa, W.Karnbrock, S.Steinbacher, A.Humm, L.Prade, T.Neuefeind, L.Moroder and r.huber,J.Mol.Biol.270:616-623 (1997). Methionine analogues with alkene or alkyne functionalities have also been effectively incorporated, which allow other modifications of proteins by chemical means. See e.g. j.c.m.vanhest and d.a.tirrell,FEBS Lett..428:68-70 (1998); J.C.M.van Hest, K.L.kiick and D.A.tirrell,J.Am.Chem.Soc122:1282-1288 (2000); K.L. kiick and D.A. tirrell, tetrahedron,56:9487-9493 (2000); U.S. patent No. 6,586,207; U.S. patent publication 2002/0042097, which is incorporated by reference herein in its entirety.
The success of this approach depends on the recognition of unnatural amino acid analogs by aminoacyl-tRNA synthetases, which generally require high selectivity to ensure fidelity of protein translation. One way to extend the scope of this approach is to relax the substrate specificity of the aminoacyl-tRNA synthetases, which has been achieved in a limited number of cases. By way of example only, substitution of Gly for Ala in E.coli phenylalanyl-tRNA synthetase (PheRS) 294 The size of the substrate binding pocket was increased and tRNAPHE was acylated with p-phenylalanine (p-Cl-Phe). See, M.Ibba, P.Kast and H.Hennecke,Biochemistry.33:7107-7112 (1994). Coli strains containing this mutant PheRS are allowed to incorporate p-phenylalanine or p-bromophenylalanine instead of phenylalanine. See e.g. m.ibba and h.hennecke,FEBS Lett..364:272-275 (1995); N.Sharma, R.Furter, P.Kast and d.a. tirrell,FEBS Lett..467:37-40 (2000). Similarly, the point mutation Phe130Ser at the amino acid binding site proximal to the escherichia coli tyrosyl-tRNA synthetase exhibits a tolerance for the more efficient incorporation of diazotyrosine than tyrosine. See, F.Hamano-Takaku, T.Iwama, S.Saito-Yano, K.Takaku, Y.Monden, M.Kitabatake, D.Soil and S.Nishimura, LBiol. Chem..275(51):40324-40328(2000)。
Another strategy for incorporating unnatural amino acids into proteins in vivo is to modify synthetases with proofreading mechanisms. These synthetases are indistinguishable and therefore activate structurally homologous natural amino acidsAmino acids. This error was corrected at the individual sites, which deacylated mismatched amino acids from the tRNA to maintain fidelity of protein translation. If the proofreading activity of the synthetase is lost, the erroneously activated structural analog may circumvent the editing function and be incorporated. This method has recently been demonstrated with valyl-tRNA synthetase (ValRS). See V.Doring, H.D.Mootz, L.A.Nangle, T.L.Hendrickson, V.de Crycy-Lagard, P.Schimmel and P.Marliere, Science.292:501-504 (2001). ValRS can be transamidated with Cys, thr, or aminobutyrate (Abu) to trnaal; these non-homologous amino acids are then hydrolyzed by the edit domain. After randomly mutagenizing the E.coli chromosome, a mutant E.coli strain having a mutation in the editing site of ValRS is selected. This edit defect ValRS error loads trnaal with Cys. Because Abu is spatially similar to Cys (in Abu Cys-SH groups are via-CH 3 Substitution), the mutant ValRS also incorporates Abu into the protein when this mutant e.coli strain is grown in the presence of Abu. Mass spectrometry showed that about 24% of valine was replaced by Abu at each valine position of the native protein.
Solid phase synthesis and semisynthetic methods also allow the synthesis of many proteins containing novel amino acids. See, for example, the following publications and references cited therein: crick, f.j.c., barrett, L.Brenner, S.Watts-Tobin, r.general nature of the genetic code for proteins.Nature,192(4809):1227-1232(1961)；Hofmann,K.,Bohn,H.Studies on polypeptides.XXXVI.The effect of pyrazole-imidazole replacements on the S-protein activating potency of an S-peptide fragment,J,Am Chem,88(24):5914-5919(1966)；Kaiser,E.T.Synthetic approaches to biologically active peptides and proteins including enyzmes,Acc Chem Res.22(2):47-54(1989)；Nakatsuka,T.,Sasaki,T.,Kaiser,E.T.Peptide segment coupling catalyzed by the semisynthetic 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 Biochemu 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 Total Synthesis of Ribonuclease A with Unnatural Catalytic Residues, Science,266,243-247(1994)。
Chemical modifications have been used to introduce a variety of unnatural side chains including cofactors, spin labels, and oligonucleotides into proteins in vitro. See, e.g., corey, D.R., schultz, P.G.Generation of a hybrid sequence-specific single-stranded deoxyribonuclease,Science,238,1401-1403(1987)；Kaiser,E.T.,Lawrence D.S.,Rokita,S.E.The chemical modification of enzymatic specificity,Ann.Rev Biochem,54,565-595(1985)；Kaiser,E.T.,Lawrence,D.S.Chemical mutation of enyzme active sites,Science,226,505-511(1984)；Neet,K.E.,Nanci A,Koshland,D.E.Properties of thiol-subtilisin,J Biol.Chem,243(24):6392-6401(1968)；Polgar,L.B.,M.L.A new enzyme containing a synthetically formed active site.Thiol-subtilisin.J.Am Chem Soc,88 (13) 3153-3154 (1966); and Pollock, S.J., nakayama, G.Schultz, P.G.Introduction of nucleophiles and spectroscopic probes into antibody combining sites,Science,1(242):1038-1040(1988)。
alternatively, biosynthetic methods using chemically modified aminoacyl-tRNA's have been used to incorporate several biophysical probes into proteins synthesized in vitro. See the following publications and references cited therein: brunner, j.new Photolabeling and crosslinking methods,Annu.Rev Biochem483-514 (1993); and Krieg, U.C., walter, P., hohnson, A.E., photoross linking of the signal sequence of nascent preprolactin of the-kilodalton polypeptide of the signal recognition particle,Proc.Natl.Acad.Sci.83,8604-8608(1986)。
previously, it has been demonstrated that chemical aminoacylation can be performed in vitro by adding a chemical aminoacylation to a protein synthesis reaction programmed with a gene containing the desired amber nonsense mutationSuppression of tRNA's to site-specifically incorporate unnatural amino acids into proteins. Using these methods, a strain that is nutritionally deficient for a particular amino acid can be used to replace a multitude of common 20 amino acids with close structural homologs, e.g., phenylalanine with fluorophenylalanine. See, e.g., noren, C.J., anthony-Call, griffith, M.C., schultz, P.G.A. general method for site-specific incorporation of unnatural amino acids into proteins, Science.244:182-188 (1989); w. nowak et al,Science 268:439-42(1995)；Bain,J.D.,Glabe,C.G.,Dix,T.A.,Chamberlin,A.R.,Diala,E.S.Biosynthetic site-specific Incoiporation of a non-natural amino acid into a polypeptide,J.Am Chem Soc111:8013-8014 (1989); n. budisk et al,FASEB J.13:41-51(1999)；Ellman,J.A.,Mendel,D.,Anthony-Cahill,S.,Noren,C.J.,Schultz,P.G.Biosynthetic method for introducing unnatural ammo acids site-specifically into proteins.Methods in Enz.,202,301-336 (1992); and Mendel, D., cornish, V.W., and Schultz, P.G.site-Directed Mutagenesis with an Expanded Genetic Code,Annu Rev Biophys.Biomol Struct.24,435-62(1995)。
for example, suppressor tRNA is prepared that recognizes the stop codon UAG and is chemically aminoacylated with an unnatural amino acid. Conventional site-directed mutagenesis is used to create the introduction of a stop codon TAG at a site of interest in a protein gene. See, e.g., sayers, J.R., schmidt, W.Eckstein, F.5',3'Exonuclease in phosphorothioate-based oligonucleotide-directed mutagenesis,Nucleic Acids Res.16 (3):791-802 (1988). When the acylation-suppressing tRNA is combined with the mutant gene in an in vitro transcription/translation system, the unnatural amino acid is incorporated in response to the UAG codon to yield a protein that contains that amino acid at the indicated position. Use [ use 3 H]Experiments with Phe and with alpha-hydroxy acids confirm that only the desired amino acid is incorporated at the position specified by the UAG codon and that this amino acid is not incorporated at any other site in the protein. See, for example, noren et al, supra; kobayashi et al, (2003) Nature Structural Biology 10 (6): 425-432; and Ellman, j.a., mendel, d., schultz, p.g., site-specific i ncorporation of novel backbone structures into proteins,Science,255,197-200(1992)。
Microinjection techniques have also been used to incorporate unnatural amino acids into proteins. See for example M.W.Nowak, P.C.Kearney, J.R.Sampson, M.E.Saks, C.G.Labarca, S.K.Silverman, W.G.Zhong, J.Thorson, J.N.Abelson, N.Davidson, P.G.Schultz, D.A.Dougherty and h.a. Lester,Science,268:439-442 (1995); d.a. Dougherty,Curr.Opin.Chem.Biol,4:645 (2000). Co-injecting into xenopus oocytes two RNA species produced in vitro: an mRNA encoding a protein of interest having a UAG stop codon at an amino acid position of interest and an amber suppressor tRNA aminoacylated with a desired unnatural amino acid. The translation mechanism of the oocyte is then inserted with unnatural amino acids at the position specified by UAG. This method allows in vivo structure-function studies of intact membrane proteins, which are generally not affected by in vitro expression systems. Examples include, but are not limited to, incorporation of fluorescent amino acids into the tachykinin neurokinin-2 receptor to measure distance by fluorescence resonance energy transfer, see, e.g., G.Turcatti, K.Nemeth, M.D.Edgerton, U.Meseth, F.Talabot, M.Peitsch, J.Knowles, H.Vogel and a. Chollet, IBiol.Chem271 (33): 19991-19998 (1996); the incorporation of biotinylated amino acids to identify surface exposed residues in ion channels, see, e.g., J.P.Gallivan, H.A.Lester and d.a. Dougherty, Chem.Biol4 (10): 739-749 (1997); the use of caged tyrosine analogues to monitor conformational changes in ion channels in real time, see for example J.C.Miller, S.K.Silverman, P.M.England, D.A.Dougherty and h.a. Lester, neuron,20:619-624 (1998); and, using alpha hydroxy amino acids that alter the ion channel backbone to probe its gating mechanism. See for example P.M.England, Y.Zhang, D.A.Dougherty and h.a. Lester,Cell96:89-98 (1999); T.Lu, A.Y.Ting, J.Mainland, L.Y.Jan, P.G.Schultz and j. Yang,Nat.Neurosci.,4(3):239-246(2001)。
the ability to incorporate unnatural amino acids directly into proteins in vivo provides the advantages of high yields of mutant proteins, ease of technology, the possibility of studying mutant proteins in cells and possibly in living organisms, and the use of these mutant proteins in therapeutic treatments. The ability to include unnatural amino acids of various sizes, acidity, nucleophilicity, hydrophobicity, and other properties into proteins can greatly extend our ability to rationally and systematically manipulate the structure of proteins to probe protein function and produce new proteins or organisms with novel properties.
In an attempt to site-specifically incorporate para-fluorophenylalanine, the yeast amber-suppressing tRNAPHUCA/phenylalanyl-tRNA synthetase pair was used in p-F-Phe resistant, phe auxotrophic E.coli strains. See for example r.furter, Protein Sci.,7:419-426(1998)。
It is also possible to use a cell-free (in vitro) translation system to obtain expression of the desired polynucleotide. In these systems, they may comprise either mRNA as a template (in vitro translation) or DNA as a template (combined in vitro transcription and translation), with in vitro synthesis being ribosome-directed. Considerable effort has been applied to develop cell-free protein expression systems. See, e.g., kim, d. -m, and j.r. swartz, biotechnology and Bio engineering,74 (4): 309-316 (2001); kim, d. -m.and j.r.swartz, biotechnology Letters,22,1537-1542, (2000); kim, d. -m.and j.r.swartz, biotechnology Progress,16,385-390, (2000); kim, d. -m.and j.r.swartz, biotechnology and Bioengineering,66 (3): 180-188, (1999); and Patnaik, R.and J.R.Swartz, biotechnology 24 (5): 862-868, (1998); U.S. patent No. 6,337,191; U.S. patent publication No. 2002/0081660; WO 00/55353; WO 90/05785, which is incorporated herein by reference in its entirety. Another method applicable to the expression of polypeptides comprising unnatural amino acids includes, but is not limited to, mRNA-peptide fusion techniques. See, e.g., r.roberts and J.Szostak, proc.Natl acad.sci. (USA) 9412297-12302 (1997); franKel et al Chemistry & Biology 10,1043-1050 (2003). In this method, the mRNA template linked to puromycin is translated into a peptide on the ribosome. Unnatural amino acids can also be incorporated into peptides if one or more tRNA molecules are modified. After reading the last mRNA codon, puromycin captures the C-terminus of the peptide. If the resulting mRNA-peptide conjugate is found to have interesting properties in an in vitro assay, its identity can be readily revealed from the mRNA sequence. In this way, libraries of polypeptides comprising one or more unnatural amino acids can be screened to identify polypeptides having the desired properties. Recently, it has been reported that in vitro ribosome translation with a purified component allows the synthesis of peptides substituted with unnatural amino acids. See, e.g., A. Forster et al, proc. Natl Acad. Sci. (USA) 100 (11): 6353-6357 (2003).
Post-translational modification of unnatural amino acid components of polypeptides
For convenience, post-translational modifications of the unnatural amino acid components of the polypeptides described in this section (XA through XJ) are generally and/or described with specific examples. However, the post-translational modification of the unnatural amino acid component of the polypeptides described in this section should not be limited to the general description or the specific examples provided in this section, but rather the post-translational modification of the unnatural amino acid component of the polypeptides described in this section is equally well suited for use with all compounds within the categories of formulas I-XVIII, XXX-XXXIV (A and B) and XXXXX-XXXXIII, including any sub-or specific compounds within the categories of formulas I-XVIII, XXX-XXXIV (A and B) and XXXXXXXXXXIII described in the specification, claims and drawings herein.
Methods, compositions, techniques and strategies have been developed to site-specifically incorporate unnatural amino acids during in vivo translation of proteins. This technique enables site-specific derivatization of recombinant proteins by incorporating unnatural amino acids with side chain chemistry orthogonal to those of naturally occurring amino acids. Thus, a major advantage of the methods, compositions, techniques and strategies described herein is that derivatized proteins can now be prepared to determine homologous products. However, the methods, compositions, reaction mixtures, techniques, and strategies described herein are not limited to unnatural amino acid polypeptides formed by in vivo protein translation techniques, but include unnatural amino acid polypeptides formed by any technique, including, by way of example only, expression protein ligation, chemical synthesis, ribozyme-based techniques (see, e.g., the section entitled "expression in alternative systems" herein).
The ability to incorporate unnatural amino acids into recombinant proteins is widely spread to enable chemistry for post-translational derivatization, where such derivatization occurs in vivo or in vitro. More specifically, derivatization of proteins that form oxime bonds on the unnatural amino acid portion of polypeptides provides several advantages. First, naturally occurring amino acids typically do not form oxime bonds and thus reagents designed to form oxime bonds will react site-specifically with the unnatural amino acid component of a polypeptide (assuming of course that the unnatural amino acid and corresponding reagents have been designed to form oxime bonds), thus the ability to site-selectively derivatize proteins provides a single homogeneous product, as opposed to mixtures of derivatized proteins produced using prior art processes. Second, oxime adducts are stable under biological conditions, suggesting that proteins derived from oxime exchange are effective candidates for therapeutic applications. Third, the stability of the resulting oxime bond can be manipulated according to the nature (i.e., functional group and/or structure) of the unnatural amino acid on which the oxime bond has been formed. Thus, as shown in example 16, the pH stability of the oxime bond of an unnatural amino acid can vary from less than 1 hour to much more than 1 week. Thus, in some embodiments, the oxime bond of the unnatural amino acid polypeptide has a half-life for decomposition of less than 1 hour, in other embodiments less than 1 day, in other embodiments less than 2 days, in other embodiments less than 1 week, and in other embodiments more than 1 week. In other embodiments, the resulting oxime is stable under moderately acidic conditions for at least two weeks, and in other embodiments, the resulting oxime is stable under moderately acidic conditions for at least 5 days. In other embodiments, the unnatural amino acid polypeptide is stable at a pH between about 2 and about 8 for at least 1 day; in other embodiments, the stabilization is at a pH of about 2 to about 6 for at least 1 day; in other embodiments, the stabilization is at a pH of about 2 to about 4 for at least 1 day. In other embodiments, using the strategies, methods, compositions, and techniques described herein, one of skill in the art should be able to synthesize oxime linkages of unnatural amino acid polypeptides having a half-life that is adjusted to the resolution desired by the skilled artisan (e.g., for therapeutic use (such as sustained release), or diagnostic use, or industrial use or military use).
The above-described unnatural amino acid polypeptides are useful for, including but not limited to, novel therapeutic agents, diagnostic agents, catalytic enzymes, industrial enzymes, binding proteins (including but not limited to antibodies and antibody fragments), and for studying, including but not limited to, protein structure and function. See, e.g., dougherty, (2000) Unnatural Amino Acids as Probes of Protein Structure and Function,Current Opinion in Chemical Biology,4:645-652. Other uses for the above-described non-natural amino acid polypeptides include, by way of example only, assay-based uses, cosmetic uses, plant biology uses, environmental uses, energy production uses, and/or military uses. However, the above-described unnatural amino acid polypeptides can undergo further modifications to incorporate new or modified functional groups, which comprise manipulating the therapeutic efficacy of the polypeptide; improving the safety profile of the polypeptide; modulating the pharmacokinetics, pharmacology, and/or potency of a polypeptide (e.g., increasing water solubility, bioavailability, increasing serum half-life, increasing therapeutic half-life, modulating immunogenicity, modulating biological activity, or extending circulation time); providing other functional groups to the polypeptide; incorporating a tag, label or detectable signal into the polypeptide; facilitating the isolation properties of the polypeptide; and any combination of the foregoing modifications.
A method of facilitating the isolation of properties of a polypeptide in certain embodiments, comprising synthesizing a non-natural amino acid polypeptide using homologous biology comprising at least one non-natural amino acid, the non-natural amino acid being selected from the group consisting of oxime-containing non-natural amino acids, carbonyl-containing non-natural amino acids, and hydroxylamine-containing non-natural amino acids. In other embodiments, these unnatural amino acids have been biosynthetically incorporated into polypeptides as described herein. In other or alternative embodiments, these unnatural amino acid polypeptides include at least one unnatural amino acid selected from the group consisting of amino acids of formulas I-XVIII, XXX-XXXIV (A and B), or XXXXX-XXXXIII.
The methods, compositions, strategies, and techniques described herein are not limited to a particular type, kind, or family of polypeptides. Indeed, almost any polypeptide may comprise at least one unnatural amino acid described herein. By way of example only, the polypeptide may be homologous to a therapeutic protein selected from the group consisting of: alpha-1 antitrypsin, angiostatin, anti-hemolytic factor, antibody, apolipoprotein, apoprotein, atrial natriuretic factor, atrial natriuretic polypeptide, atrial peptide, C-X-C chemokine, T39765, NAP-2, ENA-78, gro-a, gro-b, gro-C, IP-10, GCP-2, NAP-4, SDF-1, PF4, MIG, calcitonin, C-kit ligand, cytokine, CC chemokine, monocyte chemokine-1, monocyte chemokine-2, monocyte chemokine-3, monocyte inflammatory protein-1 alpha, monocyte inflammatory protein-iβ, RANTES, 1309, R83915, R91733, HCC1, T58847, D31065, T64262, CD40 ligand, C-kit ligand, collagen, and pharmaceutical compositions containing the same Colony Stimulating Factor (CSF), complement factor 5a, complement inhibitor, complement receptor 1, cytokine, epithelial neutrophil activating peptide-78, MIP-16, MCP-1, epidermal Growth Factor (EGF), epithelial neutrophil activating peptide, erythropoietin (EPO), epidermal exfoliative toxin, factor IX, factor VII, factor VIII, factor X, fibroblast Growth Factor (FGF), fibrinogen, fibronectin, tetrahelical bundle protein, G-CSF, glp-1, GM-CSF, glucocerebrosidase, gonadotropin, growth factor receptor, grf, hedgehog protein, hemoglobin, hepatocyte growth factor (hGF), hirudin, human growth hormone (hGH), human serum albumin, ICAM-1, ICAM-1 receptor, LFA-1 receptor, insulin-like growth factor (IGF), IGF-I, IGF-II, interferon (IFN), IFN-alpha, IFN-beta, IFN-gamma, interleukin (IL), IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, keratinocyte Growth Factor (KGF), lactoferrin, leukemia inhibitory factor, luciferase, neurogenin, neutrophil Inhibitory Factor (NIF), oncostatin M, osteogenic protein, oncogene products, paracitin, parathyroid hormone, PD-ECSF, PDGF, peptide hormones, pleiotrophin, protein A, protein G, pth, pyrogenic exotoxin A, neutropy the therapeutic agents include, but are not limited to, heat exotoxin B, heat exotoxin C, pyy, relaxin, renin, SCF, small biosynthetic proteins, soluble complement receptor I, soluble I-CAM 1, soluble interleukin receptor, soluble TNF receptor, somatostatin, growth hormone, streptokinase, superantigen, staphylococcal enterotoxin, SEA, SEB, SEC1, SEC2, SEC3, SED, SEE, steroid hormone receptor, superoxide dismutase, toxic shock syndrome toxin, thymosin alpha 1, tissue type plasminogen activator, tumor Growth Factor (TGF), tumor necrosis factor alpha, tumor necrosis factor beta, tumor Necrosis Factor Receptor (TNFR), VLA-4 protein, VCAM-1 protein, vascular Endothelial Growth Factor (VEGF), urokinase, mos, ras, raf, met, p, tat, fos, myc, jun, myb, rel, estrogen receptor, progesterone receptor, testosterone receptor, aldosterone receptor, LDL receptor, and corticosterone. The unnatural amino acid polypeptide can also be homologous to any polypeptide member of the growth hormone supergene family.
These modifications comprise binding other functional groups to the unnatural amino acid component of the polypeptide, including (but not limited to): marking; a dye; a polymer; a water-soluble polymer; derivatives of polyethylene glycol; a photocrosslinking agent; a cytotoxic compound; a drug; affinity labeling; a photoaffinity label; a reactive compound; a resin; a second protein or polypeptide analog; an antibody or antibody fragment; a metal chelator; a cofactor; a fatty acid; a carbohydrate; a polynucleotide; DNA; RNA; an antisense polynucleotide; saccharides, water-soluble dendrimers, cyclodextrins, biomaterials; a nanoparticle; spin labeling; a fluorophore-containing moiety; a radioactive portion; novel functional groups; groups that interact covalently or non-covalently with other molecules; a light-shielding portion; an actinic radiation excitable moiety; a ligand; a photoisomerizable moiety; biotin; biotin analogues; a moiety incorporating a heavy atom; a chemically cleavable group; a photocleavable group; an extended side chain; a carbon-bonded sugar; a redox active agent; an aminothiopropionic acid; a toxic moiety; isotopically labeled moieties; a biophysical probe; phosphorescent groups; a chemiluminescent group; an electron dense group; a magnetic group; an insertion group; a chromophore; an energy transfer agent; a bioactive agent; a detectable label; a small molecule; inhibitory ribonucleic acid, radionucleotide; a neutron capture agent; derivatives of biotin; a quantum dot; nano transitorin; a radiotransferrin; abzymes, activating complex activators, viruses, adjuvants, aglycones, allergens, angiostatin, anti-hormones, antioxidants, aptamers, guide RNAs, saponins, shuttle vectors, macromolecules, pseudoepitopes, receptors, reverse micelles, and any combination thereof.
In addition, the unnatural amino acid polypeptide can contain moieties that can be converted to other functional groups, such as, for example only, carbonyl, dicarbonyl, or hydroxylamine. FIG. 63A illustrates the chemical conversion of a non-natural amino acid polypeptide to a carbonyl-or dicarbonyl-containing non-natural amino acid polypeptide, while FIG. 63B illustrates the chemical conversion of a non-natural amino acid polypeptide to a hydroxylamine-containing non-natural amino acid polypeptide. The resulting carbonyl-or dicarbonyl-containing unnatural amino acid polypeptides can be used or incorporated in any of the methods, compositions, techniques, and strategies for preparing, purifying, characterizing, and using the unnatural amino acids, unnatural amino acid polypeptides, and modified unnatural amino acid polypeptides described herein. Chemical conversion of chemical moieties to other functional groups (such as, for example only, carbonyl, dicarbonyl, or hydroxylamine) can be accomplished using techniques and materials known to those skilled in the art, such as, for example, those described in March, A DVANCED O RGANIC C HEMISTRY Version 5, (Wiley 2001); and Carey and Sundberg, A DVANCED O RGANIC C HEMISTRY Volumes A and B (Plenum 2000, 2001), all of which are incorporated by reference.
Thus, by way of example only, a non-natural amino acid polypeptide containing any one of the following amino acids may be further modified using the methods and compositions described herein:
Figure BDA0001546710350001501
Wherein:
a is optional and when present is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkyl, substituted lower heterocycloalkyl, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene;
b is optional and when present is a linker selected from the group consisting of: lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O- (alkylene or substituted alkylene) -, -S- (alkylene or substituted alkylene) -, -S (O) k - (wherein k is 1, 2 or 3), -S (O) k (alkylene or substituted alkylene) -, -C (O) -, -NS (O) 2 -、-OS(O) 2 -C (O) - (alkylene or substituted alkylene) -, -C (S) - (alkylene or substituted alkylene) -, -N (R '), -NR ' - (alkylene or substituted alkylene) -, -C (O) N (R '), -CON (R ') - (alkylene or substituted alkylene) -, -CSN (R ') - (alkylene or substituted alkylene) -, -N (R ') CO- (alkylene or substituted alkylene) -, -N (R ') C (O) O-, -S (O) k N(R')-、-N(R')C(O)N(R')-、-N(R')C(S)N(R')-、-N(R')S(O) k N(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 ') -, wherein each R' is independently H, alkyl or substituted alkyl;
j is
Figure BDA0001546710350001511
R is H, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl;
each R "is independently H, alkyl, substituted alkyl, or a protecting group, or when more than one R" group is present, two R "optionally form a heterocycloalkyl;
R 1 h, amino protecting group and resin; and is also provided with
R 2 OH, an ester protecting group and a resin;
R 3 and R is R 4 Each independently is H, halogen, lower alkyl or substituted lower alkyl, or R 3 And R is R 4 Or two R 3 The radicals optionally form cycloalkyl or heterocycloalkyl;
or-a-B-J-R groups together form a bicyclic or tricyclic cycloalkyl or heterocycloalkyl group including at least one carbonyl (including dicarbonyl), protected carbonyl (including protected dicarbonyl), or masked carbonyl (including masked dicarbonyl);
or-J-R groups together form a mono-or bi-cyclic cycloalkyl or heterocycloalkyl group comprising at least one carbonyl group (including dicarbonyl), protected carbonyl group (including protected dicarbonyl), or masked carbonyl group (including masked dicarbonyl);
Figure BDA0001546710350001521
wherein:
a is optional and when present is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkyl, substituted lower heterocycloalkyl, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene;
B is optional and when present is a linker selected from the group consisting of: lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O- (alkylene or substituted alkylene) -, -S- (alkylene or substituted alkylene) -, -S (O) k - (wherein k is 1, 2 or 3), -S (O) k (alkylene or substituted alkylene) -, -C (O) -, -NS (O) 2 -、-OS(O) 2 -, -C (O) - (alkylene or substituted alkylene) -, -C (S) - (alkylene)Alkyl or substituted alkylene) -, -N (R '), -NR' - (alkylene or substituted alkylene) -, -C (O) N (R '), -CON (R') - (alkylene or substituted alkylene) -, -CSN (R '), -CSN (R') - (alkylene or substituted alkylene) -, -N (R ') CO- (alkylene or substituted alkylene) -, -N (R') C (O) O-, -S (O) k N(R')-、-N(R')C(O)N(R')-、-N(R')C(S)N(R')-、-N(R')S(O) k N(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 ') -, wherein each R' is independently H, alkyl or substituted alkyl;
r is H, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl;
R 1 h, amino protecting group and resin; and is also provided with
R 2 OH, an ester protecting group and a resin;
R 3 and R is R 4 Each independently is H, halogen, lower alkyl or substituted lower alkyl, or R 3 And R is R 4 Or two R 3 The radicals optionally form cycloalkyl or heterocycloalkyl;
R 5 is H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, alkylalkoxy, substituted alkylalkoxy, polyoxyalkylene, substituted polyoxyalkylene, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkylaryl, substituted alkylaryl, arylalkyl, substituted arylalkyl, - (alkylene or substituted alkylene) -ON (R') 2 (alkylene or substituted alkylene) -C (O) SR ', - (alkylene or substituted alkylene) -S-S- (aryl or substituted aryl), -C (O) R', -C (O) 2 R 'or-C (O) N (R') 2 Wherein each R "is independently hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, alkylaryl, substituted alkylaryl, arylalkyl, or substituted arylalkyl;
or R is 5 Is L-X, wherein X is selected from the group consisting of: marking; a dye; a polymer; water-soluble polymerizationA material; derivatives of polyethylene glycol; a photocrosslinking agent; a cytotoxic compound; a drug; affinity labeling; a photoaffinity label; a reactive compound; a resin; a second protein or polypeptide analog; an antibody or antibody fragment; a metal chelator; a cofactor; a fatty acid; a carbohydrate; a polynucleotide; DNA; RNA; an antisense polynucleotide; saccharides, water-soluble dendrimers, cyclodextrins, biomaterials; a nanoparticle; spin labeling; a fluorophore-containing moiety; a radioactive portion; novel functional groups; groups that interact covalently or non-covalently with other molecules; a light-shielding portion; an actinic radiation excitable moiety, a ligand, a photoisomerisable moiety; biotin; biotin analogues; a moiety incorporating a heavy atom; a chemically cleavable group; a photocleavable group; an extended side chain; a carbon-bonded sugar; a redox active agent; an aminothiopropionic acid; a toxic moiety; isotopically labeled moieties; a biophysical probe; phosphorescent groups; a chemiluminescent group; an electron dense group; a magnetic group; an insertion group; a chromophore; an energy transfer agent; a bioactive agent; a detectable label; a small molecule; inhibitory ribonucleic acid, radionucleotide; a neutron capture agent; derivatives of biotin; a quantum dot; nano transitorin; a radiotransferrin; abzymes, activating complex activators, viruses, adjuvants, aglycones, allergens, angiostatin, anti-hormones, antioxidants, aptamers, guide RNAs, saponins, shuttle vectors, macromolecules, pseudoepitopes, receptors, reverse micelles, and any combination thereof; and L is optional and when present is a linker selected from the group consisting of: alkylene, substituted alkylene, alkenylene, substituted alkenylene, -O- (alkylene or substituted alkylene) -, -S- (alkylene or substituted alkylene) -, -S (O) k - (wherein k is 1, 2 or 3), -S (O) k (alkylene or substituted alkylene) -, -C (O) - (alkylene or substituted alkylene) -, -C (S) - (alkylene or substituted alkylene) -, -N (R ') -, and-NR' - (alkylene or substituted alkylene) -, -C (O) N (R ') -, CON (R') - (alkylene or substituted alkylene) -, -CSN (R ') -, CSN (R') - (alkylene or substituted alkylene)Alkylene) -, -N (R ') CO- (alkylene or substituted alkylene) -, -N (R') C (O) O-, - (alkylene or substituted alkylene) -O-n=cr '-, - (alkylene or substituted alkylene) -C (O) NR' - (alkylene or substituted alkylene) -, - (alkylene or substituted alkylene) -S (O) k - (alkylene or substituted alkylene) -S-, - (alkylene or substituted alkylene) -S-S-, -S (O) k N(R')-、-N(R')C(O)N(R')-、-N(R')C(S)N(R')-、-N(R')S(O) k N(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 ') -, wherein each R' is independently H, alkyl or substituted alkyl;
Figure BDA0001546710350001531
wherein:
a is optional and when present is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkyl, substituted lower heterocycloalkyl, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene;
B is optional and when present is a linker selected from the group consisting of: lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O- (alkylene or substituted alkylene) -, -S- (alkylene or substituted alkylene) -, -S (O) k - (wherein k is 1, 2 or 3), -S (O) k (alkylene or substituted alkylene) -, -C (O) -, -NS (O) 2 -、-OS(O) 2 -C (O) - (alkylene or substituted alkylene) -, -C (S) - (alkylene or substituted alkylene) -, -N (R '), -NR ' - (alkylene or substituted alkylene) -, -C (O) N (R '), -CON (R ') - (alkylene or substituted alkylene) -, -CSN (R '), -CSN (R ') ') - (alkylene or substituted alkylene) -, -N (R ') CO- (alkylene or substituted alkylene) -, -N (R') C (O) O-, -S (O) k N(R')-、-N(R')C(O)N(R')-、-N(R')C(S)N(R')-、-N(R')S(O) k N(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 ') -, wherein each R' is independently H, alkyl or substituted alkyl;
k is-NR 6 R 7 Or-n=cr 6 R 7 ；
R is H, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl;
R 1 h, amino protecting group and resin; and is also provided with
R 2 OH, an ester protecting group and a resin;
R 3 and R is R 4 Each independently is H, halogen, lower alkyl or substituted lower alkyl, or R 3 And R is R 4 Or two R 3 The radicals optionally form cycloalkyl or heterocycloalkyl;
R 6 and R is R 7 Each independently selected from the group consisting of: H. alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, polyoxyalkylene, substituted polyoxyalkylene, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkylaryl, arylalkyl, and substituted arylalkyl, -C (O) R', -C (O) 2 R"、-C(O)N(R") 2 Wherein each R "is independently hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, alkylaryl, substituted alkylaryl, arylalkyl, or substituted arylalkyl; or R is 6 Or R is 7 Is L-X, wherein
X is selected from the group consisting of: marking; a dye; a polymer; a water-soluble polymer; derivatives of polyethylene glycol; a photocrosslinking agent; a cytotoxic compound; a drug; affinity labeling; a photoaffinity label; a reactive compound; a resin; a second protein or polypeptide analog; antibodies or anti-antibodiesA body segment; a metal chelator; a cofactor; a fatty acid; a carbohydrate; a polynucleotide; DNA; RNA; an antisense polynucleotide; saccharides, water-soluble dendrimers, cyclodextrins, biomaterials; a nanoparticle; spin labeling; a fluorophore-containing moiety; a radioactive portion; novel functional groups; groups that interact covalently or non-covalently with other molecules; a light-shielding portion; an actinic radiation excitable moiety; a ligand; a photoisomerizable moiety; biotin; biotin analogues; a moiety incorporating a heavy atom; a chemically cleavable group; a photocleavable group; an extended side chain; a carbon-bonded sugar; a redox active agent; an aminothiopropionic acid; a toxic moiety; isotopically labeled moieties; a biophysical probe; phosphorescent groups; a chemiluminescent group; an electron dense group; a magnetic group; an insertion group; a chromophore; an energy transfer agent; a bioactive agent; a detectable label; a small molecule; inhibitory ribonucleic acid, radionucleotide; a neutron capture agent; derivatives of biotin; a quantum dot; nano transitorin; a radiotransferrin; abzymes, activating complex activators, viruses, adjuvants, aglycones, allergens, angiostatin, anti-hormones, antioxidants, aptamers, guide RNAs, saponins, shuttle vectors, macromolecules, pseudoepitopes, receptors, reverse micelles, and any combination thereof; and L is optional and when present is a linker selected from the group consisting of: alkylene, substituted alkylene, alkenylene, substituted alkenylene, -O- (alkylene or substituted alkylene) -, -S- (alkylene or substituted alkylene) -, -S (O) k - (wherein k is 1, 2 or 3), -S (O) k (alkylene or substituted alkylene) -, -C (O) - (alkylene or substituted alkylene) -, -C (S) - (alkylene or substituted alkylene) -, -N (R ') -, -NR ' - (alkylene or substituted alkylene) -, -C (O) N (R ') -, CON (R ') - (alkylene or substituted alkylene) -, -CSN (R ') - (alkylene or substituted alkylene) -, -N (R ') CO- (alkylene or substituted alkylene) -, -N (R ') C (O) O-, -S (O) k N(R')-、-N(R')C(O)N(R')-、-N(R')C(S)N(R')-、-N(R')S(O) k N(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 ') -, wherein each R' is independently H, alkyl or substituted alkyl;
Figure BDA0001546710350001551
wherein:
a is optional and when present is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkyl, substituted lower heterocycloalkyl, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene;
r is H, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl;
R 1 is H, an amino protecting group, a resin, an amino acid, a polypeptide or a polynucleotide;
R 2 Is OH, an ester protecting group, a resin, an amino acid, a polypeptide or a polynucleotide;
X 1 c, S or S (O); and L is alkylene, substituted alkylene, N (R ') (alkylene), or N (R ') (substituted alkylene), wherein each R ' is independently H, alkyl, or substituted alkyl; or (b)
Figure BDA0001546710350001561
Wherein:
a is optional and when present is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkyl, substituted lower heterocycloalkyl, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene;
m is-C (R) 3 )-，
Figure BDA0001546710350001562
Figure BDA0001546710350001563
Wherein (a) indicates bonding to the A group and (b) indicates bonding to the respective carbonyl group, R 3 And R is R 4 Independently selected from H, halogen, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl, or R 3 And R is R 4 Or two R 3 Radicals or two R 4 The radicals optionally form cycloalkyl or heterocycloalkyl;
r is H, halogen, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl;
T 3 is a bond, C (R) (R), O, or S, and R is H, halogen, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;
R 1 Is H, an amino protecting group, a resin, an amino acid, a polypeptide or a polynucleotide; and is also provided with
R 2 Is OH, an ester protecting group, a resin, an amino acid, a polypeptide or a polynucleotide.
One aspect of the methods and compositions described herein is a composition comprising at least one polypeptide having at least one (including, but not limited to, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten or more) unnatural amino acids that have been post-translationally modified. The post-translationally modified unnatural amino acids can be the same or different, including, but not limited to, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more than 20 different post-translationally modified unnatural amino acids can be present in the polypeptide at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more than 20 different sites. In another aspect, the composition comprises a polypeptide in which at least one (but less than all) of the specific amino acids present in the polypeptide are replaced with post-translationally modified unnatural amino acids. For a given polypeptide having more than one post-translationally modified unnatural amino acid, the post-translationally modified unnatural amino acid can be the same or different (including, but not limited to, the polypeptide can comprise two or more different types of post-translationally modified unnatural amino acids, or can comprise two identical post-translationally modified unnatural amino acids). For a given polypeptide having more than two post-translationally modified non-natural amino acids, the post-translationally modified non-natural amino acids may be the same, different, or a combination of a plurality of post-translationally modified non-natural amino acids of the same species with at least one different post-translationally modified non-natural amino acid.
A. A method for post-translationally modifying an unnatural amino acid polypeptide: reaction of carbonyl-containing unnatural amino acids with hydroxylamine-containing reagents
The side chains of naturally occurring amino acids lack highly electrophilic sites. Thus, the incorporation of unnatural amino acids with electrophile-containing side chains, including by way of example only, amino acids containing carbonyl or dicarbonyl groups such as ketones or aldehydes, makes it possible to derivatize such side chains site-specifically by nucleophilic attack of the carbonyl or dicarbonyl groups. In the case where the attacking nucleophile is hydroxylamine, oxime-derived proteins are produced. The method for derivatization and/or further modification may be performed with the polypeptide purified prior to or after the derivatization step. In addition, the methods for derivatization and/or further modification can be carried out with synthetic polymers, polysaccharides or polynucleotides purified before or after these modifications. Furthermore, the derivatization step may occur under moderately acidic to slightly basic conditions, including, for example, at a pH between about 2-8, or at a pH between about 4-8.
The method of derivatizing polypeptides based on the reaction of carbonyl-or dicarbonyl-containing polypeptides with hydroxylamine-substituted molecules has significant advantages. First, hydroxylamine undergoes condensation with carbonyl-or dicarbonyl-containing compounds at a pH in the range of 2-8 (and in other embodiments in the pH range of 4-8) to produce oxime adducts. Under these conditions, the side chains of naturally occurring amino acids are non-reactive. Second, this selective chemistry makes it possible to site-specifically derivatize recombinant proteins: the derivatized proteins can now be prepared as defined homologous products. Third, the mild conditions required to effect the reaction of the hydroxylamine described herein with the carbonyl-or dicarbonyl-containing polypeptides described herein will not typically irreversibly disrupt the tertiary structure of the polypeptide (unless, of course, the purpose of the reaction is to disrupt such tertiary structure). Finally, although the hydroxylamine group amino appears to be metabolized by E.coli, condensation of hydroxylamine with carbonyl-or dicarbonyl-containing molecules produces oxime adducts that are stable under biological conditions.
By way of example only, the following unnatural amino acids are of the type of carbonyl-or dicarbonyl-containing amino acids that are reactive with the hydroxylamine-containing reagents described herein that can be used to further modify carbonyl-or dicarbonyl-containing unnatural amino acid polypeptides:
Figure BDA0001546710350001581
wherein:
a is optional and when present is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkyl, substituted lower heterocycloalkyl, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene;
b is optional and when present is a linker selected from the group consisting of: lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O- (alkylene or substituted alkylene) -, -S- (alkylene or substituted alkylene))-、-S(O) k - (wherein k is 1, 2 or 3), -S (O) k (alkylene or substituted alkylene) -, -C (O) -, -NS (O) 2 -、-OS(O) 2 -C (O) - (alkylene or substituted alkylene) -, -C (S) - (alkylene or substituted alkylene) -, -N (R '), -NR ' - (alkylene or substituted alkylene) -, -C (O) N (R '), -CON (R ') - (alkylene or substituted alkylene) -, -CSN (R ') - (alkylene or substituted alkylene) -, -N (R ') CO- (alkylene or substituted alkylene) -, -N (R ') C (O) O-, -S (O) k N(R')-、-N(R')C(O)N(R')-、-N(R')C(S)N(R')-、-N(R')S(O) k N(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 ') -, wherein each R' is independently H, alkyl or substituted alkyl;
j is
Figure BDA0001546710350001591
R is H, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl;
each R "is independently H, alkyl, substituted alkyl, or a protecting group, or when more than one R" group is present, two R "optionally form a heterocycloalkyl;
R 1 h, amino protecting group and resin; and is also provided with
R 2 OH, an ester protecting group and a resin;
R 3 and R is R 4 Each independently is H, halogen, lower alkyl or substituted lower alkyl, or R 3 And R is R 4 Or two R 3 The radicals optionally form cycloalkyl or heterocycloalkyl;
or-a-B-J-R groups together form a bicyclic or tricyclic cycloalkyl or heterocycloalkyl group including at least one carbonyl (including dicarbonyl), protected carbonyl (including protected dicarbonyl), or masked carbonyl (including masked dicarbonyl);
or-J-R groups together form a mono-or bi-cyclic cycloalkyl or heterocycloalkyl group comprising at least one carbonyl group (including dicarbonyl), protected carbonyl group (including protected dicarbonyl), or masked carbonyl group (including masked dicarbonyl).
In certain embodiments, the compounds of formula (I) are reactive with hydroxylamine in aqueous solution under moderately acidic conditions. In certain embodiments, the acidic condition is a pH of 2 to 8.
For the purposes described above, by way of example only, the compounds of formula (I) include compounds having the following structure:
Figure BDA0001546710350001592
wherein:
a is optional and when present is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkyl, substituted lower heterocycloalkyl, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene;
r is H, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl;
R 1 is H, an amino protecting group, a resin, an amino acid, a polypeptide or a polynucleotide;
R 2 Is OH, an ester protecting group, a resin, an amino acid, a polypeptide or a polynucleotide;
X 1 c, S or S (O); and L is a bond, alkylene, substituted alkylene, N (R ') (alkylene), or N (R ') (substituted alkylene), wherein each R ' is independently H, alkyl, or substituted alkyl.
By way of further example only, for the aforementioned purposes, the compounds of formula (I) include compounds having the structure of formula (XXXX):
Figure BDA0001546710350001601
wherein:
a is optional and when present is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkyl, substituted lower heterocycloalkyl, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene;
m is-C (R) 3 )-，
Figure BDA0001546710350001602
Figure BDA0001546710350001603
Wherein (a) indicates bonding to the A group and (b) indicates bonding to the respective carbonyl group, R 3 And R is R 4 Independently selected from H, halogen, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl, or R 3 And R is R 4 Or two R 3 Radicals or two R 4 The radicals optionally form cycloalkyl or heterocycloalkyl;
R is H, halogen, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl;
T 3 is a bond, C (R) (R), O, or S, and R is H, halogen, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;
R 1 is H, an amino protecting group, a resin, an amino acid, a polypeptide or a polynucleotide; and is also provided with
R 2 Is OH, an ester protecting group, a resin, an amino acid, a polypeptide or a polynucleotide.
The type of polypeptide comprising these carbonyl-or dicarbonyl-containing unnatural amino acids is virtually unlimited as long as the carbonyl-or dicarbonyl-containing unnatural amino acid is located on the polypeptide such that the hydroxylamine reagent can react with the carbonyl or dicarbonyl group and does not produce a resulting modified unnatural amino acid that disrupts the tertiary structure of the polypeptide (unless, of course, if such disruption is the purpose of the reaction).
By way of example only, the following hydroxylamine-containing reagents are of the type that are reactive with the carbonyl-or dicarbonyl-containing unnatural amino acids described herein and that can be used to further modify carbonyl-or dicarbonyl-containing unnatural amino acid polypeptides:
Figure BDA0001546710350001611
wherein:
x is each independently H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, alkylalkoxy, substituted alkylalkoxy, polyoxyalkylene, substituted polyoxyalkylene, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkylaryl, substituted alkylaryl, arylalkyl, substituted arylalkyl, - (alkylene, or substituted alkylene) -ON (R') 2 (alkylene or substituted alkylene) -C (O) SR ', - (alkylene or substituted alkylene) -S-S- (aryl or substituted aryl), -C (O) R', -C (O) 2 R 'or-C (O) N (R') 2 Wherein each R "is independently hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, alkylaryl, substituted alkylaryl, arylalkyl, or substituted arylalkyl;
or X are each independently selected from the group consisting of: marking; a dye; a polymer; a water-soluble polymer; derivatives of polyethylene glycol; a photocrosslinking agent; a cytotoxic compound; a drug; affinity labeling; a photoaffinity label; a reactive compound; a resin; a second protein or polypeptide analog; an antibody or antibody fragment; a metal chelator; a cofactor; a fatty acid; a carbohydrate; a polynucleotide; DNA; RNA; an antisense polynucleotide; saccharides, water-soluble dendrimers, cyclodextrins, biomaterials; a nanoparticle; spin labeling; a fluorophore-containing moiety; a radioactive portion; novel functional groups; groups that interact covalently or non-covalently with other molecules; a light-shielding portion; an actinic radiation excitable moiety; a ligand; a photoisomerizable moiety; biotin; biotin analogues; a moiety incorporating a heavy atom; a chemically cleavable group; a photocleavable group; an extended side chain; a carbon-bonded sugar; a redox active agent; an aminothiopropionic acid; a toxic moiety; isotopically labeled moieties; a biophysical probe; phosphorescent groups; a chemiluminescent group; an electron dense group; a magnetic group; an insertion group; a chromophore; an energy transfer agent; a bioactive agent; a detectable label; a small molecule; inhibitory ribonucleic acid, radionucleotide; a neutron capture agent; derivatives of biotin; a quantum dot; nano transitorin; a radiotransferrin; abzymes, activating complex activators, viruses, adjuvants, aglycones, allergens, angiostatin, anti-hormones, antioxidants, aptamers, guide RNAs, saponins, shuttle vectors, macromolecules, pseudoepitopes, receptors, reverse micelles, and any combination thereof;
Each L is independently selected from the group consisting of: alkylene, substituted alkylene, alkenylene, substituted alkenylene, -O- (alkylene or substituted alkylene) -, -S- (alkylene or substituted alkylene) -, -S (O) k - (wherein k is 1, 2 or 3), -S (O) k (alkylene or substituted alkylene) -, -C (O) - (alkylene or substituted alkylene) -, -C (S) - (alkylene or substituted alkylene) -, -N (R ') -, NR ' - (alkylene or substituted alkylene) -, -C (O) N (R ') -, CON (R ') - (alkylene or substituted alkylene) -, a- (alkylene or substituted alkylene) NR ' C (O) O- (alkylene or substituted alkylene) -, -O-CON (R ') - (alkylene or substituted alkylene) -, -CSN (R ') - (alkylene or substituted alkylene) -, -N (R ') CO- (alkylene or substituted alkylene) -, -N (R ') C (O) O- (alkylene or substituted alkylene) -, -S (O) k N (R ') -, -N (R') C (O) N (R ') -, -N (R') C (O) N (R ') - (alkylene or substituted alkylene) -, -N (R') C (S) N (R '), -N (R') S (O) k N(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 1 Is optional and when present is-C (R') p -NR' -C (O) O- (alkylene or substituted alkylene) -, wherein p is 0, 1 or 2; each R' is independently H, alkyl or substituted alkyl;
W is-N (R) 8 ) 2 Wherein R is 8 Each independently is H or an amino protecting group; and n is 1 to 3;
with the proviso that L-L 1 W together provide at least one hydroxylamine group capable of reacting with a carbonyl group (including dicarbonyl groups) on a non-natural amino acid or "modified or unmodified" non-natural amino acid polypeptide.
In certain embodiments of the compounds of formula (XIX), X is a polymer comprising alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, alkylalkoxy, substituted alkylalkoxy, polyoxyalkylene, substituted polyoxyalkylene, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkylaryl, substituted alkylaryl, arylalkyl, or substituted arylalkyl. In certain embodiments of the compounds of formula (XIX), X is a polymer comprising a polyoxyalkylene or substituted polyoxyalkylene. In certain embodiments of the compounds of formula (XIX), X is a compound comprising- [ (alkylene or substituted alkylene) -O- (hydrogen, alkyl or substituted alkyl)] x (wherein x is 20 to 10,000). In certain embodiments of the compounds of formula (XIX), X is m-PEG having a molecular weight in the range of 2KDa to 40 KDa. In certain embodiments of the compounds of formula (XIX), X is a bioactive agent selected from the group consisting of peptides, proteins, enzymes, antibodies, drugs, dyes, lipids, nucleosides, oligonucleotides, cells, viruses, liposomes, microparticles, and micelles. In certain embodiments of the compounds of formula (XIX), X is a drug selected from the group consisting of antibiotics, fungicides, antiviral agents, anti-inflammatory agents, antitumor agents, cardiovascular agents, anxiolytic agents, hormones, growth factors, and steroid agents. In certain embodiments of the compounds of formula (XIX), X is an enzyme selected from the group consisting of horseradish peroxidase, alkaline phosphatase, beta-galactosidase, and glucose oxidase. In the compounds of formula (XIX) In certain embodiments, X is a detectable label selected from the group consisting of a fluorescent moiety, a phosphorescent moiety, a chemiluminescent moiety, a chelating moiety, an electron dense moiety, a magnetic moiety, an intercalating moiety, a radioactive moiety, a chromophore moiety, and an energy transfer moiety. In certain embodiments of the compounds of formula (XIX), L is selected from the group consisting of-N (R ') CO- (alkylene or substituted alkylene) -, -CON (R ') - (alkylene or substituted alkylene) -, -N (R ') C (O) N (R ') - (alkylene or substituted alkylene) -, -O-CON (R ') - (alkylene or substituted alkylene) -, -O- (alkylene or substituted alkylene) -, -C (O) N (R ') -, and-N (R ') C (O) O- (alkylene or substituted alkylene) -.
In certain embodiments of the compounds of formula (XIX), are compounds having the structure of formula (XX):
Figure BDA0001546710350001631
in certain embodiments of the compounds of formula (XX), are compounds selected from the group consisting of:
Figure BDA0001546710350001632
wherein in other embodiments, these m-PEG or PEG groups have a molecular weight in the range of 5kDa to 30 kDa.
In certain embodiments of compounds of formula (XIX), are compounds having the structure of formula (XXI):
Figure BDA0001546710350001641
in certain embodiments of compounds of formula (XXI), are compounds selected from the group consisting of:
Figure BDA0001546710350001642
In certain embodiments of compounds of formula (XIX), are compounds having the structure of formula (XXII):
Figure BDA0001546710350001643
in certain embodiments of compounds of formula (XXII), L is- (alkylene or substituted alkylene) -N (R') C (O) O- (alkylene or substituted alkylene) -. In certain embodiments of compounds of formula (XXII), are compounds having the structure of formula (XXIII):
Figure BDA0001546710350001644
wherein in other embodiments of the compound of formula (XXII), these m-PEG groups have a molecular weight in the range of 5kDa to 30 kDa.
In certain embodiments of compounds of formula (XIX), are compounds having the structure of formula (XXIV):
Figure BDA0001546710350001651
in certain embodiments of compounds of formula (XXIV), L is- (alkylene or substituted alkylene) -N (R ') C (O) O- (alkylene or substituted alkylene) -or-N (R') C (O) O- (alkylene or substituted alkylene) -. In certain embodiments of compounds of formula (XXIV), are compounds having the structure of formula (XXV):
Figure BDA0001546710350001652
wherein in other embodiments of the compound of formula (XXIV), these m-PEG groups have a molecular weight in the range of 5kDa to 30 kDa.
In certain embodiments of compounds of formula (XIX), are compounds having the structure of formula (XXVI):
Figure BDA0001546710350001653
wherein R is 10 Each independently is H or an amino protecting group.
In certain embodiments of the compounds of formula (XXVI), the polyoxyalkylene is PEG. In other embodiments of the compound of formula (XXVI), the PEG group has a molecular weight in the range of 5kDa to 30 kDa. In another embodiment of the compounds of formula (XXVI) are compounds corresponding to the following formula:
Figure BDA0001546710350001661
three illustrative examples of methods for coupling hydroxylamine to carbonyl-containing unnatural amino acids on polypeptides are presented in FIG. 7. In these illustrative embodiments, the hydroxylamine-derived reagent is added to a buffer solution (pH 2-8) of the carbonyl-containing unnatural amino acid polypeptide. The reaction is carried out at ambient temperature for a number of hours up to days. To accelerate the bonding, additives such as those presented in fig. 8 are added; these compounds are also referred to herein as accelerators. In certain embodiments, the promoter or additive is capable of base catalysis. The resulting oxime-containing unnatural amino acid polypeptide is purified by HPLC, FPLC or size exclusion chromatography.
In one embodiment, the plurality of linker chemistries can react site-specifically with a carbonyl-or dicarbonyl-substituted unnatural amino acid polypeptide. In one embodiment, the linker methods described herein utilize a linker (mono-, di-, or multi-functional) containing a hydroxylamine functional group on at least one linker terminus. Condensation of the hydroxylamine-derived linker with the ketone-substituted protein produces a stable oxime bond. Bifunctional and/or polyfunctional linkers (e.g., hydroxylamine with one or more other bonding chemistries) allow site-specific attachment of different molecules (e.g., other proteins, polymers, or small molecules) to the unnatural amino acid polypeptide, while monofunctional linkers (substituted with hydroxylamine at all termini) promote site-specific dimerization or oligomerization of the unnatural amino acid polypeptide. By combining this linker strategy with the in vivo translation techniques described herein, it is possible to specify the three-dimensional structure of chemically refined proteins.
In certain embodiments are methods for derivatizing a polypeptide comprising an amino acid of formula I-XVIII, XXX-XXXIV (A and B) or XXXX-XXXXIII, which comprises any sub-or specific compound within the scope of formula I-XVIII, XXX-XXXIV (A and B) or XXXXXX-XXXXIII, wherein the method comprises contacting a polypeptide comprising at least one amino acid of formula I-XVIII, XXX-XXXIV (A and B) or XXXXXXXXIII with a reagent of formula (XIX). In certain embodiments, the polypeptide is purified before or after contact with the agent of formula (XIX). In other embodiments are resulting derivatized polypeptides comprising at least one oxime-containing amino acid corresponding to formula (XI), and in other embodiments, these derivatized polypeptides are stable in aqueous solution for at least 1 month under moderately acidic conditions. In other embodiments, these derivative polypeptides are stable under moderately acidic conditions for at least 2 weeks. In other embodiments, these derivative polypeptides are stable under moderately acidic conditions for at least 5 days. In other embodiments, the conditions are pH 2 to 8. In certain embodiments, the tertiary structure of the derivatized polypeptide is maintained. In other embodiments, these derivatizations of polypeptides further comprise ligating the derivatized polypeptide to another polypeptide. In other embodiments, these derivatized polypeptides are homologous to a therapeutic protein selected from the group consisting of: alpha-1 antitrypsin, angiostatin, anti-hemolytic factor, antibody, apolipoprotein, apoprotein, atrial natriuretic factor, atrial natriuretic polypeptide, atrial peptide, C-X-C chemokine, T39765, NAP-2, ENA-78, gro-a, gro-b, gro-C, IP-10, GCP-2, NAP-4, SDF-1, PF4, MIG, calcitonin, C-kit ligand, cytokine, CC chemokine, monocyte chemokine-1, monocyte chemokine-2, monocyte chemokine-3, monocyte inflammatory protein-1 alpha, monocyte inflammatory protein-iβ, RANTES, 1309, R83915, R91733, HCC1, T58847, D31065, T64262, CD40 ligand, C-kit ligand, collagen, and pharmaceutical compositions containing the same Colony Stimulating Factor (CSF), complement factor 5a, complement inhibitor, complement receptor 1, cytokine, epithelial neutrophil activating peptide-78, MIP-16, MCP-1, epidermal Growth Factor (EGF), epithelial neutrophil activating peptide, erythropoietin (EPO), epidermal exfoliative toxin, factor IX, factor VII, factor VIII, factor X, fibroblast Growth Factor (FGF), fibrinogen, fibronectin, tetrahelical bundle protein, G-CSF, glp-1, GM-CSF, glucocerebrosidase, gonadotropin, growth factor receptor, grf, hedgehog protein, hemoglobin, hepatocyte growth factor (hGF), hirudin, human growth hormone (hGH), human serum albumin, ICAM-1, ICAM-1 receptor, LFA-1 receptor, insulin-like growth factor (IGF), IGF-I, IGF-II, interferon (IFN), IFN-alpha, IFN-beta, IFN-gamma, interleukin (IL), IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, keratinocyte Growth Factor (KGF), lactoferrin, leukemia inhibitory factor, luciferase, neurogenin, neutrophil Inhibitory Factor (NIF), oncostatin M, osteogenic protein, oncogene products, paracitin, parathyroid hormone, PD-ECSF, PDGF, peptide hormones, pleiotrophin, protein A, protein G, pth, pyrogenic exotoxin A, neutropy the therapeutic agents include, but are not limited to, heat exotoxin B, heat exotoxin C, pyy, relaxin, renin, SCF, small biosynthetic proteins, soluble complement receptor I, soluble I-CAM 1, soluble interleukin receptor, soluble TNF receptor, somatostatin, growth hormone, streptokinase, superantigen, staphylococcal enterotoxin, SEA, SEB, SEC1, SEC2, SEC3, SED, SEE, steroid hormone receptor, superoxide dismutase, toxic shock syndrome toxin, thymosin alpha 1, tissue type plasminogen activator, tumor Growth Factor (TGF), tumor necrosis factor alpha, tumor necrosis factor beta, tumor Necrosis Factor Receptor (TNFR), VLA-4 protein, VCAM-1 protein, vascular Endothelial Growth Factor (VEGF), urokinase, mos, ras, raf, met, p, tat, fos, myc, jun, myb, rel, estrogen receptor, progesterone receptor, testosterone receptor, aldosterone receptor, LDL receptor, and corticosterone.
In certain embodiments is a method of producing a polypeptide dimer, wherein the method comprises:
(i) Derivatizing a first polypeptide comprising an amino acid of formula (I) with a reagent of formula (XXVI), and (ii) contacting the resulting derivatized protein of step (I) with a second protein comprising an amino acid of formula (I), thereby forming a dimer comprising the first polypeptide and the second polypeptide. In other embodiments are methods of producing a polypeptide dimer, wherein the first polypeptide and the second polypeptide comprise amino acids corresponding to formula (II). In certain embodiments, the polypeptide is purified prior to or after contact with an agent of formula (XXVI). In other embodiments, the resulting derivative protein of step (i) comprises at least one oxime-containing amino acid corresponding to formula (XXVIII):
Figure BDA0001546710350001681
B. a method for post-translationally modifying an unnatural amino acid polypeptide: reacting an oxime-containing unnatural amino acid with a carbonyl-containing reagent
Protein derivatization methods based on the exchange reaction of oxime-containing proteins with carbonyl-or dicarbonyl-substituted molecules have significant advantages. First, studies indicate that amino acid-based oxime adducts undergo oxime exchange by equilibrium with carbonyl-or dicarbonyl-containing compounds that are more reactive than the compounds used to produce the original oxime. This exchange reaction takes place in a pH range of 4-8: under these conditions, the side chains of naturally occurring amino acids are not reactive. Thus, the general methods for preparing carbonyl-or dicarbonyl-substituted molecules suitable for reaction with oxime-containing proteins can provide a way to obtain a variety of site-specifically derivatized proteins. In the context of this in vivo translation technique, the general approach for preparing carbonyl-or dicarbonyl-substituted versions of those molecules typically used to derivatize proteins, including by way of example only hydrophilic polymers such as polyethylene glycol, is valuable and would provide a route to a variety of site-specifically derivatized unnatural amino acid polypeptides. Second, this selective chemistry enables site-specific derivatization of recombinant proteins: the derived proteins can now be identified as homologous products. Third, the mild conditions required to effect the exchange reactions described herein do not typically irreversibly disrupt the tertiary structure of the polypeptide (unless, of course, the purpose of the reaction is to disrupt this tertiary structure). Finally, the exchange reaction produces a new oxime adduct that is stable under biological conditions.
By way of example only, the following unnatural amino acids are of the type of oxime-containing amino acids that are reactive with the carbonyl-or dicarbonyl-containing reagents described herein that can be used to produce the novel oxime-containing unnatural amino acid polypeptides:
Figure BDA0001546710350001691
wherein:
a is optional and when present is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkyl, substituted lower heterocycloalkyl, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene;
b is optional and when present is a linker selected from the group consisting of: lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O- (alkylene or substituted alkylene) -, -S- (alkylene or substituted alkylene) -, -S (O) k - (wherein k is 1, 2 or 3), -S (O) k (alkylene or substituted alkylene) -, -C (O) -, -NS (O) 2 -、-OS(O) 2 -C (O) - (alkylene or substituted alkylene) -, -C (S) - (alkylene or substituted alkylene) -, -N (R '), -NR ' - (alkylene or substituted alkylene) -, -C (O) N (R '), -CON (R ') - (alkylene or substituted alkylene) -, -CSN (R ') - (alkylene or substituted alkylene) -, -N (R ') CO- (alkylene or substituted alkylene) -, -N (R ') C (O) O-, -S (O) k N(R')-、-N(R')C(O)N(R')-、-N(R')C(S)N(R')-、-N(R')S(O) k N(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 ') -, wherein each R' is independently H, alkyl or substituted alkyl;
r is H, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl;
R 1 is H, an amino protecting group, a resin, an amino acid, a polypeptide or a polynucleotide; and is also provided with
R 2 Is OH, an ester protecting group, a resin, an amino acid, a polypeptide or a polynucleotide;
R 3 and R is R 4 Each independently is H, halogen, lower alkyl or substituted lower alkyl, or R 3 And R is R 4 Or two R 3 The radicals optionally form cycloalkyl or heterocycloalkyl;
R 5 is H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, alkylalkoxy, substituted alkylalkoxy, polyoxyalkylene, substituted polyoxyalkylene, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkylaryl, substituted alkylaryl, arylalkyl, substituted arylalkyl, - (alkylene or substituted alkylene) -ON (R') 2 (alkylene or substituted alkylene) -C (O) SR ', - (alkylene or substituted alkylene) -S-S- (aryl or substituted aryl), -C (O) R', -C (O) 2 R 'or-C (O) N (R') 2 Wherein each R "is independently hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, alkylaryl, substituted alkylaryl, arylalkyl, or substituted arylalkyl;
or R is 5 Is L-X, wherein X is selected from the group consisting of: marking; a dye; a polymer; a water-soluble polymer; derivatives of polyethylene glycol; a photocrosslinking agent; a cytotoxic compound; a drug; affinity labeling; a photoaffinity label; a reactive compound; a resin; a second protein or polypeptide analog; an antibody or antibody fragment; a metal chelator; a cofactor;a fatty acid; a carbohydrate; a polynucleotide; DNA; RNA; an antisense polynucleotide; saccharides, water-soluble dendrimers, cyclodextrins, biomaterials; a nanoparticle; spin labeling; a fluorophore-containing moiety; a radioactive portion; novel functional groups; groups that interact covalently or non-covalently with other molecules; a light-shielding portion; an actinic radiation excitable moiety; a ligand; a photoisomerizable moiety; biotin; biotin analogues; a moiety incorporating a heavy atom; a chemically cleavable group; a photocleavable group; an extended side chain; a carbon-bonded sugar; a redox active agent; an aminothiopropionic acid; a toxic moiety; isotopically labeled moieties; a biophysical probe; phosphorescent groups; a chemiluminescent group; an electron dense group; a magnetic group; an insertion group; a chromophore; an energy transfer agent; a bioactive agent; a detectable label; a small molecule; inhibitory ribonucleic acid, radionucleotide; a neutron capture agent; derivatives of biotin; a quantum dot; nano transitorin; a radiotransferrin; abzymes, activating complex activators, viruses, adjuvants, aglycones, allergens, angiostatin, anti-hormones, antioxidants, aptamers, guide RNAs, saponins, shuttle vectors, macromolecules, pseudoepitopes, receptors, reverse micelles, and any combination thereof; and L is optional and when present is a linker selected from the group consisting of: alkylene, substituted alkylene, alkenylene, substituted alkenylene, -O- (alkylene or substituted alkylene) -, -S- (alkylene or substituted alkylene) -, -S (O) k - (wherein k is 1, 2 or 3), -S (O) k (alkylene or substituted alkylene) -, -C (O) - (alkylene or substituted alkylene) -, -C (S) - (alkylene or substituted alkylene) -, -N (R ') -, NR' - (alkylene or substituted alkylene) -, -C (O) N (R ') -, CON (R') - (alkylene or substituted alkylene) -, -CSN (R ') -, CSN (R') - (alkylene or substituted alkylene) -, -N (R ') CO- (alkylene or substituted alkylene) -, -N (R') C (O) O-, alkylene or substituted alkylene) -O-n=cr '-, - (alkylene or substituted alkylene) -C (O) NR' - (alkylene or substituted alkylene) -, - (alkylene or substituted alkylene) -S (O) k - (alkylene or substituted alkylene) -S-, - (alkylene or substituted alkylene) -S-S-, -S (O) k N(R')-、-N(R')C(O)N(R')-、-N(R')C(S)N(R')-、-N(R')S(O) k N(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 ') -, wherein each R' is independently H, alkyl or substituted alkyl.
By way of further example only, the following unnatural amino acids are also of the type of oxime-containing amino acids that are reactive with the carbonyl-or dicarbonyl-containing reagents described herein that can be used to produce the novel oxime-containing unnatural amino acid polypeptides:
Figure BDA0001546710350001711
wherein:
a is optional and when present is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkyl, substituted lower heterocycloalkyl, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene;
B is optional and when present is a linker selected from the group consisting of: lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O- (alkylene or substituted alkylene) -, -S- (alkylene or substituted alkylene) -, -S (O) k - (wherein k is 1, 2 or 3), -S (O) k (alkylene or substituted alkylene) -, -C (O) -, -NS (O) 2 -、-OS(O) 2 -C (O) - (alkylene or substituted alkylene) -, -C (S) - (alkylene or substituted alkylene) -, -N (R '), -NR ' - (alkylene or substituted alkylene) -, -C (O) N (R '), -CON (R ') - (alkylene or substituted alkylene) -, -CSN (R ') - (alkylene)Radical or substituted alkylene) -, -N (R ') CO- (alkylene or substituted alkylene) -, -N (R') C (O) O-, -S (O) k N(R')-、-N(R')C(O)N(R')-、-N(R')C(S)N(R')-、-N(R')S(O) k N(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 ') -, wherein each R' is independently H, alkyl or substituted alkyl;
R 1 is H, an amino protecting group, a resin, an amino acid, a polypeptide or a polynucleotide; and is also provided with
R 2 Is OH, an ester protecting group, a resin, an amino acid, a polypeptide or a polynucleotide;
R 3 and R is R 4 Each independently is H, halogen, lower alkyl or substituted lower alkyl, or R 3 And R is R 4 Or two R 3 The radicals optionally form cycloalkyl or heterocycloalkyl;
R 6 and R is R 7 Each independently selected from the group consisting of: H. alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, polyoxyalkylene, substituted polyoxyalkylene, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkylaryl, arylalkyl, and substituted arylalkyl, -C (O) R', -C (O) 2 R"、-C(O)N(R") 2 Wherein each R "is independently hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, alkylaryl, substituted alkylaryl, arylalkyl, or substituted arylalkyl; or R is 6 Or R is 7 Is L-X, wherein
X is selected from the group consisting of: marking; a dye; a polymer; a water-soluble polymer; derivatives of polyethylene glycol; a photocrosslinking agent; a cytotoxic compound; a drug; affinity labeling; a photoaffinity label; a reactive compound; a resin; a second protein or polypeptide analog; an antibody or antibody fragment; a metal chelator; a cofactor; a fatty acid; a carbohydrate; a polynucleotide; DNA; RNA; an antisense polynucleotide; saccharide, water-soluble dendrimer Polymers, cyclodextrins, biological materials; a nanoparticle; spin labeling; a fluorophore-containing moiety; a radioactive portion; novel functional groups; groups that interact covalently or non-covalently with other molecules; a light-shielding portion; an actinic radiation excitable moiety; a ligand; a photoisomerizable moiety; biotin; biotin analogues; a moiety incorporating a heavy atom; a chemically cleavable group; a photocleavable group; an extended side chain; a carbon-bonded sugar; a redox active agent; an aminothiopropionic acid; a toxic moiety; isotopically labeled moieties; a biophysical probe; phosphorescent groups; a chemiluminescent group; an electron dense group; a magnetic group; an insertion group; a chromophore; an energy transfer agent; a bioactive agent; a detectable label; a small molecule; inhibitory ribonucleic acid, radionucleotide; a neutron capture agent; derivatives of biotin; a quantum dot; nano transitorin; a radiotransferrin; abzymes, activating complex activators, viruses, adjuvants, aglycones, allergens, angiostatin, anti-hormones, antioxidants, aptamers, guide RNAs, saponins, shuttle vectors, macromolecules, pseudoepitopes, receptors, reverse micelles, and any combination thereof; and L is optional and when present is a linker selected from the group consisting of: alkylene, substituted alkylene, alkenylene, substituted alkenylene, -O- (alkylene or substituted alkylene) -, -S- (alkylene or substituted alkylene) -, -S (O) k - (wherein k is 1, 2 or 3), -S (O) k (alkylene or substituted alkylene) -, -C (O) - (alkylene or substituted alkylene) -, -C (S) - (alkylene or substituted alkylene) -, -N (R ') -, -NR ' - (alkylene or substituted alkylene) -, -C (O) N (R ') -, CON (R ') - (alkylene or substituted alkylene) -, -CSN (R ') - (alkylene or substituted alkylene) -, -N (R ') CO- (alkylene or substituted alkylene) -, -N (R ') C (O) O-, -S (O) k N(R')-、-N(R')C(O)N(R')-、-N(R')C(S)N(R')-、-N(R')S(O) k N(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 ') -, wherein each R' is independently H, alkyl or substituted alkyl。
By way of example only, the following carbonyl-or dicarbonyl-containing reagents are of the type that are reactive with the oxime-containing unnatural amino acids described herein and that can be used to effect an exchange reaction to form new oxime linkages and thus modify the oxime-containing unnatural amino acid polypeptide:
Figure BDA0001546710350001721
wherein:
x is each independently H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, alkylalkoxy, substituted alkylalkoxy, polyoxyalkylene, substituted polyoxyalkylene, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkylaryl, substituted alkylaryl, arylalkyl, substituted arylalkyl, - (alkylene, or substituted alkylene) -ON (R') 2 (alkylene or substituted alkylene) -C (O) SR ', - (alkylene or substituted alkylene) -S-S- (aryl or substituted aryl), -C (O) R', -C (O) 2 R 'or-C (O) N (R') 2 Wherein each R "is independently hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, alkylaryl, substituted alkylaryl, arylalkyl, or substituted arylalkyl;
or X are each independently selected from the group consisting of: marking; a dye; a polymer; a water-soluble polymer; derivatives of polyethylene glycol; a photocrosslinking agent; a cytotoxic compound; a drug; affinity labeling; a photoaffinity label; a reactive compound; a resin; a second protein or polypeptide analog; an antibody or antibody fragment; a metal chelator; a cofactor; a fatty acid; a carbohydrate; a polynucleotide; DNA; RNA; an antisense polynucleotide; saccharides, water-soluble dendrimers, cyclodextrins, biomaterials; a nanoparticle; spin labeling; a fluorophore-containing moiety; a radioactive portion; novel functional groups; groups that interact covalently or non-covalently with other molecules; a light-shielding portion; an actinic radiation excitable moiety; a ligand; a photoisomerizable moiety; biotin; biotin analogues; a moiety incorporating a heavy atom; a chemically cleavable group; a photocleavable group; an extended side chain; a carbon-bonded sugar; a redox active agent; an aminothiopropionic acid; a toxic moiety; isotopically labeled moieties; a biophysical probe; phosphorescent groups; a chemiluminescent group; an electron dense group; a magnetic group; an insertion group; a chromophore; an energy transfer agent; a bioactive agent; a detectable label; a small molecule; inhibitory ribonucleic acid, radionucleotide; a neutron capture agent; derivatives of biotin; a quantum dot; nano transitorin; a radiotransferrin; abzymes, activating complex activators, viruses, adjuvants, aglycones, allergens, angiostatin, anti-hormones, antioxidants, aptamers, guide RNAs, saponins, shuttle vectors, macromolecules, pseudoepitopes, receptors, reverse micelles, and any combination thereof;
Each L is independently selected from the group consisting of: alkylene, substituted alkylene, alkenylene, substituted alkenylene, -O- (alkylene or substituted alkylene) -, -S- (alkylene or substituted alkylene) -, -S (O) k - (wherein k is 1, 2 or 3), -S (O) k (alkylene or substituted alkylene) -, -C (O) - (alkylene or substituted alkylene) -, -C (S) - (alkylene or substituted alkylene) -, -N (R ') -, NR ' - (alkylene or substituted alkylene) -, -C (O) N (R ') -, CON (R ') - (alkylene or substituted alkylene) -, a- (alkylene or substituted alkylene) NR ' C (O) O- (alkylene or substituted alkylene) -, -O-CON (R ') - (alkylene or substituted alkylene) -, -CSN (R ') - (alkylene or substituted alkylene) -, -N (R ') CO- (alkylene or substituted alkylene) -, -N (R ') C (O) O- (alkylene or substituted alkylene) -, -S (O) k N (R ') -, -N (R') C (O) N (R ') -, -N (R') C (O) N (R ') - (alkylene or substituted alkylene) -, -N (R') C (S) N (R '), -N (R') S (O) k N(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 1 Is optional and when present is-C (R') p -NR' -C (O) O- (alkylene or substituted alkylene) -, wherein p is 0, 1 or 2; each R' is independently H, alkyl or substituted alkyl;
W is-C (=O) R 9 Wherein R is 9 Is H OR OR'; and n is 1 to 3;
or wherein L-L 1 W together form a mono-or bi-cyclic cycloalkyl or heterocycloalkyl group comprising at least one carbonyl group (including dicarbonyl), protected carbonyl group (including protected dicarbonyl) or masked carbonyl group (including masked dicarbonyl);
with the proviso that L-L 1 W together provide at least one carbonyl group (including dicarbonyl groups) capable of undergoing an oxime exchange reaction with an oxime group on a non-natural amino acid or a "modified or unmodified" non-natural amino acid polypeptide.
Two illustrative examples of methods for effecting an oxime exchange reaction between an oxime-containing amino acid on a polypeptide and a carbonyl-containing reagent are presented in FIG. 9. In these illustrative embodiments, the carbonyl-containing reagent is added to a buffered solution (pH 2-8) of the oxime-containing unnatural amino acid polypeptide. The reaction is carried out at ambient temperature for a number of hours up to days. The modified oxime-containing unnatural amino acid polypeptide is purified by HPLC, FPLC, or size exclusion chromatography.
In one embodiment, the plurality of linker chemistries can site-specifically react with the oxime-substituted unnatural amino acid polypeptide. In one embodiment, the linker methods described herein utilize a linker (mono-, di-, or multifunctional) containing a carbonyl or dicarbonyl functional group on at least one linker terminus. Condensation of a carbonyl or dicarbonyl-derived linker with an oxime-substituted unnatural amino acid polypeptide produces a new stable oxime bond. Bifunctional and/or polyfunctional linkers (e.g., carbonyl or dicarbonyl groups with one or more other bonding chemistries) allow site-specific attachment of different molecules (e.g., other proteins, polymers, or small molecules) to the unnatural amino acid polypeptide, while monofunctional linkers (substituted with carbonyl or dicarbonyl groups on all termini) promote site-specific dimerization or oligomerization of the unnatural amino acid polypeptide. By combining this linker strategy with the in vivo translation techniques described herein, it is possible to specify the three-dimensional structure of chemically refined proteins.
C. A method for post-translationally modifying an unnatural amino acid polypeptide: reacting a hydroxylamine-containing unnatural amino acid with a carbonyl-containing reagent
The post-translational modification techniques and compositions described above can also be used to react with carbonyl-or dicarbonyl-containing reagents to produce hydroxylamine-containing unnatural amino acids of modified oxime-containing unnatural amino acid polypeptides.
Protein derivatization methods based on the reaction of hydroxylamine-containing proteins with carbonyl-or dicarbonyl-substituted molecules have significant advantages. First, hydroxylamine undergoes condensation with carbonyl or dicarbonyl containing compounds at a pH in the range of 4 to 8 to produce oxime adducts. Under these conditions, the side chains of naturally occurring amino acids are not reactive. Second, this selective chemistry enables site-specific derivatization of recombinant proteins: the derived proteins can now be prepared as defined homologous products. Third, the mild conditions required to effect the reaction of the carbonyl-containing or dicarbonyl-containing reagents described herein with the hydroxylamine-containing polypeptides described herein will not typically irreversibly disrupt the tertiary structure of the polypeptide (unless, of course, the purpose of the reaction is to disrupt such tertiary structure). Finally, although hydroxylamine amino acids appear to be metabolized by E.coli, condensation of carbonyl-or dicarbonyl-containing reagents with hydroxylamine-containing amino acids produces oxime adducts that are stable under biological conditions.
By way of example only, the following unnatural amino acids are of the type of hydroxylamine-containing amino acids that are reactive with the carbonyl-or dicarbonyl-containing reagents described herein that can be used to further modify the hydroxylamine-containing unnatural amino acid polypeptide:
Figure BDA0001546710350001751
wherein:
a is optional and when present is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkyl, substituted lower heterocycloalkyl, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene;
b is optional and when present is a linker selected from the group consisting of: lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O- (alkylene or substituted alkylene) -, -S- (alkylene or substituted alkylene) -, -S (O) k - (wherein k is 1, 2 or 3), -S (O) k (alkylene or substituted alkylene) -, -C (O) -, -NS (O) 2 -、-OS(O) 2 -C (O) - (alkylene or substituted alkylene) -, -C (S) - (alkylene or substituted alkylene) -, -N (R '), -NR ' - (alkylene or substituted alkylene) -, -C (O) N (R '), -CON (R ') - (alkylene or substituted alkylene) -, -CSN (R ') - (alkylene or substituted alkylene) -, -N (R ') CO- (alkylene or substituted alkylene) -, -N (R ') C (O) O-, -S (O) k N(R')-、-N(R')C(O)N(R')-、-N(R')C(S)N(R')-、-N(R')S(O) k N(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 ') -, wherein each R' is independently H, alkyl or substituted alkyl;
k is-NR 6 R 7 Or-n=cr 6 R 7 ；
R 1 Is H, an amino protecting group, a resin, an amino acid, a polypeptide or a polynucleotide; and is also provided with
R 2 Is OH, an ester protecting group, a resin, an amino acid, a polypeptide or a polynucleotide;
R 3 and R is R 4 Each independently is H, halogen, lower alkyl or substituted lower alkyl, or R 3 And R is R 4 Or two R 3 The radicals optionally forming cycloalkyl radicalsOr heterocycloalkyl.
In certain embodiments of the compounds of formula (XIV), K is NH 2 。
The type of polypeptide comprising these hydroxylamine-containing unnatural amino acids is virtually unlimited as long as the hydroxylamine-containing unnatural amino acid is located on the polypeptide such that the carbonyl-or dicarbonyl-containing reagent can react with the hydroxylamine group and does not produce a resulting modified unnatural amino acid that disrupts the tertiary structure of the polypeptide (unless, of course, if such disruption is the purpose of the reaction).
By way of example only, the following carbonyl-or dicarbonyl-containing reagents are of the type that are reactive with the hydroxylamine-containing unnatural amino acids described herein and that can be used to further modify the hydroxylamine-containing unnatural amino acid polypeptides:
Figure BDA0001546710350001761
wherein:
x is each independently H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, alkylalkoxy, substituted alkylalkoxy, polyoxyalkylene, substituted polyoxyalkylene, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkylaryl, substituted alkylaryl, arylalkyl, substituted arylalkyl, - (alkylene, or substituted alkylene) -ON (R') 2 (alkylene or substituted alkylene) -C (O) SR ', - (alkylene or substituted alkylene) -S-S- (aryl or substituted aryl), -C (O) R', -C (O) 2 R 'or-C (O) N (R') 2 Wherein each R "is independently hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, alkylaryl, substituted alkylaryl, arylalkyl, or substituted arylalkyl;
or X are each independently selected from the group consisting of: marking; a dye; a polymer; a water-soluble polymer; derivatives of polyethylene glycol; a photocrosslinking agent; a cytotoxic compound; a drug; affinity labeling; a photoaffinity label; a reactive compound; a resin; a second protein or polypeptide analog; an antibody or antibody fragment; a metal chelator; a cofactor; a fatty acid; a carbohydrate; a polynucleotide; DNA; RNA; an antisense polynucleotide; saccharides, water-soluble dendrimers, cyclodextrins, biomaterials; a nanoparticle; spin labeling; a fluorophore-containing moiety; a radioactive portion; novel functional groups; groups that interact covalently or non-covalently with other molecules; a light-shielding portion; an actinic radiation excitable moiety; a ligand; a photoisomerizable moiety; biotin; biotin analogues; a moiety incorporating a heavy atom; a chemically cleavable group; a photocleavable group; an extended side chain; a carbon-bonded sugar; a redox active agent; an aminothiopropionic acid; a toxic moiety; isotopically labeled moieties; a biophysical probe; phosphorescent groups; a chemiluminescent group; an electron dense group; a magnetic group; an insertion group; a chromophore; an energy transfer agent; a bioactive agent; a detectable label; a small molecule; inhibitory ribonucleic acid, radionucleotide; a neutron capture agent; derivatives of biotin; a quantum dot; nano transitorin; a radiotransferrin; abzymes, activating complex activators, viruses, adjuvants, aglycones, allergens, angiostatin, anti-hormones, antioxidants, aptamers, guide RNAs, saponins, shuttle vectors, macromolecules, pseudoepitopes, receptors, reverse micelles, and any combination thereof;
Each L is independently selected from the group consisting of: alkylene, substituted alkylene, alkenylene, substituted alkenylene, -O- (alkylene or substituted alkylene) -, -S- (alkylene or substituted alkylene) -, -S (O) k - (wherein k is 1, 2 or 3), -S (O) k (alkylene or substituted alkylene) -, -C (O) - (alkylene or substituted alkylene) -, -C (S) - (alkylene or substituted alkylene) -, -N (R ') -, -NR' - (alkylene or substituted alkylene) -, -C (O) N (R '), -CON (R') - (alkylene or substituted alkylene) -, - (alkylene or substituted alkylene) NR 'C (O) O- (alkylene or substituted alkylene) -, -O-CON (R') - (alkylene or substituted alkylene) -,-CSN (R '), -CSN (R') - (alkylene or substituted alkylene) -, -N (R ') CO- (alkylene or substituted alkylene) -, -N (R') C (O) O- (alkylene or substituted alkylene) -, -S (O) k N (R ') -, -N (R') C (O) N (R ') -, -N (R') C (O) N (R ') - (alkylene or substituted alkylene) -, -N (R') C (S) N (R '), -N (R') S (O) k N(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 1 Is optional and when present is-C (R') p -NR' -C (O) O- (alkylene or substituted alkylene) -, wherein p is 0, 1 or 2; each R' is independently H, alkyl or substituted alkyl;
W is-J-R, wherein J is
Figure BDA0001546710350001771
R is H, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl; each R "is independently H, alkyl, substituted alkyl, or a protecting group, or when more than one R" group is present, two R "optionally form a heterocycloalkyl; and n is 1 to 3;
with the proviso that L-L 1 W together provide at least one carbonyl group (including dicarbonyl groups) capable of reacting with hydroxylamine groups on a non-natural amino acid or "modified or unmodified" non-natural amino acid polypeptide.
In certain embodiments of compounds of formula (XIX) are compounds having the structure of formula (XXI):
Figure BDA0001546710350001781
an illustrative example of a method for coupling a carbonyl-containing reagent to a hydroxylamine-containing unnatural amino acid on a polypeptide is presented in fig. 10. In this illustrative example, a carbonyl-derived reagent is added to a buffer solution (pH 2-8) of a hydroxylamine-containing unnatural amino acid polypeptide. The reaction is carried out at ambient temperature for a number of hours up to days. To accelerate the bonding, additives such as those presented in fig. 8 are added. The resulting oxime-containing unnatural amino acid polypeptide is purified by HPLC, FPLC or size exclusion chromatography.
In one embodiment, the plurality of linker chemistries can site-specifically react with hydroxylamine-substituted unnatural amino acid polypeptides. In one embodiment, the linker methods described herein utilize a linker (mono-, di-, or multifunctional) containing a carbonyl or dicarbonyl functional group on at least one linker terminus. Condensation of carbonyl or dicarbonyl derived linkers with hydroxylamine substituted proteins produces stable oxime linkages. Bifunctional and/or polyfunctional linkers (e.g., carbonyl or dicarbonyl groups with one or more other bonding chemistries) allow site-specific attachment of different molecules (e.g., other proteins, polymers, or small molecules) to the unnatural amino acid polypeptide, while monofunctional linkers (substituted with carbonyl or dicarbonyl groups on all termini) promote site-specific dimerization or oligomerization of the unnatural amino acid polypeptide. By combining this linker strategy with the in vivo translation techniques described herein, it is possible to specify the three-dimensional structure of chemically refined proteins.
In certain embodiments are methods for derivatizing a polypeptide comprising an amino acid of formula XIV-XVI, which comprises any sub-or specific compound within the scope of formula XIV-XVI, wherein the method comprises contacting a polypeptide comprising at least one amino acid of formula XIV-XVI with an agent of formula (XIX). In certain embodiments, the polypeptide is purified prior to or after contact with the agent of formula (XIX). In other embodiments are the resulting derivatized polypeptides comprising at least one oxime-containing amino acid corresponding to formula (XXIX),
Figure BDA0001546710350001782
wherein:
a is optional and when present is lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, arylene, substituted arylene, heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene;
b is optional and when present is a linker selected from the group consisting of: lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, -O- (alkylene or substituted alkylene) -, -S- (alkylene or substituted alkylene) -, -S (O) k - (wherein k is 1, 2 or 3), -S (O) k (alkylene or substituted alkylene) -, -C (O) -, -NS (O) 2 -、-OS(O) 2 -C (O) - (alkylene or substituted alkylene) -, -C (S) - (alkylene or substituted alkylene) -, -N (R '), -NR ' - (alkylene or substituted alkylene) -, -C (O) N (R '), -CON (R ') - (alkylene or substituted alkylene) -, -CSN (R ') - (alkylene or substituted alkylene) -, -N (R ') CO- (alkylene or substituted alkylene) -, -N (R ') C (O) O-, -S (O) k N(R')-、-N(R')C(O)N(R')-、-N(R')C(S)N(R')-、-N(R')S(O) k N(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 ') -, wherein each R' is independently H, alkyl or substituted alkyl;
l is a linker independently selected from the group consisting of: alkylene, substituted alkylene, alkenylene, substituted alkenylene, -O- (alkylene or substituted alkylene) -, -S- (alkylene or substituted alkylene) -, -S (O) k - (wherein k is 1, 2 or 3), -S (O) k (alkylene or substituted alkylene) -, -C (O) - (alkylene or substituted alkylene) -, -C (S) - (alkylene or substituted alkylene) -, -N (R ') -, NR ' - (alkylene or substituted alkylene) -, -C (O) N (R ') -, CON (R ') - (alkylene or substituted alkylene) -, a- (alkylene or substituted alkylene) NR ' C (O) O- (alkylene or substituted alkylene) -, -O-CON (R ') - (alkylene or substituted alkylene) -, -CSN (R ') - (alkylene or substituted alkylene) -, -N (R ') CO- (alkylene or substituted alkylene) -, -N (R ') C (O) O- (alkylene or substituted alkylene) -, -S (O) k N (R ') -, -N (R') C (O) N (R ') -, -N (R') C (O) N (R ') - (alkylene or substituted alkylene) -, -N (R') C (S) N (R '), -N (R') S (O) k N(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 ') -, wherein each R' is independently H, alkyl or substituted alkyl;
R 1 is optional and when present is H, an amino protecting group, a resin, an amino acid, a polypeptide or a polynucleotide; and is also provided with
R 2 Is optional and when present is an OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide;
R 3 and R is R 4 Each independently is H, halogen, lower alkyl or substituted lower alkyl; and is also provided with
X is independently a detectable label, a bioactive agent, or a polymer.
In other embodiments, these derivatized polypeptides are stable in aqueous solution under moderately acidic conditions for at least 1 month. In other embodiments, these derivative polypeptides are stable under moderately acidic conditions for at least 2 weeks. In other embodiments, these derivative polypeptides are stable under moderately acidic conditions for at least 5 days. In other embodiments, the conditions are pH 2 to 8. In certain embodiments, the tertiary structure of the derivatized polypeptide is maintained. In other embodiments, these derivatizations of polypeptides further comprise ligating the derivatized polypeptide to another polypeptide. In other embodiments, these derivatized polypeptides are homologous to a therapeutic protein selected from the group consisting of: alpha-1 antitrypsin, angiostatin, anti-hemolytic factor, antibody, apolipoprotein, apoprotein, atrial natriuretic factor, atrial natriuretic polypeptide, atrial peptide, C-X-C chemokine, T39765, NAP-2, ENA-78, gro-a, gro-b, gro-C, IP-10, GCP-2, NAP-4, SDF-1, PF4, MIG, calcitonin, C-kit ligand, cytokine, CC chemokine, monocyte chemokine-1, monocyte chemokine-2, monocyte chemokine-3, monocyte inflammatory protein-1 alpha, monocyte inflammatory protein-iβ, RANTES, 1309, R83915, R91733, HCC1, T58847, D31065, T64262, CD40 ligand, C-kit ligand, collagen, and pharmaceutical compositions containing the same Colony Stimulating Factor (CSF), complement factor 5a, complement inhibitor, complement receptor 1, cytokine, epithelial neutrophil activating peptide-78, MIP-16, MCP-1, epidermal Growth Factor (EGF), epithelial neutrophil activating peptide, erythropoietin (EPO), epidermal exfoliative toxin, factor IX, factor VII, factor VIII, factor X, fibroblast Growth Factor (FGF), fibrinogen, fibronectin, tetrahelical bundle protein, G-CSF, glp-1, GM-CSF, glucocerebrosidase, gonadotropin, growth factor receptor, grf, hedgehog protein, hemoglobin, hepatocyte growth factor (hGF), hirudin, human growth hormone (hGH), human serum albumin, ICAM-1, ICAM-1 receptor, LFA-1 receptor, insulin-like growth factor (IGF), IGF-I, IGF-II, interferon (IFN), IFN-alpha, IFN-beta, IFN-gamma, interleukin (IL), IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, keratinocyte Growth Factor (KGF), lactoferrin, leukemia inhibitory factor, luciferase, neurogenin, neutrophil Inhibitory Factor (NIF), oncostatin M, osteogenic protein, oncogene products, paracitin, parathyroid hormone, PD-ECSF, PDGF, peptide hormones, pleiotrophin, protein A, protein G, pth, pyrogenic exotoxin A, neutropy the therapeutic agents include, but are not limited to, heat exotoxin B, heat exotoxin C, pyy, relaxin, renin, SCF, small biosynthetic proteins, soluble complement receptor I, soluble I-CAM 1, soluble interleukin receptor, soluble TNF receptor, somatostatin, growth hormone, streptokinase, superantigen, staphylococcal enterotoxin, SEA, SEB, SEC1, SEC2, SEC3, SED, SEE, steroid hormone receptor, superoxide dismutase, toxic shock syndrome toxin, thymosin alpha 1, tissue type plasminogen activator, tumor Growth Factor (TGF), tumor necrosis factor alpha, tumor necrosis factor beta, tumor Necrosis Factor Receptor (TNFR), VLA-4 protein, VCAM-1 protein, vascular Endothelial Growth Factor (VEGF), urokinase, mos, ras, raf, met, p, tat, fos, myc, jun, myb, rel, estrogen receptor, progesterone receptor, testosterone receptor, aldosterone receptor, LDL receptor, and corticosterone.
D. Examples of adding functional groups: macromolecular polymers coupled to unnatural amino acid polypeptides
Various modifications to the unnatural amino acid polypeptides described herein can be accomplished using the compositions, methods, techniques, and strategies described herein. These modifications comprise binding other functional groups to the unnatural amino acid component of the polypeptide, including (but not limited to): marking; a dye; a polymer; a water-soluble polymer; derivatives of polyethylene glycol; a photocrosslinking agent; a cytotoxic compound; a drug; affinity labeling; a photoaffinity label; a reactive compound; a resin; a second protein or polypeptide analog; an antibody or antibody fragment; a metal chelator; a cofactor; a fatty acid; a carbohydrate; a polynucleotide; DNA; RNA; an antisense polynucleotide; saccharides, water-soluble dendrimers, cyclodextrins, biomaterials; a nanoparticle; spin labeling; a fluorophore-containing moiety; a radioactive portion; novel functional groups; groups that interact covalently or non-covalently with other molecules; a light-shielding portion; an actinic radiation excitable moiety; a ligand; a photoisomerizable moiety; biotin; biotin analogues; a moiety incorporating a heavy atom; a chemically cleavable group; a photocleavable group; an extended side chain; a carbon-bonded sugar; a redox active agent; an aminothiopropionic acid; a toxic moiety; isotopically labeled moieties; a biophysical probe; phosphorescent groups; a chemiluminescent group; an electron dense group; a magnetic group; an insertion group; a chromophore; an energy transfer agent; a bioactive agent; a detectable label; a small molecule; inhibitory ribonucleic acid, radionucleotide; a neutron capture agent; derivatives of biotin; a quantum dot; nano transitorin; a radiotransferrin; abzymes, activating complex activators, viruses, adjuvants, aglycones, allergens, angiostatin, anti-hormones, antioxidants, aptamers, guide RNAs, saponins, shuttle vectors, macromolecules, pseudoepitopes, receptors, reverse micelles, and any combination thereof. As an illustrative, non-limiting example of the compositions, methods, techniques, and strategies described herein, the following description should focus on the addition of macromolecular polymers to non-natural amino acid polypeptides, while it should be understood that the additionally described compositions, methods, techniques, and strategies may also be used (with appropriate modifications, if necessary, and those skilled in the art may make use of the disclosure herein) to add other functional groups, including, but not limited to, those listed above.
A variety of macromolecular polymers and other molecules can be coupled to the unnatural amino acid polypeptides described herein to modulate the biological properties of the unnatural amino acid polypeptide (or corresponding natural amino acid polypeptide) and/or to provide novel biological properties to the unnatural amino acid polypeptide (or corresponding natural amino acid polypeptide). These macromolecular polymers may be coupled to the unnatural amino acid polypeptide via any functional substituent of the unnatural amino acid, or any substituent or functional group added to the unnatural amino acid.
The water-soluble polymer may be coupled to an unnatural amino acid that is incorporated into a polypeptide (natural or synthetic), polynucleotide, polysaccharide, or synthetic polymer described herein. The water-soluble polymer may be coupled through any functional group or substituent of the unnatural amino acid or unnatural amino acid that is incorporated into the polypeptide, or any functional group or substituent added to the unnatural amino acid. In some cases, the unnatural amino acid polypeptides described herein include one or more unnatural amino acids coupled to a water-soluble polymer and one or more naturally occurring amino acids that are linked to the water-soluble polymer. Covalent attachment of hydrophilic polymers to bioactive molecules represents a method of increasing the water solubility (such as in physiological environments), bioavailability, increasing serum half-life, increasing therapeutic half-life, modulating immunogenicity, modulating bioactivity, or prolonging circulation time of bioactive molecules (including proteins, peptides, and particularly hydrophobic molecules). Other important features of these hydrophilic polymers include biocompatibility, non-toxicity, and non-immunogenicity. For therapeutic use of the final product formulation, it is preferred that the polymer should be pharmaceutically acceptable.
Examples of hydrophilic polymers include (but are not limited to): polyalkyl ethers and alkoxy-terminated analogs thereof (e.g., polyethylene glycol, polyethylene/polypropylene glycol, and methoxy or ethoxy-terminated analogs thereof, especially polyethylene glycol, the latter also known as polyethylene glycol or PEG); polyvinylpyrrolidone; polyvinyl alkyl ether; polyoxazolines, polyalkyloxazolines, and polyhydroxyalkyl oxazolines; polyacrylamides, polyalkylacrylamides, and polyhydroxyalkyl acrylamides (e.g., polyhydroxypropyl methacrylamides and derivatives thereof); polyhydroxyalkyl acrylates; polysialic acid and analogs thereof; a hydrophilic peptide sequence; polysaccharides and derivatives thereof, including dextran and dextran derivatives, e.g., carboxymethyl dextran, dextran sulfate, aminodextran; cellulose and its derivatives, e.g., carboxymethyl cellulose, hydroxyalkyl cellulose; chitin and its derivatives, e.g., chitosan, succinyl chitosan, carboxymethyl chitin, carboxymethyl chitosan; hyaluronic acid and derivatives thereof; starch; an alginate; chondroitin sulfate; albumin; amylopectin and carboxymethyl amylopectin; polyamino acids and derivatives thereof, for example, polyglutamic acid, polylysine, polyaspartic acid; maleic anhydride copolymers such as: styrene maleic anhydride copolymer, divinyl ethyl ether maleic anhydride copolymer; polyvinyl alcohol; copolymers thereof; a terpolymer thereof; mixtures thereof; and derivatives of the foregoing. The water-soluble polymer may be in any structural form including, but not limited to, linear, forked, or branched. In some embodiments, water-soluble polymer backbones having from 2 to about 300 termini are particularly useful. Polyfunctional polymer derivatives include, but are not limited to, linear polymers having two ends, where each end is bonded to a functional group that may be the same or different. In some embodiments, the water-based polymer includes polyethylene glycol moieties. The molecular weight of the polymer may be in a wide range including, but not limited to, a range between about 100Da and about 100,000Da or higher. The molecular weight of the polymer may be between about 100Da and about 100,000Da, including, but not limited to, 100,000Da, 95,000Da, 90,000Da, 85,000Da, 80,000Da, 75,000Da, 70,000Da, 65,000Da, 60,000Da, 55,000Da, 50,000Da, 45,000Da, 40,000Da, 35,000Da, 30,000Da, 25,000Da, 20,000Da, 15,000Da, 10,000Da, 9,000Da, 8,000Da, 7,000Da, 6,000Da, 5,000Da, 4,000Da, 3,000Da, 2,000Da, 1,000Da, 900Da, 800Da, 700Da, 600Da, 500Da, 400Da, 300Da, 200Da, and 100Da. In some embodiments, the molecular weight of the polymer is between about 100Da and 50,000 Da. In some embodiments, the molecular weight of the polymer is between about 100Da and 40,000 Da. In some embodiments, the molecular weight of the polymer is between about 1,000da and 40,000 da. In some embodiments, the molecular weight of the polymer is between about 5,000da and 40,000 da. In some embodiments, the molecular weight of the polymer is between about 10,000da and 40,000 da. In some embodiments, the polyethylene glycol molecule is a branched polymer. The molecular weight of the branched PEG may be between about 1,000da and about 100,000da, including, but not limited to, 100,000da, 95,000da, 90,000da, 85,000da, 80,000da, 75,000da, 70,000da, 65,000da, 60,000da, 55,000da, 50,000da, 45,000da, 40,000da, 35,000da, 30,000da, 25,000da, 20,000da, 15,000da, 10,000da, 9,000da, 8,000da, 7,000da, 6,000da, 5,000da, 4,000da, 3,000da, 2,000da, and 1,000da. In some embodiments, the branched PEG has a molecular weight between about 1,000da and 50,000 da. In some embodiments, the branched PEG has a molecular weight between about 1,000da and 40,000 da. In some embodiments, the branched PEG has a molecular weight between about 5,000da and 40,000 da. In some embodiments, the branched PEG has a molecular weight between about 5,000da and 20,000 da. Those skilled in the art will appreciate that the foregoing list of substantially water-soluble backbones is by no means exhaustive and is merely illustrative, and that all polymeric materials having the above qualities are contemplated for use in the methods and compositions described herein.
As mentioned above, one example of a hydrophilic polymer is polyethylene glycol (abbreviated as PEG), which has been widely used in medicine, on artificial implants, and in other applications where biocompatibility, non-toxicity and non-immunogenicity are of importance. Polymer: polypeptide embodiments described herein will use PEG as an exemplary hydrophilic polymer, with the understanding that other hydrophilic polymers may be similarly used in these embodiments.
PEG is a well known water-soluble polymer that is commercially available or can be prepared by ring-opening polymerization of ethylene glycol according to methods well known in the art (Sandler and Karo, polymer Synthesis, academic Press, new York, vol.3, pages 138-161). PEG is generally transparent, colorless, odorless, soluble in water, thermally stable, inert to many chemicals, non-hydrolytic or spoiling, and generally non-toxic. Polyethylene glycol is considered biocompatible, that is, PEG can coexist with living tissue or organisms without causing damage. More specifically, PEG is non-immunogenic in nature, that is, PEG does not tend to produce an immune response in vivo. When attached to molecules (such as bioactive agents) that have some desired function in the body, PEG tends to mask the agent and may reduce or eliminate any immune response so that the organism can tolerate the presence of the agent. PEG conjugates tend not to produce a substantial immune response or cause clotting or other undesirable effects.
The term "PEG" is widely used to encompass any polyethylene glycol molecule (regardless of size or modification at the end of PEG), and when bound to an unnatural amino acid polypeptide can be represented by the following formula:
XO-(CH 2 CH 2 O) n -CH 2 CH 2 -Y
wherein n is 2 to 10,000 and X is H or a terminal modification, including but not limited to C 1-4 Alkyl, protecting group or terminal functional group. The term PEG includes, but is not limited to, any form of polyethylene glycol thereof, including difunctional PEG, multiarmed PEG, derivatized PEG, forked PEG, branched PEG (where each chain has a molecular weight of about 1kDa to about 100kDa, about 1kDa to about 50kDa, or about 1kDa to about 20 kDa), pendent PEG (i.e., PEG or related polymer having one or more functional groups pendent to the polymer backbone), or PEG with degradable linkages therein. In one embodiment, PEG wherein n is from about 20 to about 2000 is suitable for use in the methods and compositions described herein. In some embodiments, the water-based polymer includes polyethylene glycol moieties. The molecular weight of the PEG polymer can be in a wide range including, but not limited to, a range between about 100Da and about 100,000Da or higher. PEG polymersThe molecular weight of (c) may be between about 100 and about 100,000Da, including, but not limited to, 100,000Da, 95,000Da, 90,000Da, 85,000Da, 80,000Da, 75,000Da, 70,000Da, 65,000Da, 60,000Da, 55,000Da, 50,000Da, 45,000Da, 40,000Da, 35,000Da, 30,000Da, 25,000Da, 20,000Da, 15,000Da, 10,000Da, 9,000Da, 8,000Da, 7,000Da, 6,000Da, 5,000Da, 4,000Da, 3,000Da, 2,000Da, 1,000Da, 900Da, 800Da, 700Da, 600Da, 500Da, 400Da, 300Da, 200Da, and 100Da. In some embodiments, the molecular weight of the polymer is between about 100Da and 50,000 Da. In some embodiments, the molecular weight of the polymer is between about 100Da and 40,000 Da. In some embodiments, the molecular weight of the polymer is between about 1,000da and 40,000 da. In some embodiments, the molecular weight of the polymer is between about 5,000da and 40,000 da. In some embodiments, the molecular weight of the polymer is between about 10,000da and 40,000 da. In some embodiments, the polyethylene glycol molecule is a branched polymer. The molecular weight of the branched PEG may be between about 1,000da and about 100,000da, including, but not limited to, 100,000da, 95,000da, 90,000da, 85,000da, 80,000da, 75,000da, 70,000da, 65,000da, 60,000da, 55,000da, 50,000da, 45,000da, 40,000da, 35,000da, 30,000da, 25,000da, 20,000da, 15,000da, 10,000da, 9,000da, 8,000da, 7,000da, 6,000da, 5,000da, 4,000da, 3,000da, 2,000da, and 1,000da. In some embodiments, the branched PEG has a molecular weight between about 1,000da and 50,000 da. In some embodiments, the branched PEG has a molecular weight between about 1,000da and 40,000 da. In some embodiments, the branched PEG has a molecular weight between about 5,000da and 40,000 da. In some embodiments, the branched PEG has a molecular weight between about 5,000da and 20,000 da. A broad range of PEG molecules are described in (including, but not limited to) Shearwater Polymers, inc.
Specific examples of terminal functional groups in the literature include, but are not limited to, N-succinimidyl carbonate (see, e.g., U.S. Pat. Nos. 5,281,698, 5,468,478), amines (see, e.g., buckmann et al, makromol. Chem.182:1379 (1981); zalipsky et al, eur. Polym.J.19:1177 (1983)), hydrazides (see, e.g., andresz et al, makromol. Chem.179:301 (1978)), succinimidyl propionate and succinimidyl butyrate (see, e.g., olson et al, in Poly (ethylene glycol) Chemistry & Biological Applications, pages 170-181, harris and Zalipsky, ACS, washington, D.C.,1997; also see, e.g., U.S. Pat. No. 5,672,662), succinimidyl succinate (see, e.g., abuchowski et al, cancer Biochem. Biophys.7:175 (1984)), succinimidyl butyrate (see, e.g., U.S. Pat. No. 4,670,417), benzotriazolyl carbonate (see, e.g., U.S. Pat. No. 5, 5,650,234), glycidyl ether (see, e.g., eshington, etc.), biochem. 141:35:7:7:175 (1984), and so forth, e.g., biochem. 35:11, biochem. 7:175 (1989), biochem. 7:175 (see, 1984), and so forth, biochem. 7:35 (see, e.g., U.g., U.S. Pat. No. 5:35, biochem. 7:35, 7:35 (1989), J.Polym.Sci.chem.ed.22:341 (1984); U.S. patent No. 5,824,784; U.S. Pat. No. 5,252,714), maleimide (see, e.g., goodson et al, bio/Technology8:343 (1990); romani et al, chemistry of Peptides and Proteins 2:29 (1984); and Kogan, synthetic Comm.22:2417 (1992)), ortho-pyridyl disulfides (see, e.g., woghiren et al, bioconj.chem.4:314 (1993)), propenols (see, e.g., sawhney et al, macromolecules,26:581 (1993)), vinyl sulfones (see, e.g., U.S. Pat. No. 5,900,461). All of the above references and patents are incorporated by reference herein in their entirety.
In some cases, the PEG terminates at one end with a hydroxyl or methoxy group, i.e., X is H or CH 3 ("methoxy PEG"). Alternatively, PEG may terminate with a reactive group, thereby forming a difunctional polymer. Typical reactive groups may comprise functional groups commonly used in combination with groups present in 20 common amino acids, including but not limited to maleimide groups, activated carbonates, including but not limited to p-nitrophenyl esters, activated esters (inclusionThose reactive groups containing, but not limited to, N-hydroxysuccinimide, p-nitrophenyl ester, and aldehyde) and functional groups inert to 20 common amino acids but specifically reactive with complementary functional groups present in non-natural amino acids, including, but not limited to, oxime, carbonyl, or dicarbonyl groups, and hydroxylamine groups.
It should be noted that the other end of PEG represented by Y in the above formula should be directly or indirectly linked to a polypeptide (synthetic or natural), polynucleotide, polysaccharide or synthetic polymer through an unnatural amino acid. When Y is a hydroxylamine group, then the hydroxylamine-containing PEG reagent may react with a carbonyl or dicarbonyl-containing unnatural amino acid in the polypeptide to form a PEG group that is linked to the polypeptide by an oxime linkage. When Y is carbonyl or dicarbonyl, then the PEG reagent containing carbonyl or dicarbonyl can react with the hydroxylamine-containing unnatural amino acid in the polypeptide to form a PEG group that is linked to the polypeptide by an oxime linkage. When Y is carbonyl or dicarbonyl, then the PEG reagent containing the carbonyl or dicarbonyl can also react with oxime-containing unnatural amino acids in the polypeptide to form PEG groups that are linked to the polypeptide by new oxime linkages. Examples of suitable reaction conditions, purification methods and reagents are described throughout the specification and accompanying drawings. For example, fig. 7 presents three examples of carbonyl-containing unnatural amino acid polypeptides reacted with hydroxylamine-containing PEG reagents to form oxime-containing unnatural amino acid polypeptides bonded to PEG groups. In addition, fig. 9 presents two examples of oxime-containing unnatural amino acid polypeptides reacted with carbonyl-containing PEG reagents to form novel oxime-containing unnatural amino acid polypeptides that are bonded to PEG groups. And FIG. 10 presents one example of a hydroxylamine-containing unnatural amino acid polypeptide reacted with a carbonyl-containing PEG reagent to form an oxime-containing unnatural amino acid polypeptide bonded to a PEG group.
By way of example only and not as a limitation on the type or kind of PEG reagent that can be used in the compositions, methods, techniques and strategies described herein, fig. 11 presents other illustrative examples of hydroxylamine-containing PEG reagents that can react with carbonyl-containing unnatural amino acid polypeptides to form oxime-containing unnatural amino acid polypeptides that are linked to PEG groups, as well as examples of carbonyl-containing PEG reagents that can react with oxime-containing unnatural amino acid polypeptides or hydroxylamine-containing unnatural amino acid polypeptides to form novel oxime-containing unnatural amino acid polypeptides that are linked to PEG groups. Fig. 12 presents four illustrative examples of synthetic methods for forming a hydroxylamine-containing PEG reagent, or a protected form of a hydroxylamine-containing PEG reagent, or a masked form of a hydroxylamine-containing PEG reagent. Fig. 13 presents an illustrative example of a synthetic method for forming an amide-linked hydroxylamine-containing PEG reagent, or a protected form of an amide-linked hydroxylamine-containing PEG reagent, or a masked form of an amide-linked hydroxylamine-containing PEG reagent. Fig. 14 and 15 present illustrative examples of synthetic methods for forming a carbamate-linked hydroxylamine-containing PEG reagent, or a protected form of a carbamate-linked hydroxylamine-containing PEG reagent, or a masked form of a carbamate-linked hydroxylamine-containing PEG reagent. Fig. 16 presents an illustrative example of a synthetic method for forming a simple hydroxylamine-containing PEG reagent, or a protected form of a simple hydroxylamine-containing PEG reagent, or a masked form of a simple hydroxylamine-containing PEG reagent. In addition, fig. 17 presents an illustrative example of a branched hydroxylamine-containing reagent having a plurality of PEG-linked groups, and further shows the reaction of one such hydroxylamine-containing, plurality of PEG-branched reagents with carbonyl-containing, unnatural amino acid polypeptides to form oxime-containing, unnatural amino acid polypeptides having a plurality of PEG-branched groups linked.
Heterobifunctional derivatives are also particularly useful when it is desired to attach a different molecule to each end of the polymer. For example, omega-N-amino-N-azido PEG would allow for attachment of molecules with activated electrophilic groups (such as aldehydes, ketones, activated esters, activated carbonates, etc.) to one end of the PEG and molecules with an acetyl group to the other end of the PEG.
In some embodiments, strong nucleophiles (including, but not limited to, hydroxylamine) can react with aldehyde or ketone groups present in non-natural amino acids to form oximes, which in some cases can be further reduced by treatment with suitable reducing agents. Alternatively, strong nucleophiles may be incorporated into the polypeptide by unnatural amino acids and used to preferentially react with ketone or aldehyde groups present in the water-soluble polymer. Typically, at least one terminus of a PEG molecule is available for reaction with an unnatural amino acid.
Thus, in some embodiments, a polypeptide comprising an unnatural amino acid is linked to a water-soluble polymer, such as polyethylene glycol (PEG), through a side chain of the unnatural amino acid. The unnatural amino acid methods and compositions described herein provide highly efficient methods for selectively modifying proteins with PEG derivatives, including the selective incorporation of unnatural amino acids (including, but not limited to, those containing functional groups or substituents that are not found in the 20 naturally incorporated amino acids) into proteins in response to selector codons, and subsequent modification of those amino acids with suitable reactive PEG derivatives. A variety of known chemical methods are suitable for use with the unnatural amino acid methods and compositions described herein to incorporate water-soluble polymers into proteins.
The polymer backbone may be linear or branched. Branched polymer backbones are generally known in the art. Typically, branched polymers have a central branched core portion and a plurality of linear polymer chains attached to the central branched core. PEG is used in branched form and can be prepared by adding ethylene oxide to various polyols such as glycerol, glycerol oligomers, pentaerythritol, and sorbitol. The central branched moiety may also be derived from several amino acids, such as lysine. Branched polyethylene glycols can be represented in general form as R (-PEG-OH) m Wherein R is derived from a core moiety such as glycerol, glycerol oligomer or pentaerythritol and m represents the number of arms. Multi-arm PEG molecules (such as those described in U.S. Pat. Nos. 5,932,462, 5,643,575, 5,229,490, 4,289,872, U.S. patent application 2003/0143596, WO 96/21469, and WO 93/21259, each of which is incorporated herein by reference in its entirety) can also be used as the polymer backbone.
Branched PEG may also be composed of PEG (-YCHZ) 2 ) n The forked PEG form is represented, where Y is a linking group and Z is an activating end group linked to CH through an atomic chain of defined length. The other branched form overhangs the PEG along the PEG backbone rather than having a reactive group such as a carboxyl group at the end of the PEG chain.
In addition to these forms of PEG, polymers having weak or degradable linkages in the backbone can also be prepared. For example, PEG can be prepared having ester linkages in the polymer backbone that undergo hydrolysis. As shown herein, this hydrolysis causes the polymer to cleave into lower molecular weight fragments:
-PEG-CO 2 -PEG-+H 2 O→PEG-CO 2 H+HO-PEG-。
it will be appreciated by those skilled in the art that the term polyethylene glycol or PEG represents or encompasses all forms known in the art, including but not limited to those disclosed herein. The molecular weight of the polymer may be in a wide range including, but not limited to, a range between about 100Da and about 100,000Da or higher. The molecular weight of the polymer may be between about 100Da and about 100,000Da, including, but not limited to, 100,000Da, 95,000Da, 90,000Da, 85,000Da, 80,000Da, 75,000Da, 70,000Da, 65,000Da, 60,000Da, 55,000Da, 50,000Da, 45,000Da, 40,000Da, 35,000Da, 30,000Da, 25,000Da, 20,000Da, 15,000Da, 10,000Da, 9,000Da, 8,000Da, 7,000Da, 6,000Da, 5,000Da, 4,000Da, 3,000Da, 2,000Da, 1,000Da, 900Da, 800Da, 700Da, 600Da, 500Da, 400Da, 300Da, 200Da, and 100Da. In some embodiments, the molecular weight of the polymer is between about 100Da and 50,000 Da. In some embodiments, the molecular weight of the polymer is between about 100Da and 40,000 Da. In some embodiments, the molecular weight of the polymer is between about 1,000da and 40,000 da. In some embodiments, the molecular weight of the polymer is between about 5,000da and 40,000 da. In some embodiments, the molecular weight of the polymer is between about 10,000da and 40,000 da.
To maximize the desired properties of PEG, the total molecular weight of the PEG polymer or polymers attached to the bioactive molecule, as well as the hydration state, should be high enough to impart the advantageous features typically associated with PEG polymer attachment, such as increased water solubility and circulation half-life, without adversely affecting the bioactivity of the parent molecule.
The methods and compositions described herein can be used to prepare a substantially homogeneous formulation of a polymer-protein conjugate. "substantially homogeneous" as used herein means that the polymer is observed to be greater than half of the total protein in the protein conjugate molecule. Polymer protein conjugates are biologically active and the "substantially homogeneous" pegylated polypeptide formulations of the invention provided herein are those formulations that are sufficiently homogeneous to demonstrate the advantages of homogeneous formulations (e.g., ease of clinical application in predicting pharmacokinetics between batches).
Alternatively, a mixture of polymer: protein conjugate molecules may be prepared, and the advantages provided herein are that the ratio of single polymer: protein conjugate contained in the mixture may be selected. Thus, if desired, mixtures of various proteins with various numbers of polymer moieties attached (i.e., dimer moieties, trimer moieties, tetramer moieties, etc.) can be prepared and these conjugates combined with the single polymer-to-protein conjugates prepared using the methods described herein, and the mixtures made with a predetermined ratio of single polymer-to-protein conjugates.
The ratio of polyethylene glycol molecules to protein molecules will vary with their concentration in the reaction mixture. In general, the optimal ratio (in terms of reaction efficiency in that there is a minimum excess of unreacted protein or polymer) can be determined by the molecular weight of the polyethylene glycol selected and with respect to the number of available reactive groups available. With respect to molecular weight, generally the higher the molecular weight of the polymer, the fewer the number of polymer molecules that can be attached to the protein. Similarly, branching of the polymer should be considered when optimizing these parameters. In general, the higher the molecular weight (or more branches), the higher the polymer to protein ratio.
As used herein, and when covering hydrophilic polymers, polypeptide/protein conjugates, the term "therapeutically effective amount" further refers to an amount that provides the desired increase in benefit to the patient. The amount will vary from individual to individual and should depend on numerous factors, including the overall physical state of the patient and the underlying etiology of the disease, disorder, or condition to be treated. The therapeutically effective amount of the compositions of the present invention can be readily determined by one skilled in the art using publicly available materials and procedures.
The number of water-soluble polymers (i.e., the degree of pegylation or glycosylation) described herein that are linked to a "modified or unmodified" unnatural amino acid polypeptide can be adjusted to provide altered (including, but not limited to, increased or decreased) pharmacological, pharmacokinetic, or pharmacodynamic characteristics, such as in vivo half-life. In some embodiments, the half-life of the polypeptide is increased by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, two-fold, five-fold, 10-fold, 50-fold, or at least about 100-fold over the unmodified polypeptide.
In one embodiment, a polypeptide comprising a carbonyl-or dicarbonyl-containing unnatural amino acid is modified with a PEG derivative comprising a terminal hydroxylamine moiety that is directly bonded to the PEG backbone.
In some embodiments, the hydroxylamine-terminated PEG derivative has the following structure:
RO-(CH 2 CH 2 O) n -O-(CH 2 ) m -O-NH 2
where R is a simple alkyl group (methyl, ethyl, propyl, etc.), m is 2-10 and n is 100-1,000 (i.e., average molecular weight between 5kDa and 40 kDa). The molecular weight of the polymer may be in a wide range including, but not limited to, a range between about 100Da and about 100,000Da or higher. The molecular weight of the polymer may be between about 100Da and about 100,000Da, including, but not limited to, 100,000Da, 95,000Da, 90,000Da, 85,000Da, 80,000Da, 75,000Da, 70,000Da, 65,000Da, 60,000Da, 55,000Da, 50,000Da, 45,000Da, 40,000Da, 35,000Da, 30,000Da, 25,000Da, 20,000Da, 15,000Da, 10,000Da, 9,000Da, 8,000Da, 7,000Da, 6,000Da, 5,000Da, 4,000Da, 3,000Da, 2,000Da, 1,000Da, 900Da, 800Da, 700Da, 600Da, 500Da, 400Da, 300Da, 200Da, and 100Da. In some embodiments, the molecular weight of the polymer is between about 100Da and 50,000 Da. In some embodiments, the molecular weight of the polymer is between about 100Da and 40,000 Da. In some embodiments, the molecular weight of the polymer is between about 1,000da and 40,000 da. In some embodiments, the molecular weight of the polymer is between about 5,000da and 40,000 da. In some embodiments, the molecular weight of the polymer is between about 10,000da and 40,000 da.
In another embodiment, a polypeptide comprising a carbonyl-or dicarbonyl-containing amino acid is modified with a PEG derivative comprising a terminal hydroxylamine moiety, which is linked to the PEG backbone by an amide linkage.
In some embodiments, the hydroxylamine-terminated PEG derivative has the following structure:
RO-(CH 2 CH 2 O) n -O-(CH 2 ) 2 -NH-C(O)(CH 2 ) m -O-NH 2
where R is a simple alkyl group (methyl, ethyl, propyl, etc.), m is 2-10 and n is 100-1,000 (i.e., average molecular weight between 5kDa and 40 kDa). The molecular weight of the polymer may be in a wide range including, but not limited to, a range between about 100Da and about 100,000Da or higher. The molecular weight of the polymer may be between about 100Da and about 100,000Da, including, but not limited to, 100,000Da, 95,000Da, 90,000Da, 85,000Da, 80,000Da, 75,000Da, 70,000Da, 65,000Da, 60,000Da, 55,000Da, 50,000Da, 45,000Da, 40,000Da, 35,000Da, 30,000Da, 25,000Da, 20,000Da, 15,000Da, 10,000Da, 9,000Da, 8,000Da, 7,000Da, 6,000Da, 5,000Da, 4,000Da, 3,000Da, 2,000Da, 1,000Da, 900Da, 800Da, 700Da, 600Da, 500Da, 400Da, 300Da, 200Da, and 100Da. In some embodiments, the molecular weight of the polymer is between about 100Da and 50,000 Da. In some embodiments, the molecular weight of the polymer is between about 100Da and 40,000 Da. In some embodiments, the molecular weight of the polymer is between about 1,000da and 40,000 da. In some embodiments, the molecular weight of the polymer is between about 5,000da and 40,000 da. In some embodiments, the molecular weight of the polymer is between about 10,000da and 40,000 da.
In another embodiment, the polypeptide comprising carbonyl-or dicarbonyl-containing amino acids is modified with a PEG derivative comprising a terminal hydroxylamine moiety, wherein each chain of the branched PEG has a MW in the range of 10kDa-40kDa, and in other embodiments in the range of 5kDa-20 kDa. The molecular weight of the branched polymer may be in a wide range including, but not limited to, a range between about 100Da and about 100,000Da or higher. The molecular weight of the polymer may be between about 100Da and about 100,000Da, including, but not limited to, 100,000Da, 95,000Da, 90,000Da, 85,000Da, 80,000Da, 75,000Da, 70,000Da, 65,000Da, 60,000Da, 55,000Da, 50,000Da, 45,000Da, 40,000Da, 35,000Da, 30,000Da, 25,000Da, 20,000Da, 15,000Da, 10,000Da, 9,000Da, 8,000Da, 7,000Da, 6,000Da, 5,000Da, 4,000Da, 3,000Da, 2,000Da, 1,000Da, 900Da, 800Da, 700Da, 600Da, 500Da, 400Da, 300Da, 200Da, and 100Da. In some embodiments, the molecular weight of the polymer is between about 100Da and 50,000 Da. In some embodiments, the molecular weight of the polymer is between about 100Da and 40,000 Da. In some embodiments, the molecular weight of the polymer is between about 1,000da and 40,000 da. In some embodiments, the molecular weight of the polymer is between about 5,000da and 40,000 da. In some embodiments, the molecular weight of the polymer is between about 10,000da and 40,000 da.
In another embodiment, a polypeptide comprising an unnatural amino acid is modified with at least one PEG derivative having a branched structure. In some embodiments, the hydroxylamine group-containing PEG derivative has the following structure:
[RO-(CH 2 CH 2 O) n -O-(CH 2 ) 2 -C(O)-NH-CH 2 -CH 2 ] 2 CH-X-(CH 2 ) m -O-NH 2
wherein R is a simple alkyl group (methyl, ethyl, propyl, etc.), X is optionally NH, O, S, C (O) or absent, m is from 2 to 10 and n is from 100 to 1,000. The molecular weight of the polymer may be between about 1,000da and about 100,000da, including, but not limited to, 100,000da, 95,000da, 90,000da, 85,000da, 80,000da, 75,000da, 70,000da, 65,000da, 60,000da, 55,000da, 50,000da, 45,000da, 40,000da, 35,000da, 30,000da, 25,000da, 20,000da, 15,000da, 10,000da, 9,000da, 8,000da, 7,000da, 6,000da, 5,000da, 4,000da, 3,000da, 2,000da and 1,000da. In some embodiments, the branched PEG has a molecular weight between about 1,000da and 50,000 da. In some embodiments, the branched PEG has a molecular weight between about 1,000da and 40,000 da. In some embodiments, the branched PEG has a molecular weight between about 5,000da and 40,000 da. In some embodiments, the branched PEG has a molecular weight between about 5,000da and 20,000 da.
Several summaries and monographs about functionalization and conjugation of PEG are available. See, e.g., harris, macromol. Chem. Phys. C25:325-373 (1985); scouten, methods in Enzymology 135:30-65 (1987); wong et al, enzyme Microb.technology.14:866-874 (1992); delgado et al Critical Reviews in Therapeutic Drug Carrier Systems 9:249-304 (1992); zalipsky, bioconjugate chem.6:150-165 (1995).
Methods for activating polymers can also be found in WO 94/17039, U.S. Pat. No. 5,324,844, WO 94/18247, WO 94/04193, U.S. Pat. No. 5,219,564, U.S. Pat. No. 5,122,614, WO 90/13540, U.S. Pat. No. 5,281,698, and more WO 93/15189, and methods for activating the binding between a polymer and an enzyme include, but are not limited to, coagulation factor VIII (WO 94/15625), hemoglobin (WO 94/09027), oxygen-carrying molecules (U.S. Pat. No. 4,412,989), ribonuclease, and superoxide dismutase (Veronese et al, app. Biochem. Biotech.11:141-152 (1985)), all of which are incorporated herein by reference in their entirety.
If desired, the pegylated unnatural amino acid polypeptides described herein obtained from hydrophobic chromatography can be further purified by one or more procedures known to those of skill in the art, including, but not limited to, affinity chromatography; anion or cation exchange chromatography (using (including but not limited to) DEAE SEPHAROSE); silica chromatography; reversed phase HPLC; gel filtration (using (including but not limited to) SEPHADEX G-75); hydrophobic interaction chromatography; size exclusion chromatography; metal chelate chromatography; ultrafiltration/diafiltration; precipitating with ethanol; precipitating ammonium sulfate; focusing the chromatogram; displacement chromatography; electrophoresis procedures (including but not limited to preparative isoelectric focusing), differential solubility (including but not limited to ammonium sulfate precipitation), or extraction. Apparent molecular weights (Preneta AZ, PROTEIN PURIFICATION METHODS, A PRACTICAL APPROACH (Harris and Angal) IRL Press 1989, 293-306) can be estimated by GPC by comparison with globular PROTEIN standards. The purity of the unnatural amino acid polypeptide, PEG conjugate, can be assessed by proteolytic degradation, including but not limited to trypsin cleavage, followed by mass spectrometry. Pepinsky RB. et al, J.Pharmacol. & exp. Ther.297 (3): 1059-66 (2001).
The water-soluble polymers bonded to the unnatural amino acids of the polypeptides described herein can be further derivatized or substituted without limitation.
E. Enhancing affinity for serum albumin
Various molecules may also be fused to the unnatural amino acid polypeptides described herein to modulate half-life in serum. In some embodiments, the molecules are linked or fused to "modified or unmodified" unnatural amino acid polypeptides described herein to enhance affinity for endogenous serum albumin in animals.
For example, in some cases, recombinant fusion of the polypeptide with an albumin binding sequence is performed. Exemplary albumin binding sequences include, but are not limited to, albumin binding domains from streptococcal protein G (see, e.g., makrides et al, J. Pharmacol Exp. Ther.277 (l): 534-542 (1996) and Sjorander et al, J, immunol. Methods 201:115-123 (1997)), or albumin binding peptides such as, e.g., those described in Dennis et al, J. Biol. Chem.277 (38): 35035-35043 (2002).
In other embodiments, the "modified or unmodified" unnatural amino acid polypeptides described herein are acylated with fatty acids. In some cases, the fatty acid promotes binding to serum albumin. See, for example, kurtzhals et al biochem. J.312:725-731 (1995).
In other embodiments, the "modified or unmodified" unnatural amino acid polypeptides described herein are fused directly to serum albumin (including, but not limited to, human serum albumin). Those of skill in the art will appreciate that a variety of other molecules may also be linked to the modified or unmodified unnatural amino acid polypeptides as described herein to modulate binding to serum albumin or other serum components.
F. Glycosylation of unnatural amino acid polypeptides described herein
The methods and compositions described herein comprise polypeptides incorporating one or more unnatural amino acids with a sugar residue. The sugar residues may be natural (including but not limited to N-acetylglucosamine) or non-natural (including but not limited to 3-fluorogalactose). The sugar may be linked to the unnatural amino acid by an N-linked or O-linked glycosidic linkage, including but not limited to N-acetylgalactose-L-serine, or an unnatural linkage, including but not limited to an oxime or a corresponding C-linked or S-linked glycosid.
Sugar (including but not limited to glycosyl) moieties can be added to the unnatural amino acid polypeptide in vivo or in vitro. In some embodiments, polypeptides comprising carbonyl-or dicarbonyl-containing unnatural amino acids are modified with saccharides derivatized with aminooxy groups to produce corresponding glycosylated polypeptides that are linked by oxime linkages. Once attached to the unnatural amino acid, the sugar can be further refined by treatment with glycosyltransferases and other enzymes to produce an oligosaccharide that binds to the unnatural amino acid polypeptide. See, for example, H.Liu et al, J.Am.chem.Soc.125:1702-1703 (2003).
G. Use of linking groups and applications comprising polypeptide dimers and multimers
In addition to adding functional groups directly to the unnatural amino acid polypeptide, the unnatural amino acid portion of the polypeptide can be first modified with a multifunctional (e.g., difunctional, trifunctional, tetrafunctional) linker molecule, which is then further modified. That is, at least one terminus of the multifunctional linker molecule reacts with at least one unnatural amino acid in the polypeptide and at least one other terminus of the multifunctional linker is available for further functionalization. If all the termini of the multifunctional linker are identical, homomultimers of the unnatural amino acid polypeptide can be formed (depending on the stoichiometric conditions). If the ends of the multifunctional linker have different chemical reactivity, at least one end of the multifunctional linker group will bind to the unnatural amino acid polypeptide and the other end can then react with different functional groups, including (by way of example only): marking; a dye; a polymer; a water-soluble polymer; derivatives of polyethylene glycol; a photocrosslinking agent; a cytotoxic compound; a drug; affinity labeling; a photoaffinity label; a reactive compound; a resin; a second protein or polypeptide analog; an antibody or antibody fragment; a metal chelator; a cofactor; a fatty acid; a carbohydrate; a polynucleotide; DNA; RNA; an antisense polynucleotide; saccharides, water-soluble dendrimers, cyclodextrins, biomaterials; a nanoparticle; spin labeling; a fluorophore-containing moiety; a radioactive portion; novel functional groups; groups that interact covalently or non-covalently with other molecules; a light-shielding portion; an actinic radiation excitable moiety; a ligand; a photoisomerizable moiety; biotin; biotin analogues; a moiety incorporating a heavy atom; a chemically cleavable group; a photocleavable group; an extended side chain; a carbon-bonded sugar; a redox active agent; an aminothiopropionic acid; a toxic moiety; isotopically labeled moieties; a biophysical probe; phosphorescent groups; a chemiluminescent group; an electron dense group; a magnetic group; an insertion group; a chromophore; an energy transfer agent; a bioactive agent; a detectable label; a small molecule; inhibitory ribonucleic acid, radionucleotide; a neutron capture agent; derivatives of biotin; a quantum dot; nano transitorin; a radiotransferrin; abzymes, activating complex activators, viruses, adjuvants, aglycones, allergens, angiostatin, anti-hormones, antioxidants, aptamers, guide RNAs, saponins, shuttle vectors, macromolecules, pseudoepitopes, receptors, reverse micelles, and any combination thereof.
The multifunctional linker group has the following general structure:
Figure BDA0001546710350001931
wherein:
x are each independently NH 2 、-C(＝O)R 9 -SR' or-J-R, wherein R 9 Is H OR OR', wherein J is
Figure BDA0001546710350001932
R is H, alkyl, substituted alkyl, cycloalkyl or substituted cycloalkyl; each R "is independently H, alkyl, substituted alkyl, or a protecting group, or when more than one R" group is present, two R "optionally form a heterocycloalkyl;
each R' is independently H, alkyl or substituted alkyl;
each L is independently selected from the group consisting of: alkylene, substituted alkylene, alkenylene, substituted alkenylene, -O- (alkylene or substituted alkylene) -, -S- (alkylene or substituted alkylene) -, -S (O) k - (wherein k is 1, 2 or 3), -S (O) k (alkylene or substituted alkylene) -, -C (O) - (alkylene or substituted alkylene) -, -C (S) - (alkylene or substituted alkylene) -, -N (R ') -, NR ' - (alkylene or substituted alkylene) -, -C (O) N (R ') -, CON (R ') - (alkylene or substituted alkylene) -, a- (alkylene or substituted alkylene) NR ' C (O) O- (alkylene or substituted alkylene) -, -O-CON (R ') - (alkylene or substituted alkylene) -, -CSN (R ') - (alkylene or substituted alkylene) -, -N (R ') CO- (alkylene or substituted alkylene) -, -N (R ') C (O) O- (alkylene or substituted alkylene) -, -S (O) k N (R ') -, -N (R') C (O) N (R ') -, -N (R') C (O) N (R ') - (alkylene or substituted alkylene) -, -N (R') C (S) N (R '), -N (R') S (O) k N(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 1 Is optional and when present is-C (R') p -NR' -C (O) O- (alkylene or substituted alkylene) -, wherein p is 0, 1 or 2; w is NH 2 、-C(＝O)R 9 -SR' or-J-R; and n is 1 to 3;
with the proviso that X and L-L 1 -W together each independently provides at least one of the following groups: (a) Hydroxylamino groups capable of reacting with carbonyl groups (including dicarbonyl groups) on a non-natural amino acid or "modified or unmodified" non-natural amino acid polypeptide; (b) Carbonyl groups (including dicarbonyl groups) capable of reacting with hydroxylamine groups on a non-natural amino acid or "modified or unmodified" non-natural amino acid polypeptide; or (c) capable of undergoing a modification with an unnatural amino acid or "modified"Or an unmodified "carbonyl group (including dicarbonyl groups) in which an oxime group on the unnatural amino acid polypeptide undergoes an exchange reaction.
Fig. 18 presents an illustrative, non-limiting example of the synthesis of a bifunctional homotype linker (wherein the linker has two identical ends, i.e., hydroxylamine groups). This linker can be used to form homodimers of carbonyl-or dicarbonyl-containing unnatural amino acid polypeptides to form two oxime linkages. Alternatively, if one end of the linker is protected, then this portion of the protected linker can be used to bind the unprotected hydroxylamine end to a carbonyl-or dicarbonyl-containing unnatural amino acid polypeptide via an oxime linkage, leaving the other protected end available for other linkage reactions after deprotection. Alternatively, careful manipulation of the stoichiometry of the reagents may provide similar results (heterodimers), although results may be obtained in which the desired heterodimer may be similarly contaminated with some homodimer.
Fig. 19 presents an illustrative, non-limiting example of two multifunctional heteroconnectors in which each linker has more than one type of terminal reactive group (i.e., hydroxylamine, oxime, and thioester groups). Using the oxime-based chemistry discussed throughout this specification, this linker can be used to form heterodimers of unnatural amino acid polypeptides.
FIG. 20 presents a schematic, non-limiting example of the use of heterobifunctional linkers to attach PEG groups to unnatural amino acid polypeptides in a multi-step synthesis. In a first step, as depicted in such illustrative figures, a carbonyl-containing unnatural amino acid polypeptide is reacted with a hydroxylamine-containing bifunctional linker to form a modified oxime-containing unnatural amino acid polypeptide. However, the bifunctional linker still retains functional groups (illustrated herein by tangible objects) that are capable of reacting with reagents of appropriate reactivity (illustrated in the figures by objects of matching shape) to form a modified oxime-containing functionalized unnatural amino acid polypeptide. In this particular illustrative scheme, the functionalization is a PEG group, but may also include any of the foregoing functional groups, or in the case of trifunctional or tetrafunctional linkers, more than one type of functional group or types of the same functional groups. Fig. 21 presents illustrative examples of four types of linker groups for linking hydroxylamine-containing unnatural amino acid polypeptides to PEG groups. As previously mentioned, PEG functionality is provided for illustrative purposes only. Thus, the linker groups described herein provide another way to further modify the unnatural amino acid polypeptide in a site-selective manner.
The methods and compositions described herein also provide polypeptide combinations, such as homodimers, heterodimers, homomultimers, or heteromultimers (i.e., trimers, tetramers, etc.). By way of example only, the following description focuses on GH supergene family members, however, the methods, techniques, and compositions described in this section are applicable to virtually any other polypeptide in dimeric and multimeric form that may provide benefits, including (by way of example only): alpha-1 antitrypsin, angiostatin, anti-hemolytic factor, antibody, apolipoprotein, apoprotein, atrial natriuretic factor, atrial natriuretic polypeptide, atrial peptide, C-X-C chemokine, T39765, NAP-2, ENA-78, gro-a, gro-b, gro-C, IP-10, GCP-2, NAP-4, SDF-1, PF4, MIG, calcitonin, C-kit ligand, cytokine, CC chemokine, monocyte chemokine-1, monocyte chemokine-2, monocyte chemokine-3, monocyte inflammatory protein-1 alpha, monocyte inflammatory protein-iβ, RANTES, 1309, R83915, R91733, HCC1, T58847, D31065, T64262, CD40 ligand, C-kit ligand, collagen, and pharmaceutical compositions containing the same Colony Stimulating Factor (CSF), complement factor 5a, complement inhibitor, complement receptor 1, cytokine, epithelial neutrophil activating peptide-78, MIP-16, MCP-1, epidermal Growth Factor (EGF), epithelial neutrophil activating peptide, erythropoietin (EPO), epidermal exfoliative toxin, factor IX, factor VII, factor VIII, factor X, fibroblast Growth Factor (FGF), fibrinogen, fibronectin, tetrahelical bundle protein, G-CSF, glp-1, GM-CSF, glucocerebrosidase, gonadotropin, growth factor receptor, grf, hedgehog protein, hemoglobin, hepatocyte growth factor (hGF), hirudin, human growth hormone (hGH), human serum albumin, ICAM-1, ICAM-1 receptor, LFA-1 receptor, insulin-like growth factor (IGF), IGF-I, IGF-II, interferon (IFN), IFN-alpha, IFN-beta, IFN-gamma, interleukin (IL), IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, keratinocyte Growth Factor (KGF), lactoferrin, leukemia inhibitory factor, luciferase, neurogenin, neutrophil Inhibitory Factor (NIF), oncostatin M, osteogenic protein, oncogene products, paracitin, parathyroid hormone, PD-ECSF, PDGF, peptide hormones, pleiotrophin, protein A, protein G, pth, pyrogenic exotoxin A, neutropy the other ingredients include, but are not limited to, heat exotoxin B, heat exotoxin C, pyy, relaxin, renin, SCF, small biosynthetic protein, soluble complement receptor I, soluble I-CAM 1, soluble interleukin receptor, soluble TNF receptor, somatostatin, growth hormone, streptokinase, superantigen, staphylococcal enterotoxin, SEA, SEB, SEC1, SEC2, SEC3, SED, SEE, steroid hormone receptor, superoxide dismutase, toxic shock syndrome toxin, thymosin alpha 1, tissue type plasminogen activator, tumor Growth Factor (TGF), tumor necrosis factor alpha, tumor necrosis factor beta, tumor Necrosis Factor Receptor (TNFR), VLA-4 protein, VCAM-1 protein, vascular Endothelial Growth Factor (VEGF), urokinase, mos, ras, raf, met, p, tat, fos, myc, jun, myb, rel, estrogen receptor, progesterone receptor, testosterone receptor, aldosterone receptor, LDL receptor, and corticosterone.
Thus, GH supergene family member polypeptides comprising one or more unnatural amino acids, either directly bound to a polypeptide backbone or via a linker to another GH supergene family member or variant thereof or to any other polypeptide of a non-GH supergene family member, or variant thereof, are encompassed in the methods, techniques and compositions described herein. Because of its increased molecular weight as compared to the monomer, the GH supergene family member dimer or multimer conjugate may exhibit novel or desirable properties, including but not limited to, different pharmacology, pharmacokinetics, pharmacodynamics, modulated therapeutic half-life, or modulated plasma half-life relative to the monomeric GH supergene family member. In some embodiments, a GH supergene family member dimer described herein modulates dimerization of a GH supergene family member receptor. In other embodiments, a GH supergene family member dimer or multimer described herein will act as a GH supergene family member receptor antagonist, agonist, or modulator.
In some embodiments, the GH supergene family member polypeptide is directly linked, including, but not limited to, via an Asn-Lys amide linkage or a Cys-Cys disulfide linkage. In some embodiments, the linked GH supergene family member polypeptide and/or linked non-GH supergene family member will comprise a different unnatural amino acid that promotes dimerization, including, but not limited to, a first GH supergene family member, and/or a linked non-GH supergene family member, comprising a ketonic unnatural amino acid-containing polypeptide joined to a second GH supergene family member polypeptide comprising a hydroxylamine-containing unnatural amino acid and which reacts by forming the corresponding oxime.
Alternatively, two GH supergene family member polypeptides and/or linked non-GH supergene family members are linked by a linker. Any heterobifunctional linker or homobifunctional linker may be used to join two GH supergene family members, and/or linked non-GH supergene family members, polypeptides, which may have the same or different order sequences. In some cases, the linker used to bind the GH supergene family members, and/or the linked non-GH supergene family members, polypeptides together may be a bifunctional PEG reagent.
In some embodiments, the methods and compositions described herein provide water-soluble bifunctional linkers having a dumbbell structure comprising: a) An azide, alkyne, hydrazine, hydrazide, hydroxylamine or carbonyl or dicarbonyl containing moiety on at least a first end of the polymer backbone; and b) at least one second functional group on a second end of the polymer backbone. The second functional group may be the same as or different from the first functional group. In some embodiments, the second functional group is not reactive with the first functional group. In some embodiments, the methods and compositions described herein provide water-soluble compounds that include an arm of at least one branched molecular structure. For example, the branched molecular structure may be dendritic.
In some embodiments, the methods and compositions described herein provide multimers comprising one or more GH supergene family members formed by reaction with a water-soluble, activated polymer having the structure:
R-(CH 2 CH 2 O) n -O-(CH 2 ) m -X
wherein n is about 5 to 3,000, m is 2-10, X may be azide, alkyne, hydrazine, hydrazide, aminooxy, hydroxylamine, acetyl, or a moiety containing a carbonyl or dicarbonyl, and R is a capping group, a functional group, or a leaving group that may be the same as or different from X. R may be, for example, a functional group selected from the group consisting of: hydroxy, protected hydroxy, alkoxy, N-hydroxysuccinimidyl ester, 1-benzotriazole ester, N-hydroxysuccinimidyl carbonate, 1-benzotriazole ester, acetal, aldehyde, hydrated aldehyde, alkenyl, acrylate, methacrylate, acrylamide, reactive sulfone, amine, aminooxy, protected amine, hydrazide, protected thiol, carboxylic acid, protected carboxylic acid, isocyanate, isothiocyanate, maleimide, vinyl sulfone, dithiopyridine, vinyl pyridine, iodoacetamide, epoxide, dialdehyde, diketone, mesylate, tosylate, and trifluoroethyl sulfonate, alkene and ketone.
FIG. 22 presents an illustrative, non-limiting example of the formation of homodimers of non-natural amino acid polypeptides described herein using linker groups described herein. In this illustrative example, a carbonyl-containing unnatural amino acid polypeptide is reacted with a difunctional linker group having two available hydroxylamine groups under conditions suitable for forming a linked homodimeric oxime-containing unnatural amino acid polypeptide. The methods presented in the drawings are not limited to carbonyl-containing unnatural amino acid polypeptides coupled with hydroxylamine-containing bifunctional linkers. The unnatural amino acid polypeptide can further comprise an oxime group capable of undergoing an exchange reaction with the carbonyl-containing multifunctional linker group to form a homomultimer linked by a structure comprising a plurality of oxime groups, or the unnatural amino acid polypeptide can further comprise a hydroxylamine group capable of undergoing a reaction with the carbonyl-containing multifunctional linker group to form a homomultimer linked by a structure comprising a plurality of oxime groups. Of course, the homomultimer may be a homodimer, homotrimer or homotetramer.
FIG. 23 presents an illustrative, non-limiting example of the formation of a heterodimer of polypeptides using a heterofunctional linker group, wherein at least one member of the heterodimer is a non-natural amino acid polypeptide described herein and the other members are optionally non-natural amino acid polypeptides, other types of non-natural amino acid polypeptides, or naturally occurring amino acid polypeptides as described herein. In the example presented in this figure, the linker group contains two identical hydroxylamine groups and the main product of the reaction of the linker with the carbonyl-containing unnatural amino acid polypeptide is a modified oxime-containing unnatural amino acid polypeptide linked to a linker with an available hydroxylamine group by controlling the stoichiometry, temperature and other parameters of the reaction. The latter group may be further reacted with another carbonyl-or dicarbonyl-containing unnatural amino acid polypeptide to form a bifunctional heterodimer of the oxime-containing unnatural amino acid polypeptide. Of course, the functional groups on the linker need not be identical, and they need not be hydroxylamine groups. Using the chemistry detailed throughout this specification, one of skill in the art can design a linker in which at least one functional group can form an oxime group with a non-natural amino acid polypeptide; other functional groups on the linker may utilize other known chemistries, including nucleophile/electrophile based chemistries well known in the art of organic chemistry.
H. Examples of adding functional groups: isolation Properties of facilitation Polypeptides
Naturally occurring or unnatural amino acid polypeptides can be difficult to isolate from a sample for a variety of reasons, including but not limited to, the solubility or binding characteristics of the polypeptide. For example, in the preparation of a polypeptide for therapeutic use, it is possible to isolate the polypeptide from recombinant systems that have been engineered to overproduce the polypeptide. However, achieving the desired degree of purity often proves difficult due to the solubility or binding characteristics of the polypeptide. The methods, compositions, techniques and strategies described herein provide solutions to this situation.
Using the methods, compositions, techniques, and strategies described herein, one of skill in the art can prepare oxime-containing unnatural amino acid polypeptides that are homologous to a desired polypeptide, where the oxime-containing unnatural amino acid polypeptides have improved separation characteristics. In one embodiment, the homologous unnatural amino acid polypeptide is made by biosynthesis. In another or additional embodiment, the unnatural amino acid has incorporated its structure into one of the unnatural amino acids described herein. In another or additional embodiment, the unnatural amino acid is incorporated at a terminal or intermediate position and further is site-specifically incorporated.
In one embodiment, the resulting unnatural amino acid, as prepared by biosynthesis, already has the desired improved separation characteristics. In other or additional embodiments, the unnatural amino acid includes an oxime bond with a group that provides improved separation characteristics. In other or additional embodiments, the unnatural amino acid is further modified to form a modified oxime-containing unnatural amino acid polypeptide, where the modification provides an oxime bond with a group that provides improved separation characteristics. In some embodiments, this group is directly bonded to the unnatural amino acid, and in other embodiments, this group is bonded to the unnatural amino acid through a linker group. In certain embodiments, this group is attached to the unnatural amino acid by a single chemical reaction, and in other embodiments, a series of chemical reactions are required to attach this group to the unnatural amino acid. Preferably, the group that imparts improved separation characteristics is site-specifically bound to an unnatural amino acid in an unnatural amino acid polypeptide under the reaction conditions utilized, and is not bound to a naturally occurring amino acid.
In other or additional embodiments, the resulting unnatural amino acid polypeptide is homologous to GH supergene family members, however, the methods, techniques, and compositions described in this section can be applied to virtually any other polypeptide that would benefit from improved isolation characteristics, including (by way of example only): alpha-1 antitrypsin, angiostatin, anti-hemolytic factor, antibody, apolipoprotein, apoprotein, atrial natriuretic factor, atrial natriuretic polypeptide, atrial peptide, C-X-C chemokine, T39765, NAP-2, ENA-78, gro-a, gro-b, gro-C, IP-10, GCP-2, NAP-4, SDF-1, PF4, MIG, calcitonin, C-kit ligand, cytokine, CC chemokine, monocyte chemokine-1, monocyte chemokine-2, monocyte chemokine-3, monocyte inflammatory protein-1 alpha, monocyte inflammatory protein-iβ, RANTES, 1309, R83915, R91733, HCC1, T58847, D31065, T64262, CD40 ligand, C-kit ligand, collagen, and pharmaceutical compositions containing the same Colony Stimulating Factor (CSF), complement factor 5a, complement inhibitor, complement receptor 1, cytokine, epithelial neutrophil activating peptide-78, MIP-16, MCP-1, epidermal Growth Factor (EGF), epithelial neutrophil activating peptide, erythropoietin (EPO), epidermal exfoliative toxin, factor IX, factor VII, factor VIII, factor X, fibroblast Growth Factor (FGF), fibrinogen, fibronectin, tetrahelical bundle protein, G-CSF, glp-1, GM-CSF, glucocerebrosidase, gonadotropin, growth factor receptor, grf, hedgehog protein, hemoglobin, hepatocyte growth factor (hGF), hirudin, human growth hormone (hGH), human serum albumin, ICAM-1, ICAM-1 receptor, LFA-1 receptor, insulin-like growth factor (IGF), IGF-I, IGF-II, interferon (IFN), IFN-alpha, IFN-beta, IFN-gamma, interleukin (IL), IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, keratinocyte Growth Factor (KGF), lactoferrin, leukemia inhibitory factor, luciferase, neurogenin, neutrophil Inhibitory Factor (NIF), oncostatin M, osteogenic protein, oncogene products, paracitin, parathyroid hormone, PD-ECSF, PDGF, peptide hormones, pleiotrophin, protein A, protein G, pth, pyrogenic exotoxin A, neutropy the other ingredients include, but are not limited to, heat exotoxin B, heat exotoxin C, pyy, relaxin, renin, SCF, small biosynthetic protein, soluble complement receptor I, soluble I-CAM 1, soluble interleukin receptor, soluble TNF receptor, somatostatin, growth hormone, streptokinase, superantigen, staphylococcal enterotoxin, SEA, SEB, SEC1, SEC2, SEC3, SED, SEE, steroid hormone receptor, superoxide dismutase, toxic shock syndrome toxin, thymosin alpha 1, tissue type plasminogen activator, tumor Growth Factor (TGF), tumor necrosis factor alpha, tumor necrosis factor beta, tumor Necrosis Factor Receptor (TNFR), VLA-4 protein, VCAM-1 protein, vascular Endothelial Growth Factor (VEGF), urokinase, mos, ras, raf, met, p, tat, fos, myc, jun, myb, rel, estrogen receptor, progesterone receptor, testosterone receptor, aldosterone receptor, LDL receptor, and corticosterone.
In other or additional embodiments, the group that imparts improved separation characteristics improves the water solubility of the polypeptide; in other embodiments, the group improves the binding properties of the polypeptide; in other embodiments, the groups provide new binding properties to the polypeptide (including, by way of example only, biotin groups or biotin binding groups). In embodiments in which the groups improve the water solubility of the polypeptide, the groups are selected from the water-soluble polymers described herein, including, by way of example only, any of the PEG polymer groups described herein.
I. Examples of adding functional groups: detecting the presence of a polypeptide
Naturally occurring or unnatural amino acid polypeptides can be difficult to detect in samples (including in vivo samples and in vitro samples) for a variety of reasons, including, but not limited to, the lack of reagents or labels that can readily bind to the polypeptide. The methods, compositions, techniques and strategies described herein provide solutions to this situation.
Using the methods, compositions, techniques, and strategies described herein, one of skill in the art can prepare oxime-containing unnatural amino acid polypeptides that are homologous to a desired polypeptide, where the oxime-containing unnatural amino acid polypeptides allow for the detection of polypeptides in vivo and in vitro samples. In one embodiment, the homologous unnatural amino acid polypeptide is prepared by biosynthesis. In another or additional embodiment, the unnatural amino acid has incorporated its structure into one of the unnatural amino acids described herein. In another or additional embodiment, the unnatural amino acid is incorporated at a terminal or intermediate position and further is site-specifically incorporated.
In one embodiment, the resulting unnatural amino acid polypeptide, as prepared by biosynthesis, already has the desired detection characteristics. In other or additional embodiments, the unnatural amino acid polypeptide comprises at least one unnatural amino acid selected from the group consisting of an oxime-containing unnatural amino acid, a carbonyl-containing unnatural amino acid, and a hydroxylamine-containing unnatural amino acid. In other embodiments, these unnatural amino acids have been biosynthetically incorporated into polypeptides as described herein. In other or alternative embodiments, the unnatural amino acid polypeptide comprises at least one unnatural amino acid selected from the group consisting of amino acids of formulas I-XVIII, XXX-XXXIV (A and B), or XXXX-XXXXIII. In other or additional embodiments, the unnatural amino acid includes an oxime bond with a group that provides improved detection characteristics. In other or additional embodiments, the unnatural amino acid is further modified to form a modified oxime-containing unnatural amino acid polypeptide, where the modification provides an oxime bond with a group that provides improved detection characteristics. In some embodiments, this group is directly bonded to the unnatural amino acid, and in other embodiments, this group is bonded to the unnatural amino acid through a linker group. In certain embodiments, this group is attached to the unnatural amino acid by a single chemical reaction, and in other embodiments, a series of chemical reactions are required to attach this group to the unnatural amino acid. Preferably, the group that confers improved detection characteristics is site-specifically bound to an unnatural amino acid in an unnatural amino acid polypeptide under the reaction conditions utilized, and is not bound to a naturally occurring amino acid.
In other or additional embodiments, the resulting unnatural amino acid polypeptide is homologous to a GH supergene family member, however, the methods, techniques, and compositions described in this section can be applied to virtually any other polypeptide that needs to be detected in vivo samples and in vitro samples, including (by way of example only): alpha-1 antitrypsin, angiostatin, anti-hemolytic factor, antibody, apolipoprotein, apoprotein, atrial natriuretic factor, atrial natriuretic polypeptide, atrial peptide, C-X-C chemokine, T39765, NAP-2, ENA-78, gro-a, gro-b, gro-C, IP-10, GCP-2, NAP-4, SDF-1, PF4, MIG, calcitonin, C-kit ligand, cytokine, CC chemokine, monocyte chemokine-1, monocyte chemokine-2, monocyte chemokine-3, monocyte inflammatory protein-1 alpha, monocyte inflammatory protein-iβ, RANTES, 1309, R83915, R91733, HCC1, T58847, D31065, T64262, CD40 ligand, C-kit ligand, collagen, and pharmaceutical compositions containing the same Colony Stimulating Factor (CSF), complement factor 5a, complement inhibitor, complement receptor 1, cytokine, epithelial neutrophil activating peptide-78, MIP-16, MCP-1, epidermal Growth Factor (EGF), epithelial neutrophil activating peptide, erythropoietin (EPO), epidermal exfoliative toxin, factor IX, factor VII, factor VIII, factor X, fibroblast Growth Factor (FGF), fibrinogen, fibronectin, tetrahelical bundle protein, G-CSF, glp-1, GM-CSF, glucocerebrosidase, gonadotropin, growth factor receptor, grf, hedgehog protein, hemoglobin, hepatocyte growth factor (hGF), hirudin, human growth hormone (hGH), human serum albumin, ICAM-1, ICAM-1 receptor, LFA-1 receptor, insulin-like growth factor (IGF), IGF-I, IGF-II, interferon (IFN), IFN-alpha, IFN-beta, IFN-gamma, interleukin (IL), IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, keratinocyte Growth Factor (KGF), lactoferrin, leukemia inhibitory factor, luciferase, neurogenin, neutrophil Inhibitory Factor (NIF), oncostatin M, osteogenic protein, oncogene products, paracitin, parathyroid hormone, PD-ECSF, PDGF, peptide hormones, pleiotrophin, protein A, protein G, pth, pyrogenic exotoxin A, neutropy the other ingredients include, but are not limited to, heat exotoxin B, heat exotoxin C, pyy, relaxin, renin, SCF, small biosynthetic protein, soluble complement receptor I, soluble I-CAM 1, soluble interleukin receptor, soluble TNF receptor, somatostatin, growth hormone, streptokinase, superantigen, staphylococcal enterotoxin, SEA, SEB, SEC1, SEC2, SEC3, SED, SEE, steroid hormone receptor, superoxide dismutase, toxic shock syndrome toxin, thymosin alpha 1, tissue type plasminogen activator, tumor Growth Factor (TGF), tumor necrosis factor alpha, tumor necrosis factor beta, tumor Necrosis Factor Receptor (TNFR), VLA-4 protein, VCAM-1 protein, vascular Endothelial Growth Factor (VEGF), urokinase, mos, ras, raf, met, p, tat, fos, myc, jun, myb, rel, estrogen receptor, progesterone receptor, testosterone receptor, aldosterone receptor, LDL receptor, and corticosterone.
In other or additional embodiments, the group imparting improved detection characteristics is selected from the group consisting of: marking; a dye; affinity labeling; a photoaffinity label; spin labeling; a fluorophore; a radioactive portion; a moiety incorporating a heavy atom; isotopically labeled moieties; a biophysical probe; phosphorescent groups; a chemiluminescent group; an electron dense group; a magnetic group; a chromophore; an energy transfer agent; a detectable label, and any combination thereof.
J. Examples of adding functional groups: improving therapeutic properties of polypeptides
Naturally occurring or unnatural amino acid polypeptides should be able to provide a certain therapeutic benefit to a patient suffering from a particular disorder, disease or condition. This therapeutic benefit should depend on a variety of factors, including (by way of example only): the safety profile of the polypeptide, and the pharmacokinetics, pharmacology, and/or pharmacodynamics (e.g., water solubility, bioavailability, serum half-life, therapeutic half-life, immunogenicity, bioactivity, or circulation time) of the polypeptide. In addition, it may be advantageous to provide other functional groups to the polypeptide, such as linked cytotoxic compounds or drugs, or may require linking with other polypeptides to form homomultimers as well as heteromultimers described herein. Preferably, these modifications do not disrupt the activity and/or tertiary structure of the original polypeptide. The methods, compositions, techniques and strategies described herein provide solutions to these problems.
Using the methods, compositions, techniques, and strategies described herein, one of skill in the art can prepare oxime-containing unnatural amino acid polypeptides that are homologous to a desired polypeptide, where the oxime-containing unnatural amino acid polypeptides have improved therapeutic characteristics. In one embodiment, the homologous unnatural amino acid polypeptide is prepared by biosynthesis. In another or additional embodiment, the unnatural amino acid has incorporated its structure into one of the unnatural amino acids described herein. In another or additional embodiment, the unnatural amino acid is incorporated at a terminal or intermediate position and further is site-specifically incorporated.
In one embodiment, the resulting unnatural amino acid, as prepared by biosynthesis, already has the desired improved therapeutic profile. In other or additional embodiments, the unnatural amino acid includes an oxime bond with a group that provides improved therapeutic characteristics. In other or additional embodiments, the unnatural amino acid is further modified to form a modified oxime-containing unnatural amino acid polypeptide, where the modification provides an oxime bond with a group that provides improved therapeutic characteristics. In some embodiments, this group is directly bonded to the unnatural amino acid, and in other embodiments, this group is bonded to the unnatural amino acid through a linker group. In certain embodiments, this group is attached to the unnatural amino acid by a single chemical reaction, and in other embodiments, a series of chemical reactions are required to attach this group to the unnatural amino acid. Preferably, the group that imparts improved separation characteristics is site-specifically bound to an unnatural amino acid in an unnatural amino acid polypeptide under the reaction conditions utilized, and is not bound to a naturally occurring amino acid.
In other or additional embodiments, the resulting unnatural amino acid polypeptide is homologous to GH supergene family members, however, the methods, techniques, and compositions described in this section can be applied to virtually any other polypeptide that would benefit from improved therapeutic characteristics, including (by way of example only): alpha-1 antitrypsin, angiostatin, anti-hemolytic factor, antibody, apolipoprotein, apoprotein, atrial natriuretic factor, atrial natriuretic polypeptide, atrial peptide, C-X-C chemokine, T39765, NAP-2, ENA-78, gro-a, gro-b, gro-C, IP-10, GCP-2, NAP-4, SDF-1, PF4, MIG, calcitonin, C-kit ligand, cytokine, CC chemokine, monocyte chemokine-1, monocyte chemokine-2, monocyte chemokine-3, monocyte inflammatory protein-1 alpha, monocyte inflammatory protein-iβ, RANTES, 1309, R83915, R91733, HCC1, T58847, D31065, T64262, CD40 ligand, C-kit ligand, collagen, and pharmaceutical compositions containing the same Colony Stimulating Factor (CSF), complement factor 5a, complement inhibitor, complement receptor 1, cytokine, epithelial neutrophil activating peptide-78, MIP-16, MCP-1, epidermal Growth Factor (EGF), epithelial neutrophil activating peptide, erythropoietin (EPO), epidermal exfoliative toxin, factor IX, factor VII, factor VIII, factor X, fibroblast Growth Factor (FGF), fibrinogen, fibronectin, tetrahelical bundle protein, G-CSF, glp-1, GM-CSF, glucocerebrosidase, gonadotropin, growth factor receptor, grf, hedgehog protein, hemoglobin, hepatocyte growth factor (hGF), hirudin, human growth hormone (hGH), human serum albumin, ICAM-1, ICAM-1 receptor, LFA-1 receptor, insulin-like growth factor (IGF), IGF-I, IGF-II, interferon (IFN), IFN-alpha, IFN-beta, IFN-gamma, interleukin (IL), IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, keratinocyte Growth Factor (KGF), lactoferrin, leukemia inhibitory factor, luciferase, neurogenin, neutrophil Inhibitory Factor (NIF), oncostatin M, osteogenic protein, oncogene products, paracitin, parathyroid hormone, PD-ECSF, PDGF, peptide hormones, pleiotrophin, protein A, protein G, pth, pyrogenic exotoxin A, neutropy the other ingredients include, but are not limited to, heat exotoxin B, heat exotoxin C, pyy, relaxin, renin, SCF, small biosynthetic protein, soluble complement receptor I, soluble I-CAM 1, soluble interleukin receptor, soluble TNF receptor, somatostatin, growth hormone, streptokinase, superantigen, staphylococcal enterotoxin, SEA, SEB, SEC1, SEC2, SEC3, SED, SEE, steroid hormone receptor, superoxide dismutase, toxic shock syndrome toxin, thymosin alpha 1, tissue type plasminogen activator, tumor Growth Factor (TGF), tumor necrosis factor alpha, tumor necrosis factor beta, tumor Necrosis Factor Receptor (TNFR), VLA-4 protein, VCAM-1 protein, vascular Endothelial Growth Factor (VEGF), urokinase, mos, ras, raf, met, p, tat, fos, myc, jun, myb, rel, estrogen receptor, progesterone receptor, testosterone receptor, aldosterone receptor, LDL receptor, and corticosterone.
In other or additional embodiments, the group that imparts improved therapeutic characteristics improves the water solubility of the polypeptide; in other embodiments, the group improves the binding properties of the polypeptide; in other embodiments, the groups provide new binding properties to the polypeptide (including, by way of example only, biotin groups or biotin binding groups). In embodiments in which the groups improve the water solubility of the polypeptide, the groups are selected from the water-soluble polymers described herein, which include, by way of example only, PEG polymer groups. In other or additional embodiments, the moiety is a cytotoxic compound, while in other embodiments, the moiety is a drug. In other embodiments, the linked drug or cytotoxic compound can be cleaved from the unnatural amino acid polypeptide to deliver the drug or cytotoxic compound to the desired treatment site. In other embodiments, the group is a second polypeptide comprising, for example, an oxime-containing unnatural amino acid polypeptide, which further comprises, for example, a polypeptide having the same amino acid structure as the first unnatural amino acid polypeptide.
In other or additional embodiments, the oxime-containing unnatural amino acid polypeptide is a modified oxime-containing unnatural amino acid polypeptide. In other or additional embodiments, the oxime-containing non-natural amino acid polypeptide increases the bioavailability of the polypeptide relative to a homologous naturally occurring amino acid polypeptide. In other or additional embodiments, the oxime-containing non-natural amino acid polypeptide increases the safety profile of the polypeptide relative to a homologous naturally occurring amino acid polypeptide. In other or additional embodiments, the oxime-containing non-natural amino acid polypeptide increases the water solubility of the polypeptide relative to a homologous naturally occurring amino acid polypeptide. In other or additional embodiments, the oxime-containing non-natural amino acid polypeptide increases the therapeutic half-life of the polypeptide relative to a homologous naturally occurring amino acid polypeptide. In other or additional embodiments, the oxime-containing non-natural amino acid polypeptide increases the serum half-life of the polypeptide relative to a homologous naturally occurring amino acid polypeptide. In other or additional embodiments, the oxime-containing non-natural amino acid polypeptide extends the circulation time of the polypeptide relative to a homologous naturally occurring amino acid polypeptide. In other or additional embodiments, the oxime-containing non-natural amino acid polypeptide modulates the activity of the polypeptide relative to a homologous naturally occurring amino acid polypeptide. In other or additional embodiments, the oxime-containing non-natural amino acid polypeptide modulates the immunogenicity of the polypeptide relative to a homologous naturally-occurring amino acid polypeptide.
XI therapeutic uses of modified polypeptides
For convenience, the "modified or unmodified" non-native polypeptides described in this section are generally and/or described in specific examples. However, the "modified or unmodified" non-native polypeptides described in this section should not be limited to only the general descriptions or specific examples provided in this section, but the "modified or unmodified" non-native polypeptides described in this section are equally well applicable to all "modified or unmodified" non-native polypeptides comprising at least one amino acid within the scope of formulas I-XVIII, XXX-XXXIV (a and B) and XXXX-xxxiii, including any sub-or specific compounds within the scope of formulas I-XVIII, XXX-XXXIV (a and B) and XXXX-xxxiii described in the specification, claims and drawings herein.
The "modified or unmodified" non-natural amino acid polypeptides described herein (including homomultimers and heteromultimers thereof) find a variety of uses, including (but not limited to): therapeutic use, diagnostic use, assay-based use, industrial use, cosmetic use, plant biological use, environmental use, energy production use, and/or military use. By way of non-limiting illustration, the following therapeutic uses of "modified or unmodified" non-natural amino acid polypeptides are provided.
The "modified or unmodified" unnatural amino acid polypeptides described herein are useful for treating a variety of disorders, conditions, or diseases. Administration of the "modified or unmodified" non-natural amino acid polypeptide products described herein results in any activity in humans that is demonstrated by commercial polypeptide formulations. The average amount of "modified or unmodified" unnatural amino acid polypeptide product can vary and should be varied, particularly according to the recommendations and prescriptions of the qualified physician. The exact amount of the "modified or unmodified" unnatural amino acid polypeptide should be preferentially selected based on the following factors: such as the exact type of condition being treated, the condition of the patient being treated, and other ingredients in the composition. The amount to be administered can be readily determined by one of skill in the art based on the therapy using the "modified or unmodified" unnatural amino acid polypeptide.
A. Administration and pharmaceutical compositions
The "modified or unmodified" non-natural amino acid polypeptides described herein (including homomultimers and heteromultimers thereof) find a variety of uses, including (but not limited to): therapeutic use, diagnostic use, assay-based use, industrial use, cosmetic use, plant biological use, environmental use, energy production use, and/or military use. By way of non-limiting illustration, the following therapeutic uses of "modified or unmodified" non-natural amino acid polypeptides are provided.
The "modified or unmodified" unnatural amino acid polypeptides described herein are useful for treating a variety of disorders. Administration of the "modified or unmodified" non-natural amino acid polypeptide products described herein results in any activity in humans that is demonstrated by commercial polypeptide formulations. The average amount of "modified or unmodified" unnatural amino acid polypeptide product can vary and should be varied, particularly according to the recommendations and prescriptions of the qualified physician. The exact amount of the "modified or unmodified" unnatural amino acid polypeptide should be preferentially selected based on the following factors: the exact type of condition being treated, the condition of the patient being treated, and other ingredients in the composition. The amount to be administered can be readily determined by one of skill in the art based on the therapy using the "modified or unmodified" unnatural amino acid polypeptide.
Modified or unmodified non-natural amino acid polypeptides as described herein, including but not limited to synthetases, proteins comprising one or more non-natural amino acids, and the like, optionally for therapeutic use, including but not limited to in combination with a suitable pharmaceutical carrier. These compositions, for example, comprise a therapeutically effective amount of a modified or unmodified non-natural amino acid polypeptide as described herein, and a pharmaceutically acceptable carrier or excipient. The carrier or excipient includes, but is not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and/or combinations thereof. The formulation is adapted to the mode of administration. In general, methods of administering proteins are well known in the art and may be applied to the administration of modified or unmodified unnatural amino acid polypeptides as described herein.
Therapeutic compositions comprising one or more modified or unmodified unnatural amino acid polypeptides as described herein are optionally tested in one or more appropriate in vitro and/or in vivo animal models of disease to determine efficacy, tissue metabolism, and estimated dosages, according to methods well known in the art. In particular, dose initiation may be determined by the activity, stability, or other suitable measure of the unnatural amino acid relative to a natural amino acid homolog, including, but not limited to, comparing a polypeptide modified to include one or more unnatural amino acids to a natural amino acid polypeptide (i.e., in a related assay).
Administration is by any of the routes typically used to introduce molecules for eventual contact with blood or tissue cells. The modified or unmodified unnatural amino acid polypeptides as described herein are optionally administered in any suitable manner, together with one or more pharmaceutically acceptable carriers. Suitable methods of administering a modified or unmodified non-natural amino acid polypeptide as described herein to a patient are available, and while more than one route may be used to administer a particular composition, a particular route may generally provide a more direct and more effective effect or response than another route.
The pharmaceutically acceptable carrier is determined in part by the particular composition being administered and by the particular method used to administer the composition. Thus, there are a variety of suitable formulations for the pharmaceutical compositions described herein.
The unnatural amino acid polypeptides and compositions comprising these polypeptides described herein can be administered by any conventional route suitable for proteins or peptides, including but not limited to parenteral administration, e.g., injection, including but not limited to subcutaneous injection or intravenous injection or any other form of injection or infusion. Polypeptide pharmaceutical compositions (comprising the various unnatural amino acid polypeptides described herein) can be administered by a wide variety of routes, including, but not limited to, oral, intravenous, intraperitoneal, intramuscular, transdermal, subcutaneous, topical, sublingual, or rectal means. Compositions comprising modified or unmodified unnatural amino acid polypeptides as described herein can also be administered via liposomes. Such routes of administration and appropriate formulations are generally known to those skilled in the art. The unnatural amino acid polypeptides described herein can also be used alone or in combination with other suitable components, including but not limited to pharmaceutical carriers.
Modified or unmodified non-natural amino acid polypeptides as described herein, alone or in combination with other suitable components, can also be formulated into aerosol formulations that can be administered by inhalation (i.e., they can be "nebulized"). The aerosol formulation may be placed into a pressurized acceptable propellant such as dichlorodifluoromethane, propane, nitrogen, and the like.
Formulations suitable for parenteral administration (such as by intra-articular (intra-articular), intravenous, intramuscular, intradermal, intraperitoneal, and subcutaneous routes) include aqueous and non-aqueous isotonic sterile injection solutions, which may contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may contain suspending agents, solubilising agents, thickening agents, stabilisers and preservatives. The formulation of packaged nucleic acids may be present in unit-dose or multi-dose sealed containers, such as ampules and vials.
Parenteral and intravenous administration are preferred methods of administration. In particular, routes of administration for natural amino acid homolog therapeutics, including, but not limited to, those typically used for EPO, IFN, GH, G-CSF, GM-CSF, IFN, interleukins, antibodies and/or any other medical delivery protein, and formulations currently in use provide preferred routes of administration and formulations for modified or unmodified non-natural amino acid polypeptides as described herein.
In the case of the compositions and methods described herein, the dose administered to the patient is sufficient to produce a beneficial therapeutic response in the patient over time. The dosage is determined by the efficacy of the particular formulation and the activity, stability or serum half-life of the modified or unmodified unnatural amino acid polypeptide used and the patient's condition, as well as the weight or surface area of the patient to be treated. The size of the dose is also determined by the presence, nature, and extent of any adverse side effects associated with administration of a particular formulation or analog thereof in a particular patient.
In determining an effective amount of a formulation to be administered in the treatment or prevention of a disease, including but not limited to cancer, genetic disease, diabetes, AIDS, or the like, a physician evaluates circulating plasma levels, formulation toxicity, progression of the disease, and/or, if relevant, production of anti-unnatural amino acid polypeptide antibodies.
The dose administered to a patient of, for example, 70 kg is typically in the range equivalent to the dose of currently used therapeutic protein suitable for the altered activity or serum half-life of the relevant composition. The pharmaceutical formulations described herein may be supplemented with any known conventional therapies, including administration of antibodies, vaccines, cytotoxic agents, natural amino acid polypeptides, nucleic acids, nucleotide analogs, biological response modifiers, and the like.
For administration, the pharmaceutical formulations described herein are administered at a rate determined by the LD-50 or ED-50 of the relevant formulation and/or by observation of any side effects on the modified or unmodified unnatural amino acid polypeptide at various concentrations, including, but not limited to, when applied to the quality and general health of a patient. Administration may be achieved by single administration or by divided administration.
If a patient undergoing a formula infusion develops fever, coldness or muscle pain, he/she is given an appropriate dose of aspirin (aspirin), ibuprofen (ibuprofen), acetaminophen (acetaminophen) or other pain/fever controlling drugs. Patients experiencing infusion reactions such as fever, muscle pain, and coldness are given 30 minutes ahead of time prior to future infusions with aspirin, acetaminophen, or diphenhydramine (including, but not limited to). For more severe coldness and muscle pain, which do not react rapidly to antipyretics and antihistamines, meperidine (Meperidine) is used. Cell transfusion is slowed or stopped depending on the severity of the reaction.
The modified or unmodified unnatural amino acid polypeptides as described herein can be administered directly to a mammalian subject. Administration is by any of the routes commonly used to introduce polypeptides into subjects. Modified or unmodified non-natural amino acid polypeptides as described herein include those suitable for oral, rectal, topical, inhalation (including but not limited to by aerosols), buccal (including but not limited to sublingual), vaginal, parenteral (including but not limited to subcutaneous, intramuscular, intradermal, intra-articular, intrapleural, intraperitoneal, intracerebral, intraarterial or intravenous), topical (i.e., skin and mucosal surfaces, including tracheal surfaces), and transdermal administration, although the most suitable route in any given case will depend on the nature and severity of the condition being treated. Administration may be local or systemic. The formulations may be presented in unit-dose or multi-dose sealed containers, such as ampules and vials. The modified or unmodified unnatural amino acid polypeptides as described herein can be prepared in a mixture with a pharmaceutically acceptable carrier in unit dose injectable form, including but not limited to solutions, suspensions or emulsions. The modified or unmodified unnatural amino acid polypeptides as described herein can also be administered by continuous infusion (using, but not limited to, micro-vacuum pumps such as osmotic pumps), single bolus or slow release depot formulations.
Formulations suitable for administration include aqueous and non-aqueous solutions, isotonic sterile injection solutions, which may contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic; and aqueous and non-aqueous sterile suspensions which may contain suspending agents, solubilising agents, thickening agents, stabilisers and preservatives. Solutions and suspensions may be prepared from sterile powders, granules, and tablets of the kind previously described.
Lyophilization is a commonly used technique for presenting proteins that is used to remove water from protein formulations of interest. Freeze-drying (Freeze-drying) or lyophilization (lyophilization) is a process in which the material to be dried is first frozen and then the ice or frozen solvent is removed by sublimation in a vacuum environment. Excipients may be included in the pre-lyophilization formulation to increase stability during lyophilization and/or to improve stability of the lyophilized product upon storage. Pikal, M.Biopharm.3 (9) 26-30 (1990) and Arakawa et al, pharm.Res.8 (3): 285-291 (1991).
Spray drying of drugs is also known to those skilled in the art. See, for example, broadhead, J.et al, "The Spray Drying of Pharmaceuticals," Drug Dev. Ind. Pharm,18 (11 and 12), 1169-1206 (1992). In addition to small molecule drugs, a variety of biological materials have been spray dried and these materials include: enzymes, serum, microorganisms and yeasts. Spray drying is a suitable technique because it can convert liquid pharmaceutical formulations into fine, dust-free or agglomerated powders in a one-step process. The basic technology comprises the following four steps: a) Atomizing the raw material solution into spray; b) Spray-air contact; c) Spray drying; and d) separating the dried product from the drying air. U.S. patent nos. 6,235,710 and 6,001,800, which are incorporated herein by reference in their entirety, describe the preparation of recombinant erythropoietin by spray drying.
The pharmaceutical compositions described herein may include a pharmaceutically acceptable carrier, excipient, or stabilizer. The pharmaceutically acceptable carrier is determined in part by the particular composition being administered and the particular method used to administer the composition. Thus, there are a variety of suitable formulations of the pharmaceutical compositions of modified or unmodified non-natural amino acid polypeptides described herein (optionally containing pharmaceutically acceptable carriers, excipients or stabilizers), (see, e.g., remington: the Science and Practice of Pharmacy, 19 th 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. Code, pharmaceutical Dosage Forms, marcel Decker, new York, n.y., 1980), andpharmaceutical Dosage Forms and Drug Delivery Systems, 17 th edition (Lippincott Williams)&Wilkins, 1999)). Suitable carriers include buffers containing succinate, phosphate, borate, HEPES, citrate, imidazole, acetate, bicarbonate, and other organic acids; antioxidants including, but not limited to, ascorbic acid; low molecular weight polypeptides, including, but not limited to, those less than about 10 residues; proteins, including (but not limited to) serum albumin, gelatin, or immunoglobulins; hydrophilic polymers including, but not limited to, polyvinylpyrrolidone; amino acids including, but not limited to, glycine, glutamine, asparagine, arginine, histidine or histidine derivatives, methionine, glutamic acid or lysine; monosaccharides, disaccharides, and other carbohydrates including, but not limited to, trehalose, sucrose, glucose, mannose, or dextrins; chelating agents including, but not limited to, EDTA; divalent metal ions including, but not limited to, zinc, cobalt, or copper; sugar alcohols, including but not limited to mannitol or sorbitol; salt-forming counterions, including (but not limited to) sodium; and/or nonionic surfactant including (but not limited to) Tween TM (including but not limited to Tween 80 (polysorbate 80) and Tween 20 (polysorbate 20)), pluronic TM And other pluronic acids (including, but not limited to, pluronic acid F68 (poloxamer 188) or PEG). Suitable surfactants include, for example, but are not limited to, polyethers based on polyethylene oxide-polypropylene oxide-polyethylene oxide (i.e., (PEO-PPO-PEO)), or polypropylene oxide-polyethylene oxide-polypropylene oxide (i.e., (PPO-PEO-PPO)), or combinations thereof. PEO-PPO-PEO and PPO-PEO-PPO are commercially available under the trade names PluronicsTM, R-PluronicsTM, tetronicsTM and R-tetronics (BASF Wyandotte Corp., wyandotte, mich.) and are further described in U.S. Pat. No. 4,820,352, which is incorporated herein by reference in its entirety. Other ethylene/polypropylene block polymers may be suitable surfactants. The surfactant or combination of surfactants may be used to stabilize the pegylated unnatural amino acid poly against one or more stresses, including but not limited to stresses from agitationA peptide. Some of the above surfactants may be referred to as "swelling agents". Some may also be referred to as "tonicity adjusting agents".
Modified or unmodified non-natural amino acid polypeptides as described herein, including those linked to a water-soluble polymer such as PEG, can also be administered by or as part of a sustained release system. Sustained release compositions include, but are not limited to, semipermeable polymer matrices in the form of shaped articles, including, but not limited to, films or microcapsules. The sustained release matrix comprises a biocompatible material such as poly (2-hydroxyethyl methacrylate) (Langer et al, J.biomed. Mater. Res.,15:167-277 (1981); langer, chem. Tech.,12:98-105 (1982)), ethylene vinyl acetate (Langer et al, as before) or poly-D- (-) -3-hydroxybutyric acid (EP 133,988), polylactides (polylactic acid) (U.S. Pat. No. 3,773,919; EP 58,481), polyglycolides (polymers of glycolic acid), polylactide-co-glycolides (copolymers of lactic acid and glycolic acid), polyanhydrides, copolymers of L-glutamic acid and gamma-ethyl-L-glutamate (U.Sidman et al, biopolymers,22,547-556 (1983)), poly (orthoesters, polypeptides, hyaluronic acid, collagen, chondroitin sulfate, carboxylic acid, fatty acid, phospholipids, polysaccharides, nucleic acids, polyaminoacids, amino acids (such as phenylalanine, nucleotides, isoleucine, tyrosine, polyvinyl pyrrolidone, and silicone. Sustained release compositions also include liposome encapsulated compounds. Liposomes containing the compounds are prepared by methods known per se: DE 3,218,121; epstein et al, proc.Natl.Acad.Sci.U.S.A.,82:3688-3692 (1985); hwang et al, proc.Natl Acad.Sci.U.S. A.,77:4030-4034 (1980); EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP 142,641; japanese patent application 83-118008; U.S. patent nos. 4,485,045 and 4,544,545; and EP 102,324.
The liposome-encapsulated polypeptide can be prepared by the methods described in the following documents: for example, DE 3,218,121; epstein et al, proc.Natl Acad.Sci.U.S. A.,82:3688-3692 (1985); hwang et al, proc.Natl Acad.Sci.U.S. A.,77:4030-4034 (1980); EP 52,322; EP 36,676; EP 88,046; EP 143,949, a step of performing the process; EP 142,641; japanese patent application 83-118008; U.S. patent nos. 4,485,045 and 4,544,545; and EP 102,324. The composition and size of liposomes are well known or can be readily determined empirically by those skilled in the art. Some examples of liposomes are described, for example, in Park JW et al, proc.Natl. Acad.Sci. USA 92:1327-1331 (1995); lasic D and Papahadjopoulos D (code): MEDICAL A PPLICATIONS OF L IPOSOMES (1998) The method comprises the steps of carrying out a first treatment on the surface of the Drummond DC et al, liposomal drug delivery systems for cancer therapy, teicher B (ed.): CANCER DRUG DISCOVERY AND DEVELOPMENT (2002); park JW et al Clin.cancer Res.8:1172-1181 (2002); nielsen UB et al, biochim. Biophys. Acta 1591 (1-3): 109-118 (2002); mamot C et al, cancer Res.63:3154-3161 (2003).
The dosage administered to the patient in the context of the compositions, formulations and methods described herein should be sufficient to produce a beneficial response in the subject over time. Typically, the total pharmaceutically effective amount of modified or unmodified unnatural amino acid polypeptide as described herein administered per dose is in the range of about 0.01 μg to about 100 μg, or about 0.05mg to about 1mg per kilogram patient body weight per day, although it should be subjected to therapeutic judgment. The dosing frequency is also subject to therapeutic judgment and may be higher or lower frequency than that of commercially available products approved for use in humans. In general, a polymer-polypeptide conjugate (comprising, by way of example only, a PEGylated polypeptide as described herein) can be administered by any of the above routes of administration.
Examples
Example 1
This example details the synthesis of carbonyl-containing amino acids presented in fig. 4. Carbonyl-containing unnatural amino acids are prepared as described in FIG. 4.
Example 2
This example details the synthesis of the protected hydroxylamine-containing amino acid presented in fig. 5 a. The protected hydroxylamine-containing unnatural amino acid is prepared as described in fig. 5 a.
Example 3
This example details the synthesis of hydroxylamine-containing amino acids presented in fig. 5 b. The hydroxylamine-containing unnatural amino acid was prepared as described in figure 5 b.
Example 4
This example details the synthesis of hydroxylamine-containing amino acids presented in fig. 5 c. The hydroxylamine-containing unnatural amino acid was prepared as described in figure 5 c.
Example 5
This example details the synthesis of the oxime-containing amino acid presented in fig. 5 d. Oxime-containing unnatural amino acids were prepared as described in FIG. 5 d.
Example 6
This example details the synthesis of the oxime-containing amino acid presented in fig. 6 a. Oxime-containing unnatural amino acids were prepared as described in FIG. 6 a.
Example 7
This example details the synthesis of the oxime-containing amino acid presented in fig. 6 b. Oxime-containing unnatural amino acids were prepared as described in FIG. 6 b.
Example 8
This example details the synthesis of the oxime-containing amino acid presented in fig. 6 c. Oxime-containing unnatural amino acids were prepared as described in FIG. 6 c.
Example 9
This example details the synthesis of carbonyl-containing amino acids presented in figure 24.
Figure BDA0001546710350002101
Is synthesized by (a)
Diethyl ether (60 mL) was added to the NaOH solution (40 mL,25 vol%) at 0deg.C. An explosion-proof shield was placed in front of the reaction flask. To the resulting mixture was added 3 parts of N-nitroso-N-methylurea (6.0 g,57.9 mmol) over 3 minutes. The reaction was stirred at 0deg.C for 10 min. The diethyl ether layer was then separated from the sodium hydroxide layer. The organic layer was added in portions (about 6 additions) to N-Boc-4-hydroxymethylphenylpropyl over 5 minutesA solution of amino acid (7.5 g,25.4 mmol) in anhydrous THF (20 mL) was used until the starting material was completely removed (monitored by TLC). Subsequently, 5 drops of glacial acetic acid were added to terminate the reaction. After removal of the organic solvent by rotary evaporation, ethyl acetate was added. The organic layer was treated with NaHCO 3 Saturated solution, H 2 O and brine were washed sequentially, followed by anhydrous MgSO 4 Dried, filtered and concentrated to give the product as a white powder (5.9 g, 75%).
Figure BDA0001546710350002102
Is synthesized by (a)
To an alcohol (6.0 g,19.4 mmol) and pyridine (12 mL,150 mmol) at 0deg.C in CH 2 Cl 2 To the stirred solution of (400 mL) was added Dess-Martin periodate (Dess-Martin periodinane) (14.2 g,33.4 mmol). The mixture was stirred at room temperature overnight. The reaction was then carried out with Na 2 S 2 O 3 -NaHCO 3 Saturated aqueous solution (1:1, 300 mL) was quenched and quenched with CH 2 Cl 2 And (5) extracting. Combining the organic layers and using H 2 O and brine followed by anhydrous Na 2 SO 4 Dried, filtered and concentrated in vacuo. The residue was purified by flash chromatography (silica, 1:100-1:1 hexanes: etOAc) to give the aldehyde product as a white solid (5.48 g, 92%).
Figure BDA0001546710350002111
Is synthesized by (a)
To a solution of aldehyde (3.07 g,10 mmol) in EtOH (40 mL) was added acetic acid hydrazide (1.7 g,20 mmol). The reaction mixture was stirred at room temperature for 30 min and concentrated. H is added to the residue 2 O (200 mL), followed by CH addition 2 Cl 2 . The organic layer was separated and concentrated in vacuo. Purification of the residue by flash chromatography (silica, 3:7-1:9 hexanes: etOAc) afforded the product as a white solid (3.29 g, 90%).
Figure BDA0001546710350002112
Is synthesized by (a)
To a solution of the above methyl ester (3.29 g,9.1 mmol) in dioxane (10 mL) was added LiOH (10 mL,1 n) at 0 ℃. The mixture was stirred at the same temperature for 1 hour and then the reaction was stopped by adding citric acid (5 g) and taken up in H 2 O dilution. The mixture was extracted with EtOAc. The organic layer is treated with H 2 O and brine were washed sequentially, followed by anhydrous Na 2 SO 4 Dried, filtered and concentrated to give a white solid (3.05 g, 96%).
Figure BDA0001546710350002113
Is synthesized by (a)
The acid (3.02 g,8.6 mmol) mentioned above was reacted with CH at 0deg.C 2 Cl 2 Trifluoroacetic acid (20 mL) was added to the solution in (20 mL). The reaction mixture was stirred at 0 ℃ for 2 hours and concentrated. MeOH (1 mL) was added to the residue followed by HCl (2.0 mL,4n in dioxane). Diethyl ether (200 mL) was then added to precipitate the product as a yellow solid (2.07 g, 83%).
Example 10
This example details the synthesis of dicarbonyl-containing amino acids presented in fig. 25.
Figure BDA0001546710350002121
Is synthesized by (a)
To a stirred solution of amine (10 g,34 mmol) in DMF (70 mL) at 0deg.C was added pyruvic acid (5 mL,72 mmol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC, 20g,104 mmol), 1-hydroxybenzotriazole hydrate (HOBt, 85g,71 mmol) and N, N-diisopropylethylamine (DIEA, 35mL,200 mmol). The mixture was stirred at room temperature for 6 hours and then quenched with aqueous citric acid (5%, 500 mL) and extracted with EtOAc (500 mL). The organic layer is treated with H 2 O and brine were washed sequentially, followed by anhydrous Na 2 SO 4 Dried, filtered and concentrated in vacuo. Purification of the residue by flash chromatography (silica, 3:1-1:1 hexanes: etOAc) afforded the product (4.78 g, 40) as a solid％)。
Figure BDA0001546710350002122
Is synthesized by (a)
To a solution of the above methyl ester (2.96 g,8.1 mmol) in dioxane (10 mL) was added LiOH (10 mL,1 n) at 0 ℃. The mixture was stirred at the same temperature for 3 hours. The reaction was then quenched with aqueous citric acid (5%) and diluted with EtOAc. Separating the organic layer and separating the organic layer with H 2 O and brine were washed sequentially, followed by anhydrous Na 2 SO 4 Dried, filtered and concentrated to give the product as a yellow solid (2.87 g, 100%).
Figure BDA0001546710350002123
Is synthesized by (a)
The above acid (2.05 g,5.9 mmol) at 0deg.C in CH 2 Cl 2 Trifluoroacetic acid (10 mL) was added to the solution in (10 mL). The mixture was stirred for 2 hours and concentrated in vacuo. HCl (1 mL,4n in dioxane) was added to the residue followed by diethyl ether (400 mL). The precipitate was collected as a white solid (1.38 g, 82%).
Example 11
This example details the synthesis of dicarbonyl-containing amino acids presented in fig. 26.
Figure BDA0001546710350002131
Is synthesized by (a)
To a solution of 4 '-methylpropionophenone (20 g,122 mmol) and N-bromosuccinimide (NBS, 23g,130 mmol) in benzene (300 mL) was added 2,2' -azobisisobutyronitrile (AIBN, 0.6g,3.6 mmol) at 90 ℃. The resulting solution was heated to reflux overnight. The reaction was then cooled to room temperature. The brown solution was treated with H 2 O and brine were washed sequentially, followed by anhydrous Na 2 SO 4 Dried, filtered and concentrated in vacuo. The residue was crystallized from hexane to give the product as a pale yellow solid (27 g, 87%).
Figure BDA0001546710350002132
Is synthesized by (a)
To a solution of EtONa (14.5 g,203 mmol) in EtOH (400 mL) was added diethyl acetamidomalonate (39 g,180 mmol) followed by a solution of the above bromide (27 g,119 mmol) in EtOH (100 mL) at 0deg.C. The resulting mixture was heated to reflux for 1 hour and quenched with citric acid (30 g) and with H 2 O (300 mL) dilution. After removing most of the solvent in vacuo, the residue was extracted with EtOAc. The organic layer is treated with H 2 O and brine were washed sequentially, followed by anhydrous Na 2 SO 4 Dried, filtered and concentrated in vacuo. Purification of the residue by flash chromatography (silica, 10:1-3:1 hexanes: etOAc) afforded the product as a yellow solid (37 g, 88%).
Figure BDA0001546710350002141
Is synthesized by (a)
To a solution of ketone (5 g,13.8 mmol) in diethyl ether (100 mL) at 0deg.C was added Br 2 (0.8 mL,15.6 mmol). The mixture was stirred at room temperature for 3 hours and then treated with NaHCO 3 The saturated aqueous solution was stopped. By Et 2 The mixture was extracted with O. The organic layer is treated with H 2 O and brine were washed sequentially, followed by anhydrous Na 2 SO 4 Dried, filtered and concentrated in vacuo to give the product as a yellow solid (5.4 g, 88%), which was used directly in the next step without further purification.
Figure BDA0001546710350002142
Is synthesized by (a)
To alpha-bromoketone (5.4 g,12.2 mmol) and Na 2 CO 3 To a solution of (2.0 g,18.9 mmol) in DMSO (20 mL) was added KI (2.1 g,13.2 mmol). The mixture was stirred at 90 ℃ under nitrogen for 28 hours. The reaction is then carried out with H 2 O was discontinued and diluted with EtOAc. Separating the organic layer and separating the organic layer with H 2 O and brine were washed sequentially, followed by anhydrous Na 2 SO 4 Dried, filtered and concentrated in vacuo. The residue was purified by flash chromatography (silica, 6:1-1:10 hexanes: etOAc) to give the product as a solid (1.12 g, 24%).
Figure BDA0001546710350002143
Is synthesized by (a)
A solution of diketone (1.12 g,3.0 mmol) in concentrated HCl (10 mL) and dioxane (10 mL) was heated to reflux overnight. After removal of the solvent in vacuo, meOH (3 mL) was added to dissolve the residue. Diethyl ether (300 mL) was then added to precipitate the product as a pale yellow solid (302 mg, 42%).
Example 12
This example details the synthesis of dicarbonyl-containing amino acids presented in fig. 27.
Figure BDA0001546710350002151
Is synthesized by (a)
At 0 ℃ to C 3 H 7 To a solution of MgCl (2M, 50 mmol) in diethyl ether (25 mL) was added benzaldehyde (5 mL,42.5 mmol) in diethyl ether (50 mL). The resulting solution was stirred at 0℃for 30 minutes. Then the reaction is carried out with NH 4 The saturated Cl solution was stopped and diluted with diethyl ether. Separating the organic layer and separating the organic layer with H 2 O and brine were washed sequentially, followed by anhydrous Na 2 SO 4 Dried, filtered and concentrated in vacuo to give the crude product (7.2 g), which was used directly in the next reaction without purification.
Figure BDA0001546710350002152
Is synthesized by (a)
At 0deg.C on CH with the above alcohol (7.2 g,43.9 mmol) and pyridine (7 mL,86.7 mmol) 2 Cl 2 To a solution of (300 mL) was added dess-Martin periodate (19.2 g,45.3 mmol). The resulting mixture was stirred overnight and taken up in Na 2 S 2 O 3 Saturated aqueous solution and NaHCO 3 The saturated aqueous solution (1:1) was stopped. Will haveLayer H 2 O and brine were washed sequentially, followed by anhydrous Na 2 SO 4 Dried, filtered and concentrated in vacuo. Purification of the residue by flash chromatography (silica, 8:1-4:1 hexanes: etOAc) afforded the product (6.28 g, 91% for both steps) as a colorless oil.
Figure BDA0001546710350002153
Is synthesized by (a)
To a solution of the above ketone (4.43 g,27.3 mmol) and N-bromosuccinimide (NBS, 5.5g,30.9 mmol) in benzene (150 mL) was added 2,2' -azobisisobutyronitrile (AIBN, 0.2g,1.2 mmol) at 90 ℃. The resulting solution was heated to reflux overnight and then cooled to room temperature. The brown solution was treated with H 2 O and brine were washed sequentially, followed by anhydrous Na 2 SO 4 Dried, filtered and concentrated in vacuo. The residue was crystallized from hexane to give the product as a white solid (6.21 g, 95%).
Figure BDA0001546710350002161
Is synthesized by (a)
To a solution of EtONa (2.5 g,34.9 mmol) in EtOH (200 mL) was added diethyl acetamidomalonate (6.7 g,30.9 mmol) followed by a solution of the above bromide (6.2 g,25.8 mmol) in EtOH (100 mL) at 0deg.C. The resulting mixture was heated to reflux for 1 hour and then quenched with citric acid (9 g) and with H 2 O dilution. After removal of most of the solvent, the residue was extracted with EtOAc. The organic layer is treated with H 2 O and brine were washed sequentially, followed by anhydrous Na 2 SO 4 Dried, filtered and concentrated in vacuo. The residue was purified by flash chromatography (silica, 4:1-2:1 hexanes: etOAc) to give the product as a pale yellow solid (8.92 g, 92%).
Figure BDA0001546710350002162
Is synthesized by (a)
In a solution of the above ketone (1.4 g,3.71 mmol) in HOAc (50 mL)Adding Br 2 (0.7 mL,13.6 mmol). The mixture was stirred at room temperature overnight and then with NaHCO 3 The saturated aqueous solution was stopped. By Et 2 The mixture was extracted with O. The organic layer is treated with H 2 O and brine were washed sequentially, followed by anhydrous Na 2 SO 4 Dried, filtered and concentrated in vacuo. The residue was purified by flash chromatography (silica, 5:1-3:2 hexanes: etOAc) to give the product as a yellow solid (1.23 g, 73%).
Figure BDA0001546710350002163
Is synthesized by (a)
To alpha-bromoketone (1.12 g,2.46 mmol) and Na 2 CO 3 To a solution of (0.4 g,3.77 mmol) in DMSO (30 mL) was added KI (0.45 g,13.2 mmol). The mixture was stirred at 90 ℃ overnight and then with citric acid (2 g) and H 2 O (200 mL) was discontinued. The mixture was extracted with EtOAc. The organic layer is treated with H 2 O and brine were washed sequentially, followed by anhydrous Na 2 SO 4 Dried, filtered and concentrated in vacuo. Purification of the residue by flash chromatography (silica, 6:1-1:10 hexanes: etOAc) afforded the α -hydroxyketone as an oil (0.62 g, 64%).
The above alcohol (0.62 g,1.58 mmol) and pyridine (0.5 mL,6.19 mmol) at 0deg.C in CH 2 To a solution of Cl (100 mL) was added dess-Martin periodate (0.9 g,2.12 mmol). The resulting mixture was stirred overnight and then taken up in Na 2 S 2 O 3 Saturated aqueous solution and NaHCO 3 The saturated aqueous solution (1:1) was stopped. The organic layer is treated with H 2 O and brine were washed sequentially, followed by anhydrous Na 2 SO 4 Dried, filtered and concentrated in vacuo. The residue was purified by flash chromatography (silica, 9:1-3:2 hexanes: etOAc) to give the product as a yellow oil (287 mg, 30% for both steps).
Figure BDA0001546710350002171
Is synthesized by (a)
A mixture of the above diketone (272 mg,0.7 mmol) in concentrated HCl (10 mL) and dioxane (10 mL) was heated to reflux overnight. After removal of the solvent in vacuo, meOH (1 mL) was added to dissolve the residue. Diethyl ether (200 mL) was then added to precipitate the product as a yellow solid (162 mg, 81%).
Example 13
This example details the synthesis of dicarbonyl-containing amino acids presented in fig. 28. These compounds were synthesized as shown in fig. 28.
Example 14
This example details cloning and expression of modified polypeptides in E.coli. An introduced translation system including an orthogonal tRNA (O-tRNA) and an orthogonal aminoacyl tRNA synthetase (O-RS) is used to express a polypeptide that contains an unnatural amino acid. The O-RS preferentially aminoacylates the O-tRNA with the unnatural amino acid. Next, the translation system inserts unnatural amino acids into the polypeptide in response to the coding selector codon. Amino acids and polynucleotide sequences suitable for use in O-tRNA and O-RS that incorporate unnatural amino acids are described in U.S. patent application Ser. No. 10/126,927, entitled "In vivo Incorporation of Unnatural Amino Acids," and U.S. patent application Ser. No. 10/126,931, entitled "Methods and Compositions for the Production of Orthogonal tRNA-Aminoacyl tRNA Synthetase Pairs," which are incorporated herein by reference. The following O-RS and O-tRNA sequences can also be used:
Figure BDA0001546710350002181
Figure BDA0001546710350002191
Coli transformed with a plasmid containing the modified gene and an orthogonal aminoacyl tRNA synthetase/tRNA pair (specific to the desired unnatural amino acid) allows site-specific incorporation of the unnatural amino acid into the polypeptide. Transformed E.coli grown at 37℃in a medium containing between 0.01mM and 100mM of specific unnatural amino acid to a high degreeFidelity and efficiency of expression of the modified polypeptide. His-tagged polypeptides containing unnatural amino acids are produced by E.coli host cells in inclusion body or aggregate form. These aggregates were dissolved in 6M guanidine hydrochloride under denaturing conditions and affinity purified. In 50mM TRIS-HCl (pH 8.0), 40. Mu.M CuSO 4 And 2% (w/v) sodium N-dodecyl sarcosinate (Sarkosyl) was refolded by dialysis at 4℃overnight. The material was then treated with 20mM TRIS-HCl (pH 8.0), 100mM NaCl, 2mM CaCl 2 Dialysis followed by removal of His-tag. See Boissel et al, J.biol. Chem., (1993) 268:15983-93. Methods for purifying polypeptides are well known in the art and are confirmed by SDS-PAGE, western blot analysis or electrospray ionization ion trap mass spectrometry, and the like.
Example 15: testing unnatural amino acids
This example provides the results of four tests performed on certain illustrative unnatural amino acids to help predict their properties for incorporation into unnatural amino acid polypeptides.
Figure BDA0001546710350002192
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Figure BDA0001546710350002201
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Figure BDA0001546710350002211
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Figure BDA0001546710350002221
Example 16: testing unnatural amino acids
This example provides the results of pH stability tests performed on certain illustrative unnatural amino acids to help predict their properties for incorporation into unnatural amino acid polypeptides.
Figure BDA0001546710350002231
Figure BDA0001546710350002232
Figure BDA0001546710350002241
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Figure BDA0001546710350002242
Figure BDA0001546710350002243
Example 17
This example details the synthesis of dicarbonyl-containing amino acids presented in fig. 29.
Figure BDA0001546710350002244
Is synthesized by (a)
To amino acid pAF (10 g,41.1 mmol) in H 2 NaHCO was added to a solution in O-dioxane (300 mL, 1:1) 3 (12 g,142.9 mmol) and Boc 2 O (12 g,55.0 mmol). The mixture was stirred at room temperature for 7 hours and then quenched with citric acid. The mixture was extracted with EtOAc. The organic layer is treated with H 2 O and brine were washed sequentially, followed by anhydrous Na 2 SO 4 Dried, filtered and concentrated to give N-Boc-pAF (13.7 g, quantitative) as a white solid.
Figure BDA0001546710350002251
Is synthesized by (a)
NaOH (40 mL,25 volumes at 0deg.CTo the solution was added diethyl ether (60 mL). An explosion-proof shield was placed in front of the reaction flask. To the resulting mixture was added 3 parts of N-nitroso-N-methylurea (6.0 g,57.9 mmol) over 3 minutes. The reaction was stirred at 0deg.C for 10 min. The diethyl ether layer was then separated from the sodium hydroxide layer. The organic layer was added in portions (about 6 additions) to a solution of N-Boc-pAF (5.0 g,16.2 mmol) in anhydrous THF (20 mL) over 5 min until the starting material was completely disappeared (monitored by TLC). Subsequently, 5 drops of glacial acetic acid were added to terminate the reaction. After removal of the organic solvent by rotary evaporation, ethyl acetate was added. The organic layer was treated with NaHCO 3 Saturated solution, H 2 O and brine were washed sequentially, followed by anhydrous MgSO 4 Dried, filtered, and concentrated to give a white powder (4.1 g, 80%).
Figure BDA0001546710350002252
Is synthesized by (a)
To t-BuOK (60 mL,1.0M in THF) was slowly added a solution of protected pAF (3.82 g,11.9 mmol) in freshly distilled methyl propionate (20 mL,208 mmol). The resulting mixture was stirred at room temperature for 30 minutes and quenched with citric acid solution (10%, 300 mL). The mixture was extracted with EtOAc. The organic layer is treated with H 2 O and brine followed by anhydrous Na 2 SO 4 Dried, filtered and concentrated in vacuo. The residue was purified by flash chromatography (silica, 4:1-1:1 hexanes: etOAc) to give the product as a white solid (3.89 g, 87%).
Figure BDA0001546710350002261
Is synthesized by (a)
To a solution of the above methyl ester (1.12 g,2.97 mmol) in dioxane (4 mL) was added LiOH (4 mL,1 n) at 0 ℃. The mixture was stirred at 0 ℃ for 3 hours and quenched with aqueous citric acid (5%, 200 mL) and diluted with EtOAc. Separating the organic layer and separating the organic layer with H 2 O and brine were washed sequentially, followed by anhydrous Na 2 SO 4 Dried, filtered, and concentrated to give a white solid (1.02 g, 94%).
Figure BDA0001546710350002262
Is synthesized by (a)
The acid (1.0 g,2.75 mmol) was added to CH at 0deg.C 2 Cl 2 Trifluoroacetic acid (10 mL) was added to the solution in (10 mL). The mixture was stirred at 0 ℃ for 2 hours and then concentrated. MeOH (1 mL) was added to the residue followed by HCl (1.5 mL,4n in dioxane). Diethyl ether (200 mL) was then added to precipitate the product as a white solid (702 mg, 96%).
Example 18
This example details the synthesis of dicarbonyl-containing amino acids presented in figure 30.
Figure BDA0001546710350002263
Is synthesized by (a)
To t-BuOK (15 mL,1.0M in THF) was slowly added a solution of protected pAF (1.09 g,3.4 mmol) in methyl difluoroacetate (6 mL,68.7 mmol). The resulting mixture was stirred at room temperature for 30 min and quenched with citric acid (5 g,25.4 mmol) and with H 2 O dilution. The mixture was extracted with EtOAc. The organic layer is treated with H 2 O and brine were washed sequentially, followed by anhydrous Na 2 SO 4 Drying, filtering and concentrating. The residue was purified by flash chromatography (silica, 20:1-3:2 hexanes: etOAc) to give the product as a pale brown solid (1.27 g, 94%).
Figure BDA0001546710350002271
Is synthesized by (a)
To a solution of the above methyl ester (1.26 g,3.17 mmol) in dioxane (30 mL) was added LiOH (30 mL,1 n) at 0 ℃. The mixture was stirred at 0 ℃ for 0.5 hours and quenched with citric acid (10 g,51 mmol) and quenched with H 2 O dilution. The mixture was extracted with EtOAc. The organic layer is treated with H 2 O and brine were washed sequentially, followed by anhydrous Na 2 SO 4 Drying, filtering and concentrating. By flash chromatography (silica, 100:1-10:1CH 2 Cl 2 MeOH,0.5% HOAc) to give a brown oil (1.19 g, 98%).
Figure BDA0001546710350002272
Is synthesized by (a)
The above acid (1.19 g,3.1 mmol) was added to CH at 0deg.C 2 Cl 2 Trifluoroacetic acid (15 mL) was added to the solution in (15 mL). The mixture was stirred for 0.5 hours and concentrated. MeOH (2 mL) was added to the residue followed by HCl (2 mL,4n in dioxane). Diethyl ether (200 mL) was then added to precipitate the product as a white solid (0.82 mg, 82%).
Example 19
This example details the synthesis of the hydroxylamine-containing PEG reagent presented in fig. 12 a. The PEG reagent containing hydroxylamine was prepared as described in fig. 12 a.
Example 20
This example details the synthesis of the hydroxylamine-containing PEG reagent presented in fig. 12 b. The PEG reagent containing hydroxylamine was prepared as described in fig. 12 b.
Example 21
This example details the synthesis of the hydroxylamine-containing PEG reagent presented in fig. 12 c. The PEG reagent containing hydroxylamine was prepared as described in fig. 12 c.
Example 22
This example details the synthesis of the hydroxylamine-containing PEG reagent presented in fig. 12 d. The PEG reagent containing hydroxylamine was prepared as described in fig. 12 d.
EXAMPLE 23
This example details the synthesis of the hydroxylamine-containing PEG reagent presented in fig. 13. The PEG reagent containing hydroxylamine was prepared as described in fig. 13.
EXAMPLE 24
This example details the synthesis of the hydroxylamine-containing PEG reagent presented in fig. 14. The PEG reagent containing hydroxylamine was prepared as described in fig. 14.
Example 25
This example details the synthesis of the hydroxylamine-containing PEG reagent presented in fig. 15. The PEG reagent containing hydroxylamine was prepared as described in fig. 15.
EXAMPLE 26
This example details the synthesis of the hydroxylamine-containing PEG reagent presented in fig. 16 a. The PEG reagent containing hydroxylamine was prepared as described in fig. 16 a.
Example 27
This example details the synthesis of the hydroxylamine-containing PEG reagent presented in fig. 16 b. The PEG reagent containing hydroxylamine was prepared as described in fig. 16 b.
EXAMPLE 28
This example details the synthesis of the hydroxylamine-containing linker reagent presented in fig. 18. The hydroxylamine-containing linker reagent was prepared as described in fig. 18.
Example 29
This example details the synthesis of 1, 2-bis (4- (bromomethyl) phenyl) disulfane (1) presented in FIG. 36. To an oven dried round bottom flask with a stir bar was added p-tolyl disulfide (5.0 g,20.3 mmol), N-bromosuccinimide (8.6 g,48.4 mmol), and 60mL anhydrous benzene under nitrogen pressure. The solution was heated to 95 ℃. Azobisisobutyronitrile (. 106 g. Times.64 mmol) was added in one portion. The reaction was refluxed for 16 hours. The solvent was removed by rotary evaporation and the brown solid was dissolved in 100mL ethyl acetate. The reaction mixture was washed successively with saturated aqueous sodium bicarbonate (2X 50 mL), deionized water (1X 50 mL) and brine (1X 50 mL). The organic layer was separated and dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure. By using Biotage inc TM Silica chromatography of the chromatography system purified the crude product, after concentration of the appropriate fraction and removal of traces of solvent (vacuum pump), to give 1, 2-bis (4- (bromomethyl) phenyl) disulfane (2.1 g, 25%) as a white solid. Obtaining 1 H NMR spectroscopic data and mass spectra. The reaction was repeated to give (2.0 g, 23%) of the product.
Example 30
This exampleDetailed description the synthesis of 1, 2-bis (4-diethyl-2-acetamidomalonate) phenyldisulfane (2) presented in fig. 36. To an oven dried round bottom flask with a stir bar was added diethyl acetamidomalonate (6.48 g,30 mmol) and 50mL of anhydrous EtOH under nitrogen pressure. Sodium ethoxide (2.6 g,38 mmol) was added to the solution in one portion. The reaction was cooled to 0 ℃.1, 2-bis (4- (bromomethyl) phenyl) disulfane (4.1 g,10.1 mmol) was dissolved in 20mL 1:1EtOH/THF and added to the cold solution through the addition funnel over the course of 1 hour. The ice bath was removed and the reaction was stirred at room temperature for 6 hours. The solvent was removed by rotary evaporation and the red solid was dissolved in 100mL ethyl acetate. The reaction mixture was washed sequentially with 5% citric acid solution (2X 50 mL), deionized water (1X 50 mL) and brine (1X 50 mL). The organic layer was separated and dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by silica chromatography using a Biotage inc. Horizontm chromatography system, after concentration of the appropriate fractions and removal of traces of solvent (vacuum pump), to give 1, 2-bis (4-diethyl-2-acetamidomalonate) phenyldisulfane (5.0 g, 73%) as a yellow solid. Obtaining 1 H NMR spectral data, HPLC traces, and mass spectra.
Example 31
This example details the synthesis of 1, 2-bis (4- (2-amino-3-propionic acid) phenyldisulfane (3) presented in FIG. 36 to an oven dried round bottom flask with a stir bar was added 1, 2-bis (4-diethyl-2-acetamidomalonate) phenyldisulfane (1.0 g,1.4 mmol), HCl (8 mL, 12M) and 8mL of 1, 4-dioxane under nitrogen pressure, the reaction was stirred at reflux for 16 hours, solvent was removed by rotary evaporation and vacuum pump to give crude 1, 2-bis (4- (2-amino-3-propionic acid) phenyldisulfane (0.75 g, 135%) as a clear oil 1 H NMR spectroscopic data and mass spectra.
Example 32
This example details the synthesis of N, N' -di-tert-butoxycarbonyl-1, 2-bis (4- (2-amino-3-propionic acid) phenyldisulfane (4) presented in FIG. 36 to 1, 2-bis (4- (2-amino-3-propionic acid) phenyldisulfane (0.75 g,1.9 mmol) in a dry round bottom flask was added 5mL of 1, 4-bisDioxane, 5mL deionized water, di-tert-butyl dicarbonate (. 65g,3.0 mmol), and sodium bicarbonate (0.98 g,12 mmol). The reaction was stirred at room temperature for 16 hours. The solvent was removed by rotary evaporation and the clear oil was dissolved in 100mL ethyl acetate. The reaction mixture was washed sequentially with 5% citric acid solution (5 ml×2), deionized water (50 mL) and brine (50 mL). The organic layer was separated and dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure. By using Biotage inc TM Silica chromatography purification of the crude product in a chromatography system, after concentration of the appropriate fraction and removal of traces of solvent (vacuum pump), gave N, N' -di-tert-butoxycarbonyl-1, 2-bis (4- (2-amino-3-propionic acid) phenyldisulfane (0.5 g, 44% from the crude product, 52% in 2 steps) as a white solid 1 H NMR spectral data, HPLC traces, and mass spectra.
Example 33
This example details the synthesis of N-t-butoxycarbonyl-2-amino-3- (4-mercaptophenyl) propionic acid (5) represented in FIG. 36. To an oven dried round bottom flask with stirring bar was added N, N' -di-tert-butoxycarbonyl-1, 2-bis (4- (2-amino-3-propionic acid) phenyldisulfane (0.5 g,0.84 mmol), N-butylphosphine (0.6 mL,2.44 mmol) and 15mL anhydrous THF under nitrogen pressure to react with stirring at room temperature for 2 hours, solvent was removed by rotary evaporation and the clear oil was dissolved in 50mL ethyl acetate, the reaction mixture was washed sequentially with 5% citric acid solution (2X 25 mL), deionized water (25 mL) and brine (25 mL), the organic layer was separated and dried over anhydrous magnesium sulfate, filtered and concentrated under reduced pressure by using Biotage Inc TM Silica chromatography of the chromatography system purified the crude product, after concentration of the appropriate fraction and removal of traces of solvent (vacuum pump), to give N-t-butoxycarbonyl-2-amino-3- (4-mercaptophenyl) propionic acid (0.5 g, 100%) as a white solid. Obtaining 1 H NMR spectral data, HPLC traces, and mass spectra.
Example 34
This example details the synthesis of 2-amino-3- (4-mercaptophenyl) propionic acid hydrochloride (6) presented in FIG. 36. To an oven dried round bottom flask with stirring bar was added N-t-butoxycarbonyl-2-amino3- (4-mercaptophenyl) propionic acid (.5 g,1.6 mmol), 10mL of anhydrous dichloromethane, and 3mL of trifluoroacetic acid. The reaction was stirred at room temperature for 2 hours. The solvent was removed by rotary evaporation and 1mL of 4.0M hydrogen chloride in 1, 4-dioxane was added. The round bottom flask was simply swirled and 100mL of anhydrous diethyl ether was then added to precipitate 2-amino-3- (4-mercaptophenyl) propionic acid hydrochloride (0.39 g, 100%) as a white solid. Obtaining 1 H NMR spectral data, HPLC traces, and mass spectra.
Example 35
This example details the synthesis of diethyl 2- (4- (2-oxopropylsulfanyl) benzyl) -2-acetamidomalonate (7) presented in fig. 37. To an oven dried round bottom flask with stirring bar under nitrogen pressure was added (2) (1.1 g,1.6 mmol), n-Bu 3 P (1.2 mL,4.8 mmol) and 25mL dry THF (25 mL). The reaction was stirred at room temperature for 2 hours. To the reaction was added chloroacetone (0.16 mL,2.0 mmol) and NaHCO 3 (0.98 g,12 mmol). The reaction was stirred at room temperature for 2 hours. The solvent was removed by rotary evaporation and the white solid was dissolved in 100mL ethyl acetate. The reaction mixture was washed with saturated aqueous sodium bicarbonate (50 ml×2), deionized water (50 mL) and brine (50 mL) in this order. The organic layer was separated and dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure. By using Biotage inc TM Silica chromatography of the chromatography system purified the crude product, after concentration of the appropriate fraction and removal of traces of solvent (vacuum pump), to give diethyl 2- (4- (2-oxopropylsulfanyl) benzyl) -2-acetamidomalonate (0.62 g, 98%) as a white solid. Obtaining 1 H NMR spectral data, HPLC traces, and mass spectra.
Example 36
This example details the synthesis of 3- (4- (2-oxopropanethio) phenyl) -2-aminopropionic acid (8) presented in FIG. 37. To an oven dried round bottom flask with stirring bar under nitrogen pressure was added 7 (0.62 g,1.5 mmol), 10ml1, 4-dioxane and 10ml 12m HCl. The reaction was refluxed and stirred overnight. The solvent was removed by rotary evaporation to give 3- (4- (2-oxopropylsulfanyl) phenyl) -2-aminopropionic acid (0.40 g,99% crude product).
EXAMPLE 37
This example details the synthesis of N-t-butoxycarbonyl-3- (4- (2-oxopropylsulfanyl) phenyl) -2-aminopropionic acid (9) represented in FIG. 37. To 8 (0.35 g,1.3 mmol) of di-tert-butyl dicarbonate (0.63 g,3.0 mmol), sodium bicarbonate (0.98 g,12 mmol), 8mL of 1, 4-dioxane and 8mL of deionized water were added under nitrogen pressure in an oven dried round bottom flask with a stirring bar. The reaction was stirred at room temperature for 16 hours. The solvent was removed by rotary evaporation and the clear oil was dissolved in 100mL ethyl acetate. The reaction mixture was washed sequentially with 5% citric acid solution (50 ml×2), deionized water (50 mL) and brine (50 mL). The organic layer was separated and dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure. By using Biotage inc TM Silica chromatography of the chromatography system purified the crude product, after concentration of the appropriate fraction and removal of traces of solvent (vacuum pump), to give N-t-butoxycarbonyl-3- (4- (2-oxopropylsulfanyl) phenyl) -2-aminopropionic acid (0.30 g, 66% from crude product) as a white solid. Obtaining 1 H NMR spectral data, HPLC traces, and mass spectra.
Example 38
This example details the synthesis of N-t-butoxycarbonyl-3- (4- (2-oxopropylsulfinyl) phenyl) -2-aminopropionic acid (10) represented in FIG. 37. To an oven dried round bottom flask with stirring bar was added 9 (150 mg,.4 mmol), 8mL glacial acetic acid and 2mL 30% v/v hydrogen peroxide in water under nitrogen pressure. The reaction was stirred at room temperature for 2 hours. The solvent was removed by rotary evaporation and the clear oil was dissolved in 50mL ethyl acetate. The reaction mixture was washed sequentially with 5% citric acid solution (25 ml×2), deionized water (25 mL) and brine (25 mL). The organic layer was separated and dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure. By using Biotage inc TM Silica chromatography of the chromatography system purified the crude product, after concentration of the appropriate fraction and removal of traces of solvent (vacuum pump), to give N-t-butoxycarbonyl-3- (4- (2-oxopropylsulfinyl) phenyl) -2-aminopropionic acid (0.13 g,86% crude product). HPLC traces and mass spectra were obtained.
Example 39
This example details the synthesis of 3- (4- (2-oxopropylsulfinyl) phenyl) -2-aminopropionic acid (11) presented in FIG. 37. To an oven dried round bottom flask with stirring bar was added 10 (0.13 g,0.35 mmol) of N-t-butoxycarbonyl-3- (4- (2-oxopropylsulfinyl) phenyl) -2-aminopropionic acid, 10mL of anhydrous dichloromethane and 3mL of trifluoroacetic acid. The reaction was stirred at room temperature for 2 hours. The solvent was removed by rotary evaporation and 1mL of 4.0M hydrogen chloride in 1, 4-dioxane was added. The round bottom flask was simply swirled and 100mL of anhydrous diethyl ether was then added to precipitate 3- (4- (2-oxopropylsulfinyl) phenyl) -2-aminopropionic acid (0.072 g, 74% from crude product) as a white solid. Obtaining 1 H NMR spectral data, HPLC traces, and mass spectra.
Example 40
This example details the synthesis of N-t-butoxycarbonyl-3- (4- (2-oxopropylsulfonyl) phenyl) -2-aminopropionic acid (12) represented in FIG. 37. To an oven dried round bottom flask with stirring bar was added 9 (150 mg,0.4 mmol), 8mL glacial acetic acid and 2mL 30% v/v hydrogen peroxide in water under nitrogen pressure. The reaction was stirred at room temperature for 24 hours. The solvent was removed by rotary evaporation and the clear oil was dissolved in 50mL ethyl acetate. The reaction mixture was washed sequentially with 5% citric acid solution (25 ml×2), deionized water (25 mL) and brine (25 mL). The organic layer was separated and dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure. By using Biotage inc TM Silica chromatography of the chromatography system purified the crude product, after concentration of the appropriate fraction and removal of traces of solvent (vacuum pump), to give N-t-butoxycarbonyl-3- (4- (2-oxopropylsulfonyl) phenyl) -2-aminopropionic acid (12) (0.13 g,86% of crude product). HPLC traces and mass spectra were obtained.
Example 41
This example details the synthesis of 3- (4- (2-oxopropylsulfonyl) phenyl) -2-aminopropionic acid (13) presented in FIG. 37. To an oven dried round bottom flask with stirring bar was added N-t-butoxycarbonyl-3- (4- (2-oxopropylsulfonyl) phenyl) -2-aminopropionic acid 12 (0.13 g,0.35 mmol), 10mL anhydrous dichloromethane and 3mL trifluoroacetic acid. The reaction was stirred at room temperature for 2 hours. The solvent was removed by rotary evaporation and 1mL of 4.0M hydrogen chloride in 1, 4-dioxane was added. The round bottom flask was simply swirled and 100mL of anhydrous diethyl ether was then added to precipitate 3- (4- (2-oxopropylsulfonyl) phenyl) -2-aminopropionic acid (0.067 g, 65% from crude product) as a white solid. Obtaining 1 H NMR spectral data, HPLC traces, and mass spectra.
Example 42
This example details the synthesis of 3- (4- (2-oxopropanethio) phenyl) -2-aminopropionic acid (14) presented in FIG. 37. To an oven dried round bottom flask with stirring bar was added 9 (0.10 g,.28 mmol) of N-t-butoxycarbonyl-3- (4- (2-oxopropylsulfanyl) phenyl) -2-aminopropionic acid, 10mL of anhydrous dichloromethane and 3mL of trifluoroacetic acid. The reaction was stirred at room temperature for 2 hours. The solvent was removed by rotary evaporation and 1mL of 4.0M hydrogen chloride in 1, 4-dioxane was added. The round bottom flask was simply swirled and 100mL of anhydrous diethyl ether was then added to precipitate 3- (4- (2-oxopropanethio) phenyl) -2-aminopropionic acid (0.062 g, 85% from the crude product) as a white solid. Obtaining 1 H NMR spectral data, HPLC traces, and mass spectra.
EXAMPLE 43
This example details the synthesis of N-t-butoxycarbonyl-3- (4- (2-oxocyclopentylsulfanyl) phenyl) -2-aminopropionic acid (15) represented in FIG. 38. To an oven dried round bottom flask with stirring bar under nitrogen pressure was added 5 (0.15 g,0.76 mmol), 2-chlorocyclopentanone (0.12 mL,1.25 mmol), sodium bicarbonate (0.98 g,12 mmol), 15mL anhydrous THF. The reaction was stirred at room temperature for 16 hours. The solvent was removed by rotary evaporation and the white solid was dissolved in 100mL ethyl acetate. The reaction mixture was washed with saturated aqueous sodium bicarbonate (50 ml×2), deionized water (50 mL) and brine (50 mL) in this order. The organic layer was separated and dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure. By using Biotage inc TM Silica chromatography of the chromatography system purified the crude product, after concentration of the appropriate fraction and removal of traces of solvent (vacuum pump), to give N-t-butoxycarbonyl-3- (4- (2-oxocyclopentylsulfanyl) phenyl) -2-aminopropionic acid (0) as a white solid.15g, 51%). HPLC traces and mass spectra were obtained.
EXAMPLE 44
This example details the synthesis of 3- (4- (2-oxocyclopentylsulfanyl) phenyl) -2-aminopropionic acid (16) presented in FIG. 38. To an oven dried round bottom flask with stirring bar was added N-t-butoxycarbonyl-3- (4- (2-oxocyclopentylsulfanyl) phenyl) -2-aminopropionic acid 15 (0.15 g,0.39 mmol), 10mL anhydrous dichloromethane and 3mL trifluoroacetic acid. The reaction was stirred at room temperature for 2 hours. The solvent was removed by rotary evaporation and 1mL of 4.0M hydrogen chloride in 1, 4-dioxane was added. The round bottom flask was simply swirled and 100mL of anhydrous diethyl ether was then added to precipitate 3- (4- (2-oxocyclopentylsulfanyl) phenyl) -2-aminopropionic acid (0.108 g, 100%) as a white solid. Obtaining 1 H NMR spectral data, HPLC traces, and mass spectra.
Example 45
This example details the synthesis of N-t-butoxycarbonyl-3- (4- (2-oxobutylsulfanyl) phenyl) -2-aminopropionic acid (18) represented in FIG. 38. To an oven dried round bottom flask with stirring bar under nitrogen pressure was added 5 (0.15 g,0.76 mmol), 1-bromo-2-butanone (0.12 mL,1.25 mmol), sodium bicarbonate (0.98 g,12 mmol), 15mL anhydrous THF. The reaction was stirred at room temperature for 16 hours. The solvent was removed by rotary evaporation and the white solid was dissolved in 100mL ethyl acetate. The reaction mixture was washed with saturated aqueous sodium bicarbonate (50 ml×2), deionized water (50 mL) and brine (50 mL) in this order. The organic layer was separated and dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure. By using Biotage inc TM Silica chromatography of the chromatography system purified the crude product, after concentration of the appropriate fraction and removal of traces of solvent (vacuum pump), to give N-t-butoxycarbonyl-3- (4- (2-oxobutylsulfanyl) phenyl) -2-aminopropionic acid (0.15 g, 51%) as a white solid. HPLC traces and mass spectra were obtained.
Example 46
This example details the synthesis of compounds 19-22 presented in FIG. 38. Compounds 19-22 were synthesized using methods similar to those described for compounds 10-14.
Example 47
This example details the synthesis of 3- (4- (2-oxobutylsulfanyl) phenyl) -2-aminopropionic acid (23) presented in FIG. 38. To an oven dried round bottom flask with stirring bar was added N-t-butoxycarbonyl-3- (4- (2-oxobutylsulfanyl) phenyl) -2-aminopropionic acid 18 (0.15 g,.56 mmol), 10mL anhydrous dichloromethane and 3mL trifluoroacetic acid. The reaction was stirred at room temperature for 2 hours. The solvent was removed by rotary evaporation and 1mL of 4.0M hydrogen chloride in 1, 4-dioxane was added. The round bottom flask was simply swirled and 100mL of anhydrous diethyl ether was then added to precipitate 3- (4- (2-oxobutylsulfanyl) phenyl) -2-aminopropionic acid (0.149 g, 100%) as a white solid. Obtaining 1 H NMR spectral data, HPLC traces, and mass spectra.
EXAMPLE 48
This example details the synthesis of compounds 24-27 presented in FIG. 39. Compounds 24-27 were synthesized using methods similar to those described for compounds 10-14.
Example 49
This example details the synthesis of dicarbonyl-containing amino acids presented in fig. 31.
Figure BDA0001546710350002341
Is synthesized by (a)
To t-BuOK (15 mL,1.0M in THF) was slowly added a solution of protected pAF (1.0 g,3.1 mmol) in methyl trifluoroacetate (5 mL,50 mmol). The reaction mixture was stirred at room temperature for 30 min and quenched with citric acid (5 g,25.4 mmol) and diluted with EtOAc. The organic layer is treated with H 2 O and brine were washed sequentially, followed by anhydrous Na 2 SO 4 Drying, filtering and concentrating. The residue was purified by flash chromatography (silica, 20:1-3:2 hexanes: etOAc) to give the product as a pale brown solid (1.07 g, 83%).
Figure BDA0001546710350002342
Is synthesized by (a)
To a solution of the above methyl ester (1.0 g,2.4 mmol) in dioxane (30 mL) was added LiOH (30 mL,1 n) at 0 ℃. The mixture was stirred at 0 ℃ for 0.5 hours and quenched with citric acid (10 g,51 mmol) and quenched with H 2 O dilution. The mixture was extracted with EtOAc. The organic layer is treated with H 2 O and brine were washed sequentially, followed by anhydrous Na 2 SO 4 Drying, filtering and concentrating. By flash chromatography (silica, 100:1-10:1CH 2 Cl 2 MeOH,0.5% HOAc) to give a brown oil (0.87 g, 98%).
Figure BDA0001546710350002351
Is synthesized by (a)
The above acid (1.0 g,2.5 mmol) at 0deg.C in CH 2 Cl 2 Trifluoroacetic acid (15 mL) was added to the solution in (15 mL). The resulting mixture was stirred for 0.5 hours and concentrated in vacuo. MeOH (2 mL) was added to the residue followed by HCl (2 mL,4n in dioxane). Diethyl ether (200 mL) was then added to precipitate the product as a white solid (0.56 g, 75%).
Example 50
This example details the synthesis of dicarbonyl-containing amino acids presented in fig. 32.
Figure BDA0001546710350002352
Is synthesized by (a)
To t-BuOK (15 mL,1.0M in THF) was slowly added a solution of protected pAF (1.0 g,3.1 mmol) in methyl pentafluoropropionate (8 mL,62 mmol). The resulting mixture was stirred at room temperature for 1 hour and quenched with citric acid (5 g,25.4 mmol) and with H 2 O (100 mL) dilution. After removal of most of the solvent, the residue was extracted with EtOAc. The organic layer is treated with H 2 O and brine were washed sequentially, followed by anhydrous Na 2 SO 4 Drying, filtering and concentrating. The residue was purified by flash chromatography (silica, 20:1-3:2 hexanes: etOAc) to give the product as a pale brown solid (1.1 g, 76%).
Figure BDA0001546710350002353
Is synthesized by (a)
To a solution of the above methyl ester (1.0 g,2.1 mmol) in dioxane (30 mL) was added LiOH (30 mL,1 n) at 0 ℃. The resulting mixture was stirred at 0deg.C for 0.5 hours and with citric acid (10 g,51 mmol) and H 2 O is discontinued. The mixture was extracted with EtOAc. The organic layer is treated with H 2 O and brine were washed sequentially, followed by anhydrous Na 2 SO 4 Drying, filtering and concentrating. By flash chromatography (silica, 100:1-10:1CH 2 Cl 2 MeOH,0.5% HOAc) to give the product as a yellow solid (0.8 g, 84%).
Figure BDA0001546710350002361
Is synthesized by (a)
The above acid (0.7 g,2.5 mmol) at 0deg.C in CH 2 Cl 2 Trifluoroacetic acid (15 mL) was added to the solution in (15 mL). The mixture was stirred at the same temperature for 0.5 hours and concentrated. MeOH (2 mL) was added to the residue followed by HCl (2 mL,4n in dioxane). Diethyl ether (200 mL) was then added to precipitate the product as a white solid (0.62 g, 70%).
Example 51
This example details the synthesis of dicarbonyl-containing amino acids presented in fig. 33.
Figure BDA0001546710350002362
Is synthesized by (a)
To alcohol 1 (2.35 g,7.6 mmol) and pyridine (1.5 mL,18.6 mmol) at 0deg.C in CH 2 Cl 2 To the stirred solution in (150 mL) was added dess-martin periodate (3.5 g,8.3 mmol). The mixture was stirred at room temperature overnight and taken up in Na 2 S 2 O 3 -NaHCO 3 Saturated aqueous solution (1:1, 100 mL) was stopped and taken up in CH 2 Cl 2 And (5) diluting. Separating the organic layer and separating the organic layer with H 2 O and brine followed by anhydrous Na 2 SO 4 Dried, filtered and concentrated in vacuo. The residue was purified by flash chromatography (silica, 20:1-3:1 hexanes: etOAc) to give aldehyde 2 (2.15 g, 92%) as a white solid. ESI-MS, M/z 292 (M) + -OH),232,204,175,131,115(100)。
Figure BDA0001546710350002371
Is synthesized by (a)
To a stirred solution of diisopropylamine (0.33 mL,2.33 mmol) in THF (60 mL) at 0deg.C was added n-butyllithium (1.46 mL,2.34 mmol). The mixture was stirred at 0 ℃ for 20 minutes and cooled to-78 ℃ and then cyclopentanone was added. After stirring the mixture at-78 ℃ for 20 minutes, a solution of aldehyde 2 (0.5 g,1.63 mmol) in THF (20 mL, washing with 20 mL) was added. The resulting mixture was stirred at-78℃for 1.0 h and with NH 4 The saturated aqueous solution of Cl was stopped. After removal of most of the solvent, the residue was extracted with EtOAc. The organic layer is treated with H 2 O and brine followed by anhydrous Na 2 SO 4 Dried, filtered and concentrated in vacuo. Purification of the residue by flash chromatography (silica, 10:1-1:1 hexanes: etOAc) afforded 3 (480 mg, 80%) as a colorless oil.
Figure BDA0001546710350002372
Is synthesized by (a)
To alcohol 3 (0.44 g,1.13 mmol) and pyridine (0.6 mL,7.44 mmol) at 0deg.C in CH 2 Cl 2 To the stirred solution in (150 mL) was added dess-Martin periodate (0.6 g,1.41 mmol). The mixture was stirred at room temperature overnight. The reaction is carried out with Na 2 S 2 O 3 -NaHCO 3 Saturated aqueous solution (1:1, 100 mL) was stopped and taken up in CH 2 Cl 2 And (5) extracting. Combining the organic layers and using H 2 O and brine followed by anhydrous Na 2 SO 4 Dried, filtered and concentrated in vacuo. Purification of the residue by flash chromatography (silica, 20:1-2:1 hexanes: etOAc) afforded diketone 4 (349mg, 78%) as a colorless oil. ESI-MS, M/z:412 (M + +Na),356,312,230,212,184,146(100)。
Figure BDA0001546710350002373
Is synthesized by (a)
To a solution of methyl ester 4 (330 mg,0.85 mmol) in dioxane (4 mL) was added LiOH (4 mL,1 n) at 0 ℃. The resulting mixture was stirred at 0deg.C for 30 min and quenched with aqueous citric acid (5%, 100 mL). The mixture was extracted with EtOAc. The organic layer is treated with H 2 O and brine were washed sequentially, followed by anhydrous Na 2 SO 4 Dried, filtered, and concentrated to give a white solid (310 mg, 97%). ESI-MS, M/z 342,330 (M) + -COOH),298,230,185,119(100)。
Figure BDA0001546710350002381
Is synthesized by (a)
To acid 5 (310 mg,0.83 mmol) at 0deg.C in CH 2 Cl 2 To the solution in (4 mL) was added trifluoroacetic acid (4 mL). The mixture was stirred at 0 ℃ for 30 min and concentrated in vacuo. MeOH (1 mL) was added to the residue followed by HCl (1 mL,4n in dioxane). Diethyl ether (100 mL) was then added to precipitate the product as a white solid (241 mg, 94%). ESI-MS, M/z 298 (M + +Na),276(M + +1),230(M + -COOH),184,119(100)。
Example 52
This example details the synthesis of dicarbonyl-containing amino acids presented in fig. 34.
Figure BDA0001546710350002382
Is synthesized by (a)
To an alcohol (6.0 g,19.4 mmol) and pyridine (12 mL,150 mmol) at 0deg.C in CH 2 Cl 2 To the stirred solution in (400 mL) was added dess-martin periodate (14.2 g,33.4 mmol). The mixture was stirred at room temperature overnight. The reaction is carried out with Na 2 S 2 O 3 -NaHCO 3 Saturated aqueous solution (1:1, 300 mL) was stopped and taken up in CH 2 Cl 2 And (5) extracting. Will haveCombined with the machine layer and by H 2 O and brine followed by anhydrous Na 2 SO 4 Dried, filtered and concentrated in vacuo. The residue was purified by flash chromatography (silica, 1:100-1:1 hexanes: etOAc) to afford the aldehyde as a white solid (5.48 g, 92%).
Figure BDA0001546710350002391
Is synthesized by (a)
To a solution of the above aldehyde (3.41 g,11.1 mmol) in acetone (70 mL) was added H 2 KMnO in O (10 mL) 4 (2.5 g,15.8 mmol). The resulting mixture was stirred at room temperature overnight. After removal of most of the solvent, the residue was dissolved in aqueous citric acid (5%, 300 mL) and extracted with EtOAc. Combining the organic layers and using H 2 O and brine followed by anhydrous Na 2 SO 4 Dried, filtered and concentrated in vacuo to give the product as a white solid (2.83 g, 79%) which was used directly in the next step without further purification.
Figure BDA0001546710350002392
Is synthesized by (a)
To a solution of the above acid (2.83 g,8.76 mmol) in DMF (60 mL) was added 1-amino-2-propanol (1.4 mL,17.9 mmol), 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride (EDC, 4.1g,21.4 mmol), 1-hydroxybenzotriazole hydrate (HOBt, 2.2g,18.5 mmol) and N, N-diisopropylethylamine (DIEA, 9mL,51.6 mmol) at 0deg.C. The mixture was stirred at room temperature overnight and then quenched with aqueous citric acid (5%, 200 mL) and extracted with EtOAc. Combining the organic layers and using H 2 O and brine followed by anhydrous Na 2 SO 4 Dried, filtered and concentrated in vacuo. Purification of the residue by flash chromatography (silica, 10:1-1:1 hexanes: etOAc) afforded the product as a white foam (2.45 g, 74%).
Figure BDA0001546710350002393
Is synthesized by (a)
At 0deg.C on CH, the above alcohol (2.44 g,6.4 mmol) and pyridine (4 mL,49.6 mmol) 2 Cl 2 To the stirred solution in (100 mL) was added dess-Martin periodate (4.1 g,9.7 mmol). The mixture was stirred at room temperature overnight. The reaction is carried out with Na 2 S 2 O 3 -NaHCO 3 Saturated aqueous solution (1:1, 300 mL) was stopped and taken up in CH 2 Cl 2 And (5) extracting. The organic layer is treated with H 2 O and brine followed by anhydrous Na 2 SO 4 Dried, filtered and concentrated in vacuo. The residue was purified by flash chromatography (silica, 1:1-1:3 hexanes: etOAc) to give the product as a yellow solid (1.84 g, 76%).
Figure BDA0001546710350002401
Is synthesized by (a)
To a solution of the above methyl ester (1.72 g,4.6 mmol) in dioxane (10 mL) was added LiOH (10 mL,1 n) at 0 ℃. The mixture was stirred at the same temperature for 3 hours and quenched with aqueous citric acid (5%). The mixture was extracted with EtOAc. The organic layer is treated with H 2 O and brine were washed sequentially, followed by anhydrous Na 2 SO 4 Dried, filtered and concentrated in vacuo to give the product (1.7 g) as a solid, which was used directly in the next step without purification.
At 0deg.C, the above acid (1.7 g,4.7 mmol) was added to CH 2 Cl 2 Trifluoroacetic acid (15 mL) was added to the solution in (15 mL). The mixture was stirred at the same temperature for 2 hours and concentrated in vacuo. HCl (1.5 mL,4n in dioxane) was added to the residue followed by diethyl ether (400 mL). The precipitated product was collected as a white solid (1.52 g, 90% for 2 steps).
Example 53
This example details the synthesis of the hydrazide-containing amino acids presented in figure 44.
Figure BDA0001546710350002402
Is synthesized by (a)
To a solution of aldehyde (410 mg,1.34 mmol) in EtOH (15 mL) was added formic acid hydrazide (170 mg,2.83 mmol). The reaction mixture was stirred at room temperature for 1 hour. After removal of the majority of the solvent, with CH 2 Cl 2 The residue was extracted. The organic layers were combined and concentrated in vacuo. The residue was purified by flash chromatography (silica, 1:6-1:1 hexanes: etOAc) to give a white solid (390 mg, 83%).
Figure BDA0001546710350002411
Is synthesized by (a)
To a solution of the above methyl ester (349 mg,1 mmol) in dioxane (7 mL) was added LiOH (7 mL,1 n) at 0 ℃. The mixture was stirred at the same temperature for 10 minutes and quenched with citric acid (2.5 g) and with H 2 And O extraction. The mixture was extracted with EtOAc. The organic layer is treated with H 2 O and brine were washed sequentially, followed by anhydrous Na 2 SO 4 Dried, filtered, and concentrated to give a white solid (290 mg, 87%).
Figure BDA0001546710350002412
Is synthesized by (a)
The above acid (290 mg,0.87 mmol) was added to CH at 0deg.C 2 Cl 2 Trifluoroacetic acid (20 mL) was added to the solution in (20 mL). The mixture was stirred for 20 minutes and concentrated. MeOH (1 mL) was added to the residue followed by HCl (1.0 mL,4n in dioxane). Diethyl ether (200 mL) was then added to precipitate the product as a pale yellow solid (195 mg, 83%).
Example 54
This example details the synthesis of the hydrazide-containing amino acids presented in fig. 45.
Figure BDA0001546710350002413
Is synthesized by (a)
To a solution of N-t-butoxycarbonyl-4-hydroxymethylphenylalanine (11.73 g,39.8 mmol) in DMF (100 mL) was added alanineMethyl ester hydrochloride (9.0 g,64.5 mmol), 1- (3-dimethylaminopropyl) 3-ethylcarbodiimide hydrochloride (EDC, 15.4g,80.3 mmol), N-diisopropylethylamine (DIEA, 30mL,172 mmol) and 1-hydroxybenzotriazole hydrate (HOBt, 8.4g,70.6 mmol). The reaction mixture was stirred at room temperature overnight and then diluted with EtOAc. Separating the organic layer and separating the organic layer with H 2 O, citric acid (5%), H 2 O、NaHCO 3 、H 2 O and brine were washed sequentially, followed by anhydrous Na 2 SO 4 Dried, filtered, and concentrated to give protected dipeptide as a white solid (13.74 g, 91%).
Figure BDA0001546710350002421
Is synthesized by (a)
The protected dipeptide (10.33 g,27.2 mmol) described above was cleaved at 0℃in CH 2 Cl 2 Pyridine (8 mL,99.1 mmol) and dess-Martin periodate (14 g,33.0 mmol) were added to a solution in (300 mL). The reaction mixture was stirred overnight and then taken up with NaHCO 3 /Na 2 S 2 O 3 The saturated aqueous solution (1:1) was stopped. The organic layer is treated with H 2 O, citric acid, H 2 O and brine were washed sequentially, followed by anhydrous Na 2 SO 4 Drying, filtering and concentrating. The residue was purified by flash chromatography (silica, 9:1-1:1 hexanes: etOAc) to give the product as a white solid (10.12 g, 98%).
Figure BDA0001546710350002422
Is synthesized by (a)
To a solution of dipeptide aldehyde (10.1 g,26.7 mmol) in EtOH (200 mL) was added acetic acid hydrazide (3.7 g,45 mmol). The reaction mixture was stirred at room temperature for 30 min and concentrated. H is added to the residue 2 O (1L) and CH 2 Cl 2 (500 mL). The organic layer was separated and concentrated to give a white solid (11.21 g, 97%).
Figure BDA0001546710350002423
Is synthesized by (a)
To a solution of the above methyl ester (11.1 g,25.6 mmol) in dioxane (50 mL) was added LiOH (50 mL,1 n) at 0 ℃. The mixture was stirred at the same temperature for 30 minutes and then quenched with citric acid (20 g) and with H 2 O (200 mL) dilution. The mixture was extracted with EtOAc. The organic layer is treated with H 2 O and brine were washed sequentially, followed by anhydrous Na 2 SO 4 Dried, filtered, and concentrated to give a white solid (9.52 g, 88%).
Figure BDA0001546710350002431
Is synthesized by (a)
The acid (9.5 g,22.6 mmol) was added to CH at 0deg.C 2 Cl 2 Trifluoroacetic acid (50 mL) was added to the solution in (50 mL). The mixture was stirred at 0 ℃ for 1 hour and concentrated in vacuo. HCl (7 mL,4n in dioxane) was added to the residue followed by diethyl ether (500 mL). The precipitate was collected as a white solid (7.25 g, 90%).
Example 55
This example details the synthesis of the oxime-containing amino acid presented in figure 46A.
Figure BDA0001546710350002432
Is synthesized by (a)
Aldehyde (3.0 g) in MeOH/H + To the solution in (2) was added hydroxylamine hydrochloride. The reaction mixture was stirred at room temperature for 2 hours and concentrated. H is added to the residue 2 O (200 mL), followed by CH addition 2 Cl 2 . The organic layer was separated and concentrated in vacuo. Purification of the residue by flash chromatography (silica, 3:7-1:9 hexanes: etOAc) afforded the product as a solid (96%).
Figure BDA0001546710350002433
Is synthesized by (a)
To a solution of the above methyl ester (3.0 g) in dioxane (10 mL) was added LiO at 0deg.CH (10 mL, 1N). The mixture was stirred at the same temperature for 3 hours and then quenched by the addition of citric acid (5 g) and taken up with H 2 O dilution. The mixture was extracted with EtOAc. The organic layer is treated with H 2 O and brine were washed sequentially, followed by anhydrous Na 2 SO 4 Dried, filtered, and concentrated to give a solid. Subsequently, the acid obtained is reacted with CH at 0 DEG C 2 Cl 2 Trifluoroacetic acid (20 mL) was added to the solution in (20 mL). The reaction mixture was stirred at 0 ℃ for 1 hour and concentrated. MeOH (1 mL) was added to the residue followed by HCl (2.0 mL,4n in dioxane). Diethyl ether (200 mL) was then added to precipitate the product as a solid (87%).
Example 56
This example details the synthesis of the oxime-containing amino acid presented in fig. 46B.
Figure BDA0001546710350002441
Is synthesized by (a)
Aldehyde (3.0 g) in MeOH/H + To the solution in (2) was added 2 equivalents of methoxyamine hydrochloride. The reaction mixture was stirred at room temperature for 2 hours and concentrated. H is added to the residue 2 O (200 mL), followed by CH addition 2 Cl 2 . The organic layer was separated and concentrated in vacuo. Purification of the residue by flash chromatography (silica, 3:7-1:9 hexanes: etOAc) afforded the product as a solid (93%).
Figure BDA0001546710350002442
Is synthesized by (a)
To a solution of the above methyl ester (3.0 g) in dioxane (10 mL) was added LiOH (10 mL,1 n) at 0 ℃. The mixture was stirred at the same temperature for 3 hours and then quenched by the addition of citric acid (5 g) and taken up with H 2 O dilution. The mixture was extracted with EtOAc. The organic layer is treated with H 2 O and brine were washed sequentially, followed by anhydrous Na 2 SO 4 Dried, filtered, and concentrated to give a solid. Subsequently, the acid obtained is reacted with CH at 0 DEG C 2 Cl 2 Addition of trifluoro to the solution in (20 mL)Acetic acid (20 mL). The reaction mixture was stirred at 0 ℃ for 1 hour and concentrated. MeOH (1 mL) was added to the residue followed by HCl (2.0 mL,4n in dioxane). Diethyl ether (200 mL) was then added to precipitate the product as a solid (89%).
Example 57
This example details the synthesis of hydrazine-containing amino acids presented in fig. 46C.
Figure BDA0001546710350002451
Is synthesized by (a)
Aldehyde (3.0 g) in MeOH/H + To the solution in (2) was added 2 equivalents of methyl hydrazine. The reaction mixture was stirred at room temperature for 2 hours and concentrated. H is added to the residue 2 O (200 mL), followed by CH addition 2 Cl 2 . The organic layer was separated and concentrated in vacuo. Purification of the residue by flash chromatography (silica, 3:7-1:9 hexanes: etOAc) afforded the product as a solid (93%).
Figure BDA0001546710350002452
Is synthesized by (a)
To a solution of the above methyl ester (3.0 g) in dioxane (10 mL) was added LiOH (10 mL,1 n) at 0 ℃. The mixture was stirred at the same temperature for 3 hours and then quenched by the addition of citric acid (5 g) and taken up with H 2 O dilution. The mixture was extracted with EtOAc. The organic layer is treated with H 2 O and brine were washed sequentially, followed by anhydrous Na 2 SO 4 Dried, filtered, and concentrated to give a solid. Subsequently, the acid obtained is reacted with CH at 0 DEG C 2 Cl 2 Trifluoroacetic acid (20 mL) was added to the solution in (20 mL). The reaction mixture was stirred at 0 ℃ for 1 hour and concentrated. MeOH (1 mL) was added to the residue followed by HCl (2.0 mL,4n in dioxane). Diethyl ether (200 mL) was then added to precipitate the product as a solid (89%).
Example 58
This example details the synthesis of mPEG-hydroxylamine presented in fig. 48A.
Figure BDA0001546710350002453
Is synthesized by (a)
To mPEG (30K) -OH (1.0 g,0.033 mmol) in dry CH 2 Cl 2 To the solution in (10 mL) was added p-nitrophenol chloroformate (60 mg,0.28 mmol). The mixture was stirred at room temperature for 15 hours. Diethyl ether (200 mL) was added. The precipitate was filtered, washed with diethyl ether and dried in vacuo to give the product as a white powder (1.0 g, 100%).
Figure BDA0001546710350002461
Is synthesized by (a)
To a solution of tert-butyl 3-hydroxyethylcarbamate (1.75 g,10 mmol) in THF (60 mL) was added N-hydroxyphthalimide (3.2 g,20 mmol), triphenylphosphine (2.0 g,15 mmol). The reaction mixture was stirred at room temperature for 10 minutes and then cooled to 0 ℃. Diisopropylazodicarbonate (DIAD, 2.0mL,10.5 mmol) was added dropwise via syringe over 1 hour. The ice bath was removed and the mixture was stirred overnight and concentrated. The white solid was dissolved in ethyl acetate (100 mL). The reaction mixture was treated with saturated aqueous sodium hydrogencarbonate (100 mL) and H 2 O (100 mL) and brine (100 mL) followed by anhydrous MgSO 4 Dried, filtered and concentrated in vacuo. The crude product was purified by flash chromatography (silica, 100:1-10:1 hexanes: etOAc) to give the title compound (2.6 g, 81%) as a white solid.
Figure BDA0001546710350002462
Is synthesized by (a)
To Jing Shuding oxycarbonyl-protected linker (2.0 g,9.1 mmol) on CH 2 Cl 2 To the solution in (5 mL) was added trifluoroacetic acid (5 mL). The resulting mixture was stirred at room temperature for 1 hour and concentrated. To the residue was added HCl (4N in dioxane, 1.5 mL), followed by Et 2 O (150 mL). The precipitate was filtered, washed with diethyl ether and dried in vacuo to give Amine linker (1.1 g, 85%) as a white solid.
Figure BDA0001546710350002463
Is synthesized by (a)
To mPEG (30K) p-nitrophenol carbonate (1.0 g,0.033 mmol) and amine linker (53 mg,0.21 mmol) in DMF-CH 2 Cl 2 Diisopropylethylamine (50. Mu.L, 0.28 mmol) and DMAP (5 mg,0.041 mmol) were added to the mixture in (10 mL, 1:2). The resulting mixture was stirred at room temperature for 15 hours. Diethyl ether (200 mL) was added. The precipitate was filtered, washed with diethyl ether and dried in vacuo to give the product as a white powder (0.83 g, 83%).
Figure BDA0001546710350002471
Is synthesized by (a)
To a solution of mPEG phthalimide (30K, 0.8g,0.0266 mmol) in MeOH (5 mL) was added hydrazine (8.5. Mu.L, 0.27 mmol). The resulting mixture was stirred at 45℃for 1.0 hour. After the reaction cooled to room temperature, CH was added 2 Cl 2 (150 mL) and the solution was washed with aqueous HCl (0.1N, 100 mL). By CH 2 Cl 2 (150 mL) the aqueous layer was extracted. Combining the organic layers and using H 2 O (100 mL) followed by anhydrous Na 2 SO 4 Drying, filtering and concentrating. Dissolving the residue in CH 2 Cl 2 (5 mL). Diethyl ether (200 mL) was added to precipitate hydroxylamine product (0.72 g, 90%) as a white powder.
Example 59
This example details the synthesis of mPEG-hydroxylamine presented in fig. 48B.
Figure BDA0001546710350002472
Is synthesized by (a)
To mPEG (30K) -OH (1.0 g,0.033 mmol) in dry CH 2 Cl 2 To the solution in (10 mL) was added p-nitrophenol chloroformate (60 mg,0.28 mmol). The mixture 1 was stirred at room temperatureAnd 5 hours. Diethyl ether (200 mL) was added. The precipitate was filtered, washed with diethyl ether and dried in vacuo to give the product as a white powder (1.0 g, 100%).
Figure BDA0001546710350002473
Is synthesized by (a)
To a solution of tert-butyl 2-hydroxyethylcarbamate (2.8 mL,18 mmol) in THF (60 mL) was added N-hydroxyphthalimide (5.8 g,36 mmol), triphenylphosphine (3.6 g,27 mmol). The reaction mixture was stirred at room temperature for 10 minutes and then cooled to 0 ℃. Diisopropylazodicarbonate (DIAD, 3.6ml,19 mmol) was added dropwise via syringe over 1 hour. The ice bath was removed and the mixture was stirred overnight and concentrated. The white solid was dissolved in ethyl acetate (100 mL). The reaction mixture was treated with saturated aqueous sodium hydrogencarbonate (2X 50 mL) and H 2 O (50 mL) and brine (50 mL) were washed sequentially followed by anhydrous MgSO 4 Dried, filtered and concentrated in vacuo. By using Biotage inc TM Flash chromatography of the chromatographic system purified the crude product to give the title compound (12 g, 206%) as a white solid containing impurities.
Figure BDA0001546710350002481
Is synthesized by (a)
To crude Jing Shuding oxycarbonyl-protected linker (12 g) to CH 2 Cl 2 To the solution in (5 mL) was added trifluoroacetic acid (5 mL). The resulting mixture was stirred at room temperature for 1 hour and concentrated. To the residue was added HCl (4N in dioxane, 1.5 mL), followed by Et 2 O (150 mL). The precipitate was filtered, washed with diethyl ether and dried in vacuo to give the amine linker as a white solid (3.0 g, 68% for both steps).
Figure BDA0001546710350002482
Is synthesized by (a)
To mPEG (30K) p-nitrophenol carbonate (1.0 g,0.033 mmol) and amineLinker (50 mg,0.21 mmol) in DMF-CH 2 Cl 2 Diisopropylethylamine (50. Mu.L, 0.28 mmol) and 4-dimethylaminopyridine (4 mg,0.033 mmol) were added to the mixture in (10 mL, 1:2). The resulting mixture was stirred at room temperature for 15 hours. Diethyl ether (200 mL) was added. The precipitate was filtered, washed with diethyl ether and dried in vacuo to give the product as a white powder (0.81 g, 81%).
Figure BDA0001546710350002483
Is synthesized by (a)
To a solution of mPEG (30K) phthalimide (0.8 g,0.0266 mmol) in MeOH (5 mL) was added hydrazine (8.5 μl,0.27 mmol). The resulting mixture was stirred at 45℃for 1.0 hour. After the reaction cooled to room temperature, CH was added 2 Cl 2 (150 mL) and the solution was washed with aqueous HCl (0.1N, 100 mL). By CH 2 Cl 2 (150 mL) the aqueous layer was extracted. Combining the organic layers and using H 2 O (100 mL) followed by anhydrous Na 2 SO 4 Drying, filtering and concentrating. Dissolving the residue in CH 2 Cl 2 (5 mL). Diethyl ether (200 mL) was added to precipitate hydroxylamine product (0.68 g, 85%) as a white powder.
Example 60
This example details the synthesis of mPEG-hydroxylamine presented in fig. 49A.
Figure BDA0001546710350002491
Is synthesized by (a)
To a solution of N- (3-bromopropyl) phthalimide (4.0 g,15.0 mmol) in DMF (50 mL) at 0deg.C was added K 2 CO 3 (10 g,73 mmol) and t-butyl N-hydroxycarbamate (2.5 g,18.8 mmol). The reaction mixture was stirred at room temperature for 3 hours. The mixture is treated with H 2 O was diluted (200 mL) and extracted with EtOAc (200 mL). The organic layer is treated with H 2 O and brine followed by anhydrous Na 2 SO 4 Dried, filtered and concentrated in vacuo. The residue was purified by flash chromatography (silica, 20:1-3:1 hexanes: etOAc)The residue gave the product (3.5 g, 72%) as a colourless oil.
Figure BDA0001546710350002492
Is synthesized by (a)
To a solution of the above phthalimide (500 mg,1.6 mmol) in EtOH (10 mL) was added hydrazine (0.25 mL,8.0 mmol). The resulting mixture was stirred at room temperature for 3 hours. After removing the precipitate, the filtrate was concentrated. The residue was left under high vacuum overnight. Purification of the residue by flash chromatography (silica, 3:1-1:1EtOAc: meOH) afforded the amine linker as a white solid (252 mg, 85%).
Figure BDA0001546710350002493
Is synthesized by (a)
To mPEG (30K) -OH (1.0 g,0.033 mmol) in dry CH 2 Cl 2 To the solution in (10 mL) was added p-nitrophenol chloroformate (60 mg,0.28 mmol). The mixture was stirred at room temperature for 15 hours. Diethyl ether (200 mL) was added. The precipitate was filtered, washed with diethyl ether and dried in vacuo to give the product as a white solid (1.0 g, 100%).
Figure BDA0001546710350002501
Is synthesized by (a)
The above activated mPEG (30K) (3.0 g,0.1 mmol) in dry CH 2 Cl 2 To a solution in (30 mL) were added diisopropylethylamine (88. Mu.L, 0.5 mmol) and an amine linker (76 mg,0.4 mmol). The resulting mixture was stirred at room temperature for 15 hours. Diethyl ether (700 mL) was added to the reaction mixture. The precipitate was filtered, washed with diethyl ether and dried in vacuo to give a white powder (2.8 g, 93%).
Figure BDA0001546710350002502
Is synthesized by (a)
To Jing Shuding oxycarbonyl-protected mPEG (30K) (2.0 g,0.067 mmol) in anhydrous CH at 0deg.C 2 Cl 2 Trifluoroacetic acid (10 mL) was added to the solution in (10 mL). The resulting mixture was stirred at room temperature for 5 hours. Diethyl ether (500 mL) was added to the reaction mixture. The precipitate was filtered, washed with diethyl ether and dried in vacuo to give a white powder (1.8 g, 90%).
Example 61
This example details the synthesis of mPEG-hydroxylamine presented in fig. 49B.
Figure BDA0001546710350002503
Is synthesized by (a)
To a solution of tert-butyl N-hydroxycarbamate (5.0 g,37.6 mmol) in DMF (30 mL) was added K at 0deg.C 2 CO 3 (12 g,87.6 mmol) and N- (2-bromoethyl) phthalimide (10.0 g,39.7 mmol). The reaction mixture was stirred at room temperature for 3 hours. The mixture is treated with H 2 O was diluted (200 mL) and extracted with EtOAc (200 mL). The organic layer is treated with H 2 O and brine followed by anhydrous Na 2 SO 4 Dried, filtered and concentrated in vacuo. Purification of the residue by flash chromatography (silica, 20:1-1:1 hexanes: etOAc) afforded the product as a white solid (5.2 g, 55%).
Figure BDA0001546710350002511
Is synthesized by (a)
To a solution of the above phthalimide (500 mg,1.6 mmol) in EtOH (10 mL) was added hydrazine (0.25 mL,8.0 mmol). The resulting mixture was stirred at room temperature for 3 hours. After removing the precipitate, the filtrate was concentrated. The residue was left under high vacuum overnight. Purification of the residue by flash chromatography (silica, 3:1-1:1EtOAc: meOH) afforded the amine linker as a white solid (301 mg, 86%).
Figure BDA0001546710350002512
Is synthesized by (a)
The above activated mPEG (30K) (1.0 g,0.033 mmol) in dry CH 2 Cl 2 (1To a solution of 0 mL) was added diisopropylethylamine (58. Mu.L, 0.33 mmol), 4-dimethylaminopyridine (4 mg,0.033 mmol) and the amine linker (64 mg,0.31 mmol). The resulting mixture was stirred at room temperature for 15 hours. Diethyl ether (200 mL) was added to the reaction mixture. The precipitate was filtered, washed with diethyl ether and dried in vacuo to give a white powder (0.85 g, 85%).
Figure BDA0001546710350002513
Is synthesized by (a)
To Jing Shuding oxycarbonyl-protected mPEG (30K) (0.85 g,0.028 mmol) in anhydrous CH at 0deg.C 2 Cl 2 Trifluoroacetic acid (10 mL) was added to the solution in (10 mL). The resulting mixture was stirred at room temperature for 5 hours. Diethyl ether (200 mL) was added to the reaction mixture. The precipitate was filtered, washed with diethyl ether and dried in vacuo to give a white powder (0.68 g, 80%).
Example 62
This example details the synthesis of mPEG-hydroxylamine presented in fig. 50A.
Figure BDA0001546710350002521
Is synthesized by (a)
To a solution of mono-tert-butyloxycarbonyl phthalimide (2.1 g,6.9 mmol) in pyridine (50 mL) at 0deg.C was added Boc 2 O (3.3 g,15.1 mmol). The resulting mixture was heated to 60 ℃ overnight. After removal of the solvent in vacuo, the residue was diluted with EtOAc (200 mL) and washed with citric acid (5%, 200 mL), water (200 mL) and brine (200 mL), followed by anhydrous Na 2 SO 4 Dried, filtered and concentrated in vacuo. Purification of the residue by flash chromatography (silica, 20:1-3:1 hexanes: etOAc) afforded the product as a yellow oil (2.37 g, 85%).
Figure BDA0001546710350002522
Is synthesized by (a)
To a solution of di-tert-butoxycarbonyl phthalimide (1.21 g,2.98 mmol) in MeOH (15 mL) was added ammonia (15 mL,7N,105 mmol) in MeOH at 0deg.C. The resulting mixture was stirred at room temperature overnight. The precipitate was filtered off and the filtrate was concentrated in vacuo. Purification of the residue by flash chromatography (silica, 10:1-6:4EtOAc: meOH) afforded the amine linker as a white solid (0.61 g, 74%).
Figure BDA0001546710350002523
Is synthesized by (a)
To mPEG (30K) -OH (1.0 g,0.033 mmol) in dry CH 2 Cl 2 To the solution in (10 mL) was added p-nitrophenol chloroformate (60 mg,0.28 mmol). The mixture was stirred at room temperature for 15 hours. Diethyl ether (200 mL) was added to precipitate mPEG (30K) product. The product was filtered, washed with diethyl ether and dried in vacuo (1.0 g, 100%).
Figure BDA0001546710350002524
Is synthesized by (a)
The above activated mPEG (30K) (1.0 g,0.033 mmol) in dry CH 2 Cl 2 To a solution of (10 mL) was added diisopropylethylamine (58. Mu.L, 0.33 mmol), 4-dimethylaminopyridine (5 mg,0.041 mmol) and the above-mentioned di-t-butoxycarbonyl amine linker (90 mg,0.33 mmol). The resulting mixture was stirred at room temperature for 15 hours. Diethyl ether (200 mL) was added to the reaction mixture. The precipitate was filtered, washed with diethyl ether and dried in vacuo to give a white powder (0.82 g, 82%).
Figure BDA0001546710350002531
Is synthesized by (a)
To Jing Shuding oxycarbonyl-protected mPEG (30K) (0.82 g,0.027 mmol) in anhydrous CH at 0deg.C 2 Cl 2 Trifluoroacetic acid (8 mL) was added to the solution in (8 mL). The resulting mixture was stirred at room temperature for 5 hours. Diethyl ether (200 mL) was added to the reaction mixture. The precipitate was filtered, washed with diethyl ether and dried in vacuo to give a white powder (0.57 g, 70%).
Example 63
This example details the synthesis of mPEG-hydroxylamine presented in fig. 50B.
Figure BDA0001546710350002532
Is synthesized by (a)
To a solution of mono-tert-butyloxycarbonyl phthalimide (1.5 g,4.7 mmol) in pyridine was added Boc at 0deg.C 2 O (2.2 g,10.0 mmol). The resulting mixture was heated to 60 ℃ overnight. After removal of the solvent in vacuo, the residue was diluted with EtOAc (200 mL) and washed with citric acid (5%, 200 mL), water (200 mL) and brine (200 mL), followed by anhydrous Na 2 SO 4 Dried, filtered and concentrated in vacuo. The residue was purified by flash chromatography (silica, 20:1-3:1 hexanes: etOAc) to give the product as an oil (1.6 g, 81%).
Figure BDA0001546710350002533
Synthesis of->
To a solution of di-tert-butoxycarbonyl phthalimide (1.5 g,3.6 mmol) in MeOH (15 mL) was added ammonia (15 mL,7N,105 mmol) in MeOH at 0deg.C. The resulting mixture was stirred at room temperature overnight. The precipitate was filtered off and the filtrate was concentrated in vacuo. Purification of the residue by flash chromatography (silica, 10:1-6:4EtOAc: meOH) afforded the amine linker as a white solid (0.85 g, 82%).
Figure BDA0001546710350002541
Is synthesized by (a)
To mPEG (30K) -OH (1.0 g,0.033 mmol) in dry CH 2 Cl 2 To the solution in (10 mL) was added p-nitrophenol chloroformate (60 mg,0.28 mmol). The mixture was stirred at room temperature for 15 hours. Diethyl ether (200 mL) was added. The precipitate was filtered, washed with diethyl ether and dried in vacuo to give the product as a white powder (1.0 g, 100%).
Figure BDA0001546710350002542
Is synthesized by (a)
The above activated mPEG (30K) (1.0 g,0.033 mmol) in dry CH 2 Cl 2 To a solution of (10 mL) was added diisopropylethylamine (58. Mu.L, 0.33 mmol), 4-dimethylaminopyridine (5 mg,0.041 mmol) and the above-mentioned di-t-butoxycarbonyl amine linker (100 mg,0.34 mmol). The resulting mixture was stirred at room temperature for 15 hours. Diethyl ether (200 mL) was added to the reaction mixture. The precipitate was filtered, washed with diethyl ether and dried in vacuo to give a white powder (0.89 g, 89%).
Figure BDA0001546710350002543
Is synthesized by (a)
To Jing Shuding oxycarbonyl-protected mPEG (30K) (0.89 g,0.030 mmol) in anhydrous CH at 0deg.C 2 Cl 2 Trifluoroacetic acid (8 mL) was added to the solution in (8 mL). The resulting mixture was stirred at room temperature for 5 hours. Diethyl ether (200 mL) was added to the reaction mixture. The precipitate was filtered, washed with diethyl ether and dried in vacuo to give a white powder (0.65 g, 73%).
Example 64
This example details the synthesis of mPEG-hydroxylamine presented in fig. 51A.
Figure BDA0001546710350002544
Is synthesized by (a)
To a mixture of mPEG (30K) propanal (0.5 g,0.0166 mmol) and an amine linker (73 mg,0.25 mmol) in MeOH (10 mL) was added NaCNBH 3 (20 mg,0.30 mmol). The resulting mixture was stirred at room temperature for 48 hours. After removing most of the solvent, diethyl ether (200 mL) was added. The precipitate was filtered, washed with diethyl ether and dried in vacuo to give a white powder (0.43 g, 86%).
Figure BDA0001546710350002551
Is synthesized by (a)
To di-tert-butoxycarbonyl protected mPEG (30K) (0.42 g,0.014 mmol) in anhydrous CH at 0deg.C 2 Cl 2 To the solution in (4 mL) was added trifluoroacetic acid (4 mL). The resulting mixture was stirred at room temperature for 8 hours. Diethyl ether (100 mL) was added to the reaction mixture. The precipitate was filtered, washed with diethyl ether and dried in vacuo to give a white powder (0.35 g, 83%).
Example 65
This example details the synthesis of mPEG-hydroxylamine presented in fig. 51B.
Figure BDA0001546710350002552
Is synthesized by (a)
To mPEG (30K) (6.0 g,0.2 mmol) and pyridine (0.1 mL,1.2 mmol) at 0deg.C in CH 2 Cl 2 To the stirred solution in (60 mL) was added dess-Martin periodate (0.2 g,0.47 mmol). The mixture was stirred at room temperature overnight. The reaction was then carried out with Na 2 S 2 O 3 -NaHCO 3 Saturated aqueous solution (1:1, 100 mL) was stopped and taken up in CH 2 Cl 2 (500 mL. Times.2) extraction. The organic layers were combined and washed with water and brine, followed by anhydrous Na 2 SO 4 Dried, filtered and concentrated in vacuo. Dissolving the residue in CH 2 Cl 2 (50 mL). Diethyl ether (1L) was added to the solution. The precipitate was filtered, washed with diethyl ether and dried in vacuo to give a white powder (4.9 g, 82%).
Figure BDA0001546710350002553
Is synthesized by (a)
To a mixture of mPEG (30K) acetaldehyde (0.5 g,0.0166 mmol) and an amine linker (73 mg,0.25 mmol) in MeOH (10 mL) was added NaCNBH 3 (20 mg,0.30 mmol). The resulting mixture was stirred at room temperature for 48 hours. After removing most of the solvent, diethyl ether (200 mL) was added. The precipitate was filtered, washed with diethyl ether and dried in vacuo to give a white powder (0.43 g, 85%).
Figure BDA0001546710350002561
Is synthesized by (a)
To di-tert-butoxycarbonyl protected mPEG (30K) (0.42 g,0.014 mmol) in anhydrous CH at 0deg.C 2 Cl 2 To the solution in (4 mL) was added trifluoroacetic acid (4 mL). The resulting mixture was stirred at room temperature for 8 hours. Diethyl ether (100 mL) was added to the reaction mixture. The precipitate was filtered, washed with diethyl ether and dried in vacuo to give a white powder (0.35 g, 83%).
Example 66
This example details the synthesis of mPEG-hydroxylamine presented in fig. 52A.
Figure BDA0001546710350002562
Is synthesized by (a)
To mPEG (30K) -NH 2 To a solution of (6.0 g,0.2 mmol) in DMF (60 mL) was added Boc-Ser-OH (205 mg,1.0 mmol), 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC, 190mg,1.0 mmol) and N, N' -diisopropylethylamine (0.17 mL,1.0 mmol). The mixture was stirred at room temperature for 10 hours and diluted with EtOAc (500 mL). The mixture was treated with NaHCO 3 Saturated aqueous solution (300 mL), H 2 O (300 mL) and brine (300 mL) followed by anhydrous Na 2 SO 4 Dried, filtered and concentrated in vacuo. Dissolving the residue in CH 2 Cl 2 (50 mL). Diethyl ether (700 mL) was added. The precipitate was filtered, washed with diethyl ether and dried in vacuo to give a white powder (5.1 g, 82%).
Figure BDA0001546710350002563
Is synthesized by (a)
At 0deg.C, the above mPEG (30K) (3.0 g,0.1 mmol) in CH 2 Cl 2 Trifluoroacetic acid (15 mL) was added to the solution in (15 mL). The resulting mixture was stirred at room temperature for 3 hours and concentrated. Addition of CH to the residue 2 Cl 2 (5 mL) followed by addition of HCl(4N in dioxane, 2 mL). Diethyl ether (400 mL) was added to precipitate the dihydroxyamine (2.6 g, 85%) as a white solid.
Figure BDA0001546710350002571
Is synthesized by (a)
The above mPEG (30K) (2.0 g,0.067 mmol) in H 2 O-CH 3 NaIO was added to a solution in CN (1:1, 20 mL) 4 (15 mg,0.07 mmol). The mixture was stirred at room temperature for 4.0 hours and with CH 2 Cl 2 (500 mL) dilution. The resulting mixture was washed with water (100 mL) and brine (100 mL), followed by anhydrous Na 2 SO 4 Dried, filtered and concentrated in vacuo. Dissolving the residue in CH 2 Cl 2 (50 mL). Diethyl ether (700 mL) was added. The precipitate was filtered, washed with diethyl ether and dried in vacuo to give a white powder (1.8 g, 90%).
Figure BDA0001546710350002572
Is synthesized by (a)
To a mixture of mPEG (30K) acetaldehyde (0.5 g,0.0166 mmol) and an amine linker (73 mg,0.25 mmol) in MeOH (10 mL) was added NaCNBH 3 (20 mg,0.30 mmol). The resulting mixture was stirred at room temperature for 48 hours. After removing most of the solvent, diethyl ether (200 mL) was added. The precipitate was filtered, washed with diethyl ether and dried in vacuo to give a white powder (0.43 g, 86%).
Figure BDA0001546710350002573
Is synthesized by (a)
To di-tert-butoxycarbonyl protected mPEG (30K) (0.42 g,0.014 mmol) in anhydrous CH at 0deg.C 2 Cl 2 To the solution in (4 mL) was added trifluoroacetic acid (4 mL). The resulting mixture was stirred at room temperature for 8 hours. Diethyl ether (100 mL) was added to the reaction mixture. The precipitate was filtered, washed with diethyl ether and dried in vacuo to give a white powder (0.35 g, 83%).
Example 67
This example details the synthesis of mPEG 4-hydroxylamine presented in fig. 52B.
Figure BDA0001546710350002574
Is synthesized by (a)
To a mixture of mPEG propionaldehyde (30K, 0.5g,0.0166 mmol) and amine linker (70 mg,0.25 mmol) in MeOH (10 mL) was added NaCNBH 3 (20 mg,0.30 mmol). The resulting mixture was stirred at room temperature for 48 hours. After removing most of the solvent, diethyl ether (200 mL) was added. The precipitate was filtered, washed with diethyl ether and dried in vacuo to give a white powder (0.40 g, 80%).
Figure BDA0001546710350002581
Is synthesized by (a)
To di-tert-butoxycarbonyl protected mPEG (30K) (0.40 g,0.013 mmol) in anhydrous CH at 0deg.C 2 Cl 2 To the solution in (4 mL) was added trifluoroacetic acid (4 mL). The resulting mixture was stirred at room temperature for 8 hours. Diethyl ether (100 mL) was added to the reaction mixture. The precipitate was filtered, washed with diethyl ether and dried in vacuo to give a white powder (0.35 g, 87%).
Example 68
This example details the synthesis of mPEG-hydroxylamine presented in fig. 53A.
Figure BDA0001546710350002582
Is synthesized by (a)
To mPEG (30K) (6.0 g,0.2 mmol) and pyridine (0.1 mL,1.2 mmol) at 0deg.C in CH 2 Cl 2 To the stirred solution in (60 mL) was added dess-Martin periodate (0.2 g,0.47 mmol). The mixture was stirred at room temperature overnight. The reaction is carried out with Na 2 S 2 O 3 -NaHCO 3 Saturated aqueous solution (1:1, 100 mL) was stopped and taken up in CH 2 Cl 2 (500 mL. Times.2) extraction. Combining the organic layers and using H 2 O and brine followed by anhydrous Na 2 SO 4 Dried, filtered and concentrated in vacuo. Dissolving the residue in CH 2 Cl 2 (50 mL). Diethyl ether (1L) was added to the solution. The precipitate was filtered, washed with diethyl ether and dried in vacuo to give a white powder (4.9 g, 82%).
Figure BDA0001546710350002583
Is synthesized by (a)
To a mixture of mPEG (30K) acetaldehyde (0.5 g,0.0166 mmol) and an amine linker (70 mg,0.25 mmol) in MeOH (10 mL) was added NaCNBH 3 (20 mg,0.30 mmol). The resulting mixture was stirred at room temperature for 48 hours. After removing most of the solvent, diethyl ether (200 mL) was added. The precipitate was filtered, washed with diethyl ether and dried in vacuo to give a white powder (0.40 g, 80%).
Figure BDA0001546710350002591
Is synthesized by (a)
To di-tert-butoxycarbonyl protected mPEG (30K) (0.40 g,0.013 mmol) in anhydrous CH at 0deg.C 2 Cl 2 To the solution in (4 mL) was added trifluoroacetic acid (4 mL). The resulting mixture was stirred at room temperature for 8 hours. Diethyl ether (100 mL) was added to the reaction mixture. The precipitate was filtered, washed with diethyl ether and dried in vacuo to give a white powder (0.35 g, 87%).
Example 69
This example details the synthesis of mPEG-hydroxylamine presented in fig. 53B.
Figure BDA0001546710350002592
Is synthesized by (a)
To mPEG (30K) -NH 2 To a solution of (6.0 g,0.2 mmol) in DMF (60 mL) was added Boc-Ser-OH (205 mg,1.0 mmol), 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC, 190mg,1.0 mmol) and N, N' -diisopropylethylamine (0.17 mL,1.0 mmol). The mixture was stirred at room temperature for 10 hours and with EtOAc (500 mL) And (5) diluting. The mixture was treated with NaHCO 3 Saturated aqueous solution (300 mL), H 2 O (300 mL) and brine (300 mL) followed by anhydrous Na 2 SO 4 Dried, filtered and concentrated in vacuo. Dissolving the residue in CH 2 Cl 2 (50 mL). Diethyl ether (700 mL) was added. The precipitate was filtered, washed with diethyl ether and dried in vacuo to give a white powder (5.1 g, 82%).
Figure BDA0001546710350002593
Is synthesized by (a)
At 0deg.C, the above mPEG (30K) (3.0 g,0.1 mmol) in CH 2 Cl 2 Trifluoroacetic acid (15 mL) was added to the solution in (15 mL). The resulting mixture was stirred at room temperature for 3 hours and concentrated. Addition of CH to the residue 2 Cl 2 (5 mL) followed by HCl (4N in dioxane, 2 mL). Diethyl ether (400 mL) was added to precipitate the dihydroxyamine (2.6 g, 85%) as a white solid.
Figure BDA0001546710350002601
Is synthesized by (a)
The above mPEG (30K) (2.0 g,0.067 mmol) in H 2 O-CH 3 NaIO was added to a solution in CN (1:1, 20 mL) 4 (15 mg,0.07 mmol). The mixture was stirred at room temperature for 4.0 hours and with CH 2 Cl 2 (500 mL) dilution. The mixture obtained is treated with H 2 O (100 mL) and brine (100 mL) followed by anhydrous Na 2 SO 4 Dried, filtered and concentrated in vacuo. Dissolving the residue in CH 2 Cl 2 (50 mL). Diethyl ether (700 mL) was added. The precipitate was filtered, washed with diethyl ether and dried in vacuo to give a white powder (1.8 g, 90%).
Figure BDA0001546710350002602
Is synthesized by (a)
To mPEG (30K) acetaldehyde (0.5 g,0.0166 mmol) and an amine linker (70 mg,0.25 mmol) in MeOH (10 mL)Adding NaCNBH to the mixture 3 (20 mg,0.30 mmol). The resulting mixture was stirred at room temperature for 48 hours. After removing most of the solvent, diethyl ether (200 mL) was added. The precipitate was filtered, washed with diethyl ether and dried in vacuo to give a white powder (0.40 g, 80%).
Figure BDA0001546710350002603
Is synthesized by (a)
To di-tert-butoxycarbonyl protected mPEG (30K) (0.40 g,0.013 mmol) in anhydrous CH at 0deg.C 2 Cl 2 To the solution in (4 mL) was added trifluoroacetic acid (4 mL). The resulting mixture was stirred at room temperature for 8 hours. Diethyl ether (100 mL) was added to the reaction mixture. The precipitate was filtered, washed with diethyl ether and dried in vacuo to give a white powder (0.35 g, 87%).
Example 70
This example details the synthesis of mPEG-hydroxylamine presented in fig. 54A.
Figure BDA0001546710350002611
Is synthesized by (a)
To a mixture of mPEG (30K) propanal (0.5 g,0.0166 mmol) and amine linker (40 mg,0.16 mmol) in MeOH (10 mL) was added NaCNBH 3 (12 mg,0.17 mmol). The resulting mixture was stirred at room temperature for 60 hours. After removal of the majority of the solvent, the residue was dissolved in CH 2 Cl 2 (200 mL) and washed with citric acid (5%, 100 mL). The organic layer was treated with anhydrous Na 2 SO 4 Dried, filtered and concentrated in vacuo to give a white solid (0.42 g, 84%).
Figure BDA0001546710350002612
Is synthesized by (a)
To a mixture of mPEG (30K) phthalimide (0.4 g,0.013 mmol) in MeOH (4 mL) was added H 2 NNH 2 (4.2. Mu.L, 0.13 mmol). The mixture was stirred at 45 ℃ for 1.0 hour and concentrated. The residue is taken upDissolved in CH 2 Cl 2 (100 mL) and washed with HCl (0.1N, 100 mL). By CH 2 Cl 2 (100 mL) the aqueous layer was extracted. Combining the organic layers and using H 2 O washing followed by anhydrous Na 2 SO 4 Drying, filtering and concentrating. Dissolving the residue in CH 2 Cl 2 (5 mL). Diethyl ether (200 mL) was added to precipitate hydroxylamine product (0.34 g, 85%) as a white powder.
Example 71
This example details the synthesis of mPEG-hydroxylamine presented in fig. 54B.
Figure BDA0001546710350002613
Is synthesized by (a)
To mPEG (30K) (6.0 g,0.2 mmol) and pyridine (0.1 mL,1.2 mmol) at 0deg.C in CH 2 Cl 2 To the stirred solution in (60 mL) was added dess-Martin periodate (0.2 g,0.47 mmol). The mixture was stirred at room temperature overnight. The reaction was then carried out with Na 2 S 2 O 3 -NaHCO 3 Saturated aqueous solution (1:1, 100 mL) was stopped and taken up in CH 2 Cl 2 (500 mL. Times.2) extraction. Combining the organic layers and using H 2 O and brine followed by anhydrous Na 2 SO 4 Dried, filtered and concentrated in vacuo. Dissolving the residue in CH 2 Cl 2 (50 mL). Diethyl ether (1L) was added to the solution. The precipitate was filtered, washed with diethyl ether and dried in vacuo to give a white powder (4.9 g, 82%).
Figure BDA0001546710350002621
Is synthesized by (a)
To a mixture of mPEG (30K) acetaldehyde (0.5 g,0.0166 mmol) and an amine linker (40 mg,0.16 mmol) in MeOH (10 mL) was added NaCNBH 3 (12 mg,0.17 mmol). The resulting mixture was stirred at room temperature for 60 hours. After removal of the majority of the solvent, the residue was dissolved in CH 2 Cl 2 (200 mL) and washed with citric acid (5%, 100 mL). The organic layer was treated with anhydrous Na 2 SO 4 Dried, filtered and concentrated in vacuo to give a white solid (0.42 g, 84%).
Figure BDA0001546710350002622
Is synthesized by (a)
To a mixture of mPEG (30K) phthalimide (0.4 g,0.013 mmol) in MeOH (4 mL) was added H 2 NNH 2 (4.2. Mu.L, 0.13 mmol). The mixture was stirred at 45 ℃ for 1.0 hour and concentrated. Dissolving the residue in CH 2 Cl 2 (100 mL) and washed with HCl (0.1N, 100 mL). By CH 2 Cl 2 (100 mL) the aqueous layer was extracted. Combining the organic layers and using H 2 O washing followed by anhydrous Na 2 SO 4 Drying, filtering and concentrating. Dissolving the residue in CH 2 Cl 2 (5 mL). Diethyl ether (200 mL) was added to precipitate hydroxylamine product (0.34 g, 85%) as a white powder.
Example 72
This example details the synthesis of mPEG-hydroxylamine presented in fig. 55.
Figure BDA0001546710350002623
Is synthesized by (a)
To mPEG (30K) -NH 2 To a solution of (6.0 g,0.2 mmol) in DMF (60 mL) was added Boc-Ser-OH (205 mg,1.0 mmol), 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC, 190mg,1.0 mmol) and N, N' -diisopropylethylamine (0.17 mL,1.0 mmol). The mixture was stirred at room temperature for 10 hours and diluted with EtOAc (500 mL). The mixture was treated with NaHCO 3 Saturated aqueous solution (300 mL), H 2 O (300 mL) and brine (300 mL) followed by anhydrous Na 2 SO 4 Dried, filtered and concentrated in vacuo. Dissolving the residue in CH 2 Cl 2 (50 mL). Diethyl ether (700 mL) was added. The precipitate was filtered, washed with diethyl ether and dried in vacuo to give a white powder (5.1 g, 82%).
Figure BDA0001546710350002631
Is synthesized by (a)
At 0deg.C, the above mPEG (30K) (3.0 g,0.1 mmol) in CH 2 Cl 2 Trifluoroacetic acid (15 mL) was added to the solution in (15 mL). The resulting mixture was stirred at room temperature for 3 hours and concentrated. Addition of CH to the residue 2 Cl 2 (5 mL) followed by HCl (4N in dioxane, 2 mL). Diethyl ether (400 mL) was added to precipitate the dihydroxyamine (2.6 g, 85%) as a white solid.
Figure BDA0001546710350002632
Is synthesized by (a)
The above mPEG (30K) (2.0 g,0.067 mmol) in H 2 O-CH 3 NaIO was added to a solution in CN (1:1, 20 mL) 4 (15 mg,0.07 mmol). The mixture was stirred at room temperature for 4.0 hours and with CH 2 Cl 2 (500 mL) dilution. The mixture obtained is treated with H 2 O (100 mL) and brine (100 mL) followed by anhydrous Na 2 SO 4 Dried, filtered and concentrated in vacuo. Dissolving the residue in CH 2 Cl 2 (50 mL). Diethyl ether (700 mL) was added. The precipitate was filtered, washed with diethyl ether and dried in vacuo to give a white powder (1.8 g, 90%).
Figure BDA0001546710350002633
Is synthesized by (a)
To a mixture of mPEG (30K) acetaldehyde (0.5 g,0.0166 mmol) and an amine linker (40 mg,0.16 mmol) in MeOH (10 mL) was added NaCNBH 3 (12 mg,0.17 mmol). The resulting mixture was stirred at room temperature for 60 hours. After removal of the majority of the solvent, the residue was dissolved in CH 2 Cl 2 (200 mL) and washed with citric acid (5%, 100 mL). The organic layer was treated with anhydrous Na 2 SO 4 Dried, filtered and concentrated in vacuo to give a white solid (0.42 g, 84%).
Figure BDA0001546710350002641
Is synthesized by (a)
To a mixture of mPEG (30K) phthalimide (0.4 g,0.013 mmol) in MeOH (4 mL) was added H 2 NNH 2 (4.2. Mu.L, 0.13 mmol). The mixture was stirred at 45 ℃ for 1.0 hour and concentrated. Dissolving the residue in CH 2 Cl 2 (100 mL) and washed with HCl (0.1N, 100 mL). By CH 2 Cl 2 (100 mL) the aqueous layer was extracted. Combining the organic layers and using H 2 O washing followed by anhydrous Na 2 SO 4 Drying, filtering and concentrating. Dissolving the residue in CH 2 Cl 2 (5 mL). Diethyl ether (200 mL) was added to precipitate hydroxylamine product (0.34 g, 85%) as a white powder.
EXAMPLE 73
This example details the synthesis of mPEG-hydroxylamine presented in fig. 56A.
Figure BDA0001546710350002642
Is synthesized by (a)
To a mixture of mPEG (30K) propanal (0.5 g,0.0166 mmol) and amine linker (40 mg,0.16 mmol) in MeOH (10 mL) was added NaCNBH 3 (12 mg,0.17 mmol). The resulting mixture was stirred at room temperature for 60 hours. After removal of the majority of the solvent, the residue was dissolved in CH 2 Cl 2 (200 mL) and washed with citric acid (5%, 100 mL). The organic layer was treated with anhydrous Na 2 SO 4 Dried, filtered and concentrated in vacuo to give a white solid (0.41 g, 82%).
Figure BDA0001546710350002643
Is synthesized by (a)
To a mixture of mPEG (30K) phthalimide (0.4 g,0.013 mmol) in MeOH (4 mL) was added H 2 NNH 2 (4.2. Mu.L, 0.13 mmol). The mixture was stirred at 45 ℃ for 1.0 hour and concentrated. Dissolving the residue in CH 2 Cl 2 (100 mL) and washed with HCl (0.1N, 100 mL). By CH 2 Cl 2 (100mL) of the aqueous layer. Combining the organic layers and using H 2 O washing followed by anhydrous Na 2 SO 4 Drying, filtering and concentrating. Dissolving the residue in CH 2 Cl 2 (5 mL). Diethyl ether (200 mL) was added to precipitate hydroxylamine product (0.34 g, 83%) as a white powder.
Example 74
This example details the synthesis of mPEG-hydroxylamine presented in fig. 56B.
Figure BDA0001546710350002651
Is synthesized by (a)
To mPEG (30K) (6.0 g,0.2 mmol) and pyridine (0.1 mL,1.2 mmol) at 0deg.C in CH 2 Cl 2 To the stirred solution in (60 mL) was added dess-Martin periodate (0.2 g,0.47 mmol). The mixture was stirred at room temperature overnight. The reaction was then carried out with Na 2 S 2 O 3 -NaHCO 3 Saturated aqueous solution (1:1, 100 mL) was stopped and taken up in CH 2 Cl 2 (500 mL. Times.2) extraction. Combining the organic layers and using H 2 O and brine followed by anhydrous Na 2 SO 4 Dried, filtered and concentrated in vacuo. Dissolving the residue in CH 2 Cl 2 (50 mL). Diethyl ether (1L) was added to the solution. The precipitate was filtered, washed with diethyl ether and dried in vacuo to give a white powder (4.9 g, 82%).
Figure BDA0001546710350002652
Is synthesized by (a)
To a mixture of mPEG (30K) acetaldehyde (0.5 g,0.0166 mmol) and an amine linker (40 mg,0.16 mmol) in MeOH (10 mL) was added NaCNBH 3 (12 mg,0.17 mmol). The resulting mixture was stirred at room temperature for 60 hours. After removal of the majority of the solvent, the residue was dissolved in CH 2 Cl 2 (200 mL) and washed with citric acid (5%, 100 mL). The organic layer was treated with anhydrous Na 2 SO 4 Dried, filtered and concentrated in vacuo to give a white solid (0.41 g, 82%).
Figure BDA0001546710350002653
Is synthesized by (a)
To a mixture of mPEG (30K) phthalimide (0.4 g,0.013 mmol) in MeOH (4 mL) was added H 2 NNH 2 (4.2. Mu.L, 0.13 mmol). The mixture was stirred at 45 ℃ for 1.0 hour and concentrated. Dissolving the residue in CH 2 Cl 2 (100 mL) and washed with HCl (0.1N, 100 mL). By CH 2 Cl 2 (100 mL) the aqueous layer was extracted. Combining the organic layers and using H 2 O washing followed by anhydrous Na 2 SO 4 Drying, filtering and concentrating. Dissolving the residue in CH 2 Cl 2 (5 mL). Diethyl ether (200 mL) was added to precipitate hydroxylamine product (0.34 g, 83%) as a white powder.
Example 75
This example details the synthesis of mPEG-hydroxylamine presented in fig. 57.
Figure BDA0001546710350002661
Is synthesized by (a)
To mPEG (30K) -NH 2 To a solution of (6.0 g,0.2 mmol) in DMF (60 mL) was added Boc-Ser-OH (205 mg,1.0 mmol), 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC, 190mg,1.0 mmol) and N, N' -diisopropylethylamine (0.17 mL,1.0 mmol). The mixture was stirred at room temperature for 10 hours and diluted with EtOAc (500 mL). The mixture was treated with NaHCO 3 Saturated aqueous solution (300 mL), H 2 O (300 mL) and brine (300 mL) followed by anhydrous Na 2 SO 4 Dried, filtered and concentrated in vacuo. Dissolving the residue in CH 2 Cl 2 (50 mL). Diethyl ether (700 mL) was added. The precipitate was filtered, washed with diethyl ether and dried in vacuo to give a white powder (5.1 g, 82%).
Figure BDA0001546710350002662
Is synthesized by (a)
At 0deg.C, the above mPEG (30K) (3.0 g,0.1 mmol) in CH 2 Cl 2 Trifluoroacetic acid (15 mL) was added to the solution in (15 mL). The resulting mixture was stirred at room temperature for 3 hours and concentrated. Addition of CH to the residue 2 Cl 2 (5 mL) followed by HCl (4N in dioxane, 2 mL). Diethyl ether (400 mL) was added to precipitate the dihydroxyamine (2.6 g, 85%) as a white solid.
Figure BDA0001546710350002663
Is synthesized by (a)
The above mPEG (30K) (2.0 g,0.067 mmol) in H 2 O-CH 3 NaIO was added to a solution in CN (1:1, 20 mL) 4 (15 mg,0.07 mmol). The mixture was stirred at room temperature for 4.0 hours and with CH 2 Cl 2 (500 mL) dilution. The mixture obtained is treated with H 2 O (100 mL) and brine (100 mL) followed by anhydrous Na 2 SO 4 Dried, filtered and concentrated in vacuo. Dissolving the residue in CH 2 Cl 2 (50 mL). Diethyl ether (700 mL) was added. The precipitate was filtered, washed with diethyl ether and dried in vacuo to give a white powder (1.8 g, 90%).
Figure BDA0001546710350002671
Is synthesized by (a)
To a mixture of mPEG (30K) acetaldehyde (0.5 g,0.0166 mmol) and an amine linker (40 mg,0.16 mmol) in MeOH (10 mL) was added NaCNBH 3 (12 mg,0.17 mmol). The resulting mixture was stirred at room temperature for 60 hours. After removal of the majority of the solvent, the residue was dissolved in CH 2 Cl 2 (200 mL) and washed with citric acid (5%, 100 mL). The organic layer was treated with anhydrous Na 2 SO 4 Dried, filtered and concentrated in vacuo to give a white solid (0.41 g, 82%).
Figure BDA0001546710350002672
Is synthesized by (a)
Ortho to mPEG (30K)To a mixture of phthalimide (0.4 g,0.013 mmol) in MeOH (4 mL) was added H 2 NNH 2 (4.2. Mu.L, 0.13 mmol). The mixture was stirred at 45 ℃ for 1.0 hour and concentrated. Dissolving the residue in CH 2 Cl 2 (100 mL) and washed with HCl (0.1N, 100 mL). By CH 2 Cl 2 (100 mL) the aqueous layer was extracted. Combining the organic layers and using H 2 O washing followed by anhydrous Na 2 SO 4 Drying, filtering and concentrating. Dissolving the residue in CH 2 Cl 2 (5 mL). Diethyl ether (200 mL) was added to precipitate hydroxylamine product (0.34 g, 83%) as a white powder.
Example 76
This example details the synthesis of mPEG-hydroxylamine presented in fig. 58A.
Synthesis of mPEG (5K) -OMs
To mPEG (5K) -OH (1.0 g,0.2 mmol) in CH 2 Cl 2 To a solution in (40 mL) were added triethylamine (110. Mu.L, 0.79 mmol) and MsCl (50. Mu.L, 0.64 mmol). The mixture was stirred at room temperature for 10 hours and concentrated. The crude product (1.0 g) was used directly in the next step without purification.
mPEG(5K)-O-NHBocIs synthesized by (a)
To crude mPEG (5K) -OMs (1.0 g,0.2 mmol) in CH 2 Cl 2 To a solution of (10 mL) was added tert-butyl-N-hydroxyurethane (0.3 g,2.2 mmol) and triethylamine (0.4 mL,2.9 mmol). The resulting mixture was stirred at 45 ℃ for 10 hours and cooled to room temperature. Diethyl ether (200 mL) was added. The precipitate was filtered, washed and dried in vacuo to give the product as a white solid (0.42, 42%).
3 + - mPEG(5K)-O-NHClIs synthesized by (a)
To mPEG (5K) -ONHBoc (0.2 g,0.04 mmol) at CH at℃ 2 Cl 2 To the solution in (3 mL) was added trifluoroacetic acid (7 mL). The resulting mixture was stirred at room temperature for 1 hour and concentrated. Addition of CH to the residue 2 Cl 2 (5 mL) followed by HCl (4N in dioxane, 2 mL). Diethyl ether (300 mL) was added to precipitate PEG dihydroxyamine as a white solidDerivative (170 mg, 85%).
Example 77
This example details the synthesis of mPEG-hydroxylamine presented in fig. 58B.
mPEG(30K)-OTfIs synthesized by (a)
To mPEG (30K) -OH (3.0 g,0.1 mmol) in CH 2 Cl 2 To a solution in (30 mL) were added 2, 6-lutidine (60. Mu.L, 0.5 mmol) and Tf 2 O (65. Mu.L, 0.4 mmol). The mixture was stirred at room temperature for 10 hours. Diethyl ether (400 mL) was added to the mixture. The precipitate was filtered, washed with diethyl ether and dried in vacuo to give a white powder (2.7 g, 90%).
mPEG(30K)-O-NHBocIs synthesized by (a)
To mPEG (30K) -OTf (2.5 g,0.083 mmol) on CH 2 Cl 2 To a solution of (25 mL) was added tert-butyl N-hydroxycarbamate (110 mg,0.84 mmol) and diisopropylethylamine (0.2 mL,1 mmol). The mixture was stirred at room temperature overnight. Diethyl ether (200 mL) was added to precipitate the product as a white powder (2.2 g, 88%).
3 + - mPEG(30K)-O-NHClIs synthesized by (a)
At 0deg.C above mPEG (30K) -ONHBoc (2.0 g,0.067 mmol) on CH 2 Cl 2 Trifluoroacetic acid (15 mL) was added to the solution in (15 mL). The resulting mixture was stirred at room temperature for 3 hours and concentrated. Addition of CH to the residue 2 Cl 2 (5 mL) followed by HCl (4N in dioxane, 2 mL). Diethyl ether (300 mL) was added to precipitate PEG dihydroxyamine derivative (1.72 g, 86%) as a white solid.
Example 78
This example details the synthesis of mPEG-hydroxylamine presented in fig. 59A.
Figure BDA0001546710350002691
Synthesis of->
To 2- (2-hydroxyethoxy) phthalimide (0.5 g,2.4 mmol) in CH 2 Cl 2 Phosgene (20% (in toluene), 8.0mL,15.0 mmol) was added to the solution in (20 mL). The reaction mixture was stirred at room temperature for 10 hours and concentrated in vacuo. The residue (0.45 g, 70%) was used directly in the next reaction without purification.
Figure BDA0001546710350002692
Is synthesized by (a)
To mPEG (30K) -NH 2 (3 g,0.1 mmol) and chloroformate linker (0.27 g,1.0 mmol) to CH 2 Cl 2 Diisopropylethylamine (0.2 mL,1.1 mmol) was added to the mixture in (30 mL). The resulting mixture was stirred at room temperature for 15 hours. Diethyl ether (500 mL) was added to the mixture. The precipitate was filtered, washed and dried in vacuo to give the product as a white solid (2.7 g, 90%).
Figure BDA0001546710350002693
Is synthesized by (a)
To a solution of mPEG (30K) phthalimide (2.1 g,0.07 mmol) in MeOH (15 mL) was added ammonia (7N in methanol, 15 mL). The resulting mixture was stirred at room temperature for 15 hours and concentrated. Addition of CH to the residue 2 Cl 2 (5 mL) followed by HCl (4N in dioxane, 1 mL). Diethyl ether (300 mL) was added to precipitate the product as a white solid (2.4 g, 89%).
Example 79
This example details the synthesis of mPEG-hydroxylamine presented in fig. 59B.
Figure BDA0001546710350002701
Is synthesized by (a)
To (tert-butoxycarbonyl-aminooxy) acetic acid (3.0 g,16 mmol) in CH 2 Cl 2 N, N' -diisopropylcarbodiimide (DIC, 1.3mL,8 mmol) was added to the solution in (80 mL). The mixture was stirred at room temperature for 1 hour and concentrated in vacuo. The crude product (4.9 g, 84%) was purified without further purificationCan be directly used in the next step.
Figure BDA0001546710350002702
Is synthesized by (a)
To a solution of anhydride (7.3 g,20 mmol) in DMF (20 mL) was added mPEG (5K) -NH 2 (20 g,4 mmol). The mixture was stirred at room temperature for 10 hours and taken up in H 2 O dilution (200 mL). By CH 2 Cl 2 (500 mL) the mixture was extracted. The organic layer is treated with H 2 O and brine (100 mL) followed by anhydrous Na 2 SO 4 Dried, filtered and concentrated in vacuo. Addition of CH to the residue 2 Cl 2 (10 mL) followed by diethyl ether (500 mL). The precipitate was filtered, washed with diethyl ether and dried in vacuo to give the product as a white powder (19.8 g, 99%).
Figure BDA0001546710350002703
Is synthesized by (a)
To Jing Shuding oxycarbonyl-protected mPEG (5K) (1.0 g,0.2 mmol) on CH 2 Cl 2 To the solution in (5 mL) was added trifluoroacetic acid (5 mL). The resulting mixture was stirred at room temperature for 1 hour and concentrated. Addition of CH to the residue 2 Cl 2 (2 mL) followed by HCl (4N in dioxane, 0.1 mL) and diethyl ether (40 mL). The precipitate was filtered, washed with diethyl ether and dried in vacuo to give the product (0.75 g, 75%).
Example 80
This example details the synthesis of mPEG-hydroxylamine as presented in fig. 60A.
Figure BDA0001546710350002711
Is synthesized by (a)
To mPEG (30K) -OH (4.5 g,0.15 mmol) on CH 2 Cl 2 Phosgene (20%, 1.6mL,3.0mmol in toluene) was added to the solution in (50 mL). The reaction mixture was stirred at room temperature for 10 hours and concentrated in vacuo. Residue (4.2 g,93%) Can be used in the next reaction without purification.
Figure BDA0001546710350002712
Is synthesized by (a)
The above activated mPEG (30K) II (4.2 g,0.14 mmol) and amine linker VIII (67 mg,0.28 mmol) in DMF-CH 2 Cl 2 Diisopropylethylamine (75. Mu.L, 0.42 mmol) was added to the mixture in (10 mL, 1:2). After stirring the resulting mixture at room temperature for 15 hours, diethyl ether (200 mL) was added. The precipitate was filtered, washed with diethyl ether and dried in vacuo to give the product as a white powder (3.8 g, 90%).
Figure BDA0001546710350002713
Is synthesized by (a)
To a solution of mPEG (30K) phthalimide III (3.5 g,0.12 mmol) in MeOH (15 mL) was added ammonia (7N in methanol, 15 mL). The resulting mixture was stirred at room temperature for 15 hours and concentrated. Addition of CH to the residue 2 Cl 2 (5 mL) followed by HCl (4N in dioxane, 1 mL). Diethyl ether (300 mL) was added to precipitate the product as a white solid (3.0 g, 86%).
Figure BDA0001546710350002714
Is synthesized by (a)
To a solution of tert-butyl 2-hydroxyethylcarbamate (2.8 mL,18 mmol) in THF (60 mL) was added N-hydroxyphthalimide (5.8 g,36 mmol), triphenylphosphine (3.6 g,27 mmol). The reaction mixture was stirred at room temperature for 10 minutes and then cooled to 0 ℃. Diisopropylazodicarbonate (DIAD, 3.6ml,19 mmol) was added dropwise via syringe over 1 hour. The ice bath was removed and the mixture was stirred overnight and concentrated. The white solid was dissolved in ethyl acetate (100 mL). The reaction mixture was washed successively with a saturated aqueous solution of sodium hydrogencarbonate (2X 50 mL), deionized water (50 mL) and brine (50 mL), followed by anhydrous MgSO 4 Dried, filtered and concentrated in vacuo. By usingBiotage Inc.HORIZON TM Flash chromatography of the chromatographic system purified the crude product to give the title compound (12 g, 206%) as a white solid containing impurities.
Figure BDA0001546710350002721
Is synthesized by (a)
To crude Jing Shuding oxycarbonyl-protected linker (12 g) to CH 2 Cl 2 To the solution in (5 mL) was added trifluoroacetic acid (5 mL). The resulting mixture was stirred at room temperature for 1 hour and concentrated. To the residue was added HCl (4N in dioxane, 1.5 mL), followed by Et 2 O (150 mL). The precipitate was filtered, washed with diethyl ether and dried in vacuo to give the amine linker as a white solid (3.0 g, 68% for both steps).
Example 81
This example details the synthesis of mPEG-hydroxylamine presented in fig. 60B.
Figure BDA0001546710350002722
Is synthesized by (a)
To mPEG (5K) -OH (10 g,2.0 mmol), ph at 0deg.C 3 P (79mg, 3.0 mmol) and N-hydroxyphthalimide (0.49 g,3.0 mmol) in CH 2 Cl 2 To a solution in THF (2:3, 45 mL) was added diisopropyl azodicarboxylate (DIAD, 409. Mu.L, 2.0 mmol). After the mixture was stirred at room temperature for 15 hours, diethyl ether (1L) was added to the mixture. The precipitate was filtered, washed with diethyl ether and dried in vacuo to give mPEG (5K) -phthalimide (9.8 g, 98%) as a white powder.
2 mPEG(5K)-O-NHIs synthesized by (a)
To a solution of mPEG (5K) phthalimide (3 g,0.6 mmol) in MeOH (25 mL) was added ammonia (7N in methanol, 25 mL). The resulting mixture was stirred at room temperature for 15 hours and concentrated. Addition of CH to the residue 2 Cl 2 (5 mL) followed by HCl (4N in dioxane, 1 mL). Diethyl ether (300 mL) was added toHydroxylamine (2.5 g, 83%) precipitated as a white solid.
Figure BDA0001546710350002731
Is synthesized by (a)
To mPEG (30K) -OH (6 g,0.2 mmol), ph at 0deg.C 3 P (80 mg,0.3 mmol) and N-hydroxyphthalimide (49 mg,0.3 mmol) on CH 2 Cl 2 To a solution in THF (2:3, 45 mL) was added diisopropyl azodicarboxylate (DIAD, 41. Mu.L, 0.2 mmol). The mixture was stirred at room temperature for 15 hours. Diethyl ether (200 mL) was added to the mixture. The precipitate was washed with diethyl ether and dried in vacuo to give mPEG (30K) -phthalimide product (5.8 g, 96%) as a white powder.
2 mPEG(30K)-O-NHIs synthesized by (a)
To a solution of mPEG (30K) phthalimide (3 g,0.1 mmol) in MeOH (20 mL) was added ammonia (7N in methanol, 20 mL). The resulting mixture was stirred at room temperature for 15 hours and concentrated. Addition of CH to the residue 2 Cl 2 (5 mL) followed by HCl (4N in dioxane, 1 mL). Diethyl ether (300 mL) was added to precipitate mPEG (30K) -ONH as a white solid 2 (2.6g，87％)。
Example 82
This example details the synthesis of mPEG-hydroxylamine presented in fig. 61A.
Figure BDA0001546710350002732
Is synthesized by (a)
To a solution of 2- (2- (2-aminoethoxy) ethoxy) ethylamine (5.0 g,33.8 mmol) in DMF (100 mL) was added (t-butoxycarbonyl-aminooxy) acetic acid (14.2 g,74.2 mmol), 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC, 28.5g,0.15 mol) and N, N' -diisopropylethylamine (26 mL,0.15 mol). The mixture was stirred at room temperature for 10 hours and diluted with EtOAc (500 mL). The mixture was treated with NaHCO 3 Saturated aqueous solution (300 mL), H 2 O (300 mL) and brine (300 mL) in this orderWashing with anhydrous Na 2 SO 4 Dried, filtered and concentrated in vacuo. The crude product (14.2 g, 85%) was used directly in the next reaction without purification.
Figure BDA0001546710350002741
Is synthesized by (a)
The above di-t-butoxycarbonyl linker (3.0 g,6.1 mmol) was attached to CH at 0deg.C 2 Cl 2 Trifluoroacetic acid (15 mL) was added to the solution in (15 mL). The resulting mixture was stirred at room temperature for 3 hours and concentrated. Addition of CH to the residue 2 Cl 2 (5 mL) followed by HCl (4N in dioxane, 2 mL). Diethyl ether (400 mL) was added to precipitate the dihydroxyamine (1.47 g, 82%) as a white solid.
Example 83
This example details the synthesis of mPEG-hydroxylamine presented in fig. 61B.
Figure BDA0001546710350002742
Is synthesized by (a)
To a solution of tris (ethylene glycol) (1.5 g,10 mmol) in THF (100 mL) at 0deg.C was added Ph 3 P (8.0 g,30 mmol) and N-hydroxyphthalimide (4.9 g,30 mmol). Diisopropyl azodicarboxylate (DIAD, 4.08mL,20 mmol) was slowly added to the mixture. The resulting mixture was stirred at 0 ℃ for 4 hours and at room temperature for 2 days. Diethyl ether (25 mL) was added to the reaction mixture. The precipitate was washed with diethyl ether and dried in vacuo to give the diphthalimide product (3.72 g, 82%) as a white powder.
Figure BDA0001546710350002743
Is synthesized by (a)
To a solution of diphthalimide (2.2 g,5.0 mmol) in MeOH (20 mL) was added ammonia (7N in methanol, 20 mL). The resulting mixture was stirred at room temperature for 15 hours and concentrated. Addition of CH to the residue 2 Cl 2 (5 mL) followed by HCl (4N in dioxane, 1 mL). Diethyl ether (300 mL) was added to precipitate the tris (ethylene glycol) dihydroxyamine linker (1.1 g, 87%) as a white solid.
Example 84
This example details the synthesis of mPEG-hydroxylamine presented in fig. 61C.
Figure BDA0001546710350002751
Is synthesized by (a)
To a solution of tetra (ethylene glycol) (1.94 g,10 mmol) in THF (100 mL) at 0deg.C was added Ph 3 P (8.0 g,30 mmol) and N-hydroxyphthalimide (4.9 g,30 mmol). Diisopropyl azodicarboxylate (DIAD, 4.08mL,20 mmol) was slowly added to the mixture. The resulting mixture was stirred at 0 ℃ for 4 hours and at room temperature for 2 days. Diethyl ether (25 mL) was added to the reaction mixture. The precipitate was washed with diethyl ether and dried in vacuo to give the diphthalimide product (3.58 g, 74%) as a white powder.
Figure BDA0001546710350002752
Is synthesized by (a)
To a solution of diphthalimide (2.42 g,5.0 mmol) in MeOH (20 mL) was added ammonia (7N in methanol, 20 mL). The resulting mixture was stirred at room temperature for 15 hours and concentrated. Addition of CH to the residue 2 Cl 2 (5 mL) followed by HCl (4N in dioxane, 1 mL). Diethyl ether (300 mL) was added to precipitate the tetrakis (ethylene glycol) dihydroxyamine linker (1.27 g, 85%) as a white solid.
Example 85
This example details the synthesis of mPEG-hydroxylamine presented in fig. 62A.
Figure BDA0001546710350002753
Is synthesized by (a)
To hexa (ethylene glycol) at 0 ℃ (2.82 g,10 mmol) in THF (100 mL) was added Ph 3 P (8.0 g,30 mmol) and N-hydroxyphthalimide (4.9 g,30 mmol). Diisopropyl azodicarboxylate (DIAD, 4.08mL,20 mmol) was slowly added to the mixture. The resulting mixture was stirred at 0 ℃ for 4 hours and at room temperature for 2 days. Diethyl ether (25 mL) was added to the reaction mixture. The precipitate was washed with diethyl ether and dried in vacuo to give the diphthalimide product (4.40 g, 77%) as a white powder.
Figure BDA0001546710350002761
Is synthesized by (a)
To a solution of diphthalimide (2.86 g,5.0 mmol) in MeOH (20 mL) was added ammonia (7N in methanol, 20 mL). The resulting mixture was stirred at room temperature for 15 hours and concentrated. Addition of CH to the residue 2 Cl 2 (5 mL) followed by HCl (4N in dioxane, 1 mL). Diethyl ether (300 mL) was added to precipitate the hexa (ethylene glycol) dihydroxyamine linker (1.68 g, 87%) as a white solid.
Example 86
This example details the synthesis of mPEG compounds presented in fig. 62B.
Figure BDA0001546710350002762
Is synthesized by (a)
To mPEG-OH (30K, 3.0g,0.1 mmol) in dry CH 2 Cl 2 Diphosgene (63. Mu.L, 0.5 mmol) was added to the solution in (30 mL). The mixture was stirred at room temperature overnight. Diethyl ether (700 mL) was added to precipitate mPEG. The product was filtered, washed with diethyl ether and dried in vacuo (3.0 g, 100%).
The following examples describe methods for measuring and comparing in vitro and in vivo activity of modified therapeutically active unnatural amino acid polypeptides with in vitro and in vivo activity of therapeutically active natural amino acid polypeptides.
Example 87: cell binding assays
At 0 ℃ in the absence or presence ofIn the case of unlabeled GH, hGH or GM-CSF in various concentrations (volume: 10. Mu.l) and in the case of 125 I-GH (about 100,000cpm or 1 ng) cells (3×l0) 6 ) Incubation (in duplicate) in PBS/1% BSA (100 μl) for 90 min (total volume: 120 μl). Cells were then suspended and layered on 200. Mu.l ice-cold FCS in 350. Mu.l plastic centrifuge tubes and centrifuged (1000 g;1 min). The centrifugation block was collected by cutting the bottom of the tube and the centrifugation block and supernatant were counted separately with a gamma counter (Packard).
Specific binding (cpm) was determined as the total binding in the absence of competitors (average of duplicate) minus the binding in the presence of 100-fold excess unlabeled GH (cpm) (non-specific binding). Nonspecific binding was measured for each of the cell types used. The experiment was carried out using the same 125 The I-GH formulations were run on separate days and should show internal consistency. 125 I-GH demonstrates binding to GH receptor producing cells. Binding is inhibited in a dose-dependent manner by unlabeled native GH or hGH, but not by GM-CSF or other negative control. hGH Competition for naturalness 125 The ability of I-GH (similar to native GH) to bind suggests that the receptor recognizes both forms equally well.
Example 88:in vivo studies of PEGylated hGH
PEG-hGH, unmodified hGH and buffer solution were administered to mice or rats. The results should show that the pegylated hGH of the present invention has superior activity and prolonged half-life compared to unmodified hGH, as indicated by a significant increase in body weight.
Example 89:measurement of in vivo half-life of conjugated and non-conjugated hGH and variants thereof
All animal experiments were performed in AAALAC quality qualification equipment and with protocols approved by the laboratory animal management and use committee (Institutional Animal Care and Use Committee) of the University of st. Rats were individually housed in cages in a 12 hour light/dark cycle in the room. Animals were allowed access to a qualified purena rodent chow 5001 and water ad libitum. For hypophysectomized rats, the drinking water additionally contained 5% glucose.
Example 90:pharmacokinetic studies
The quality of each PEGylated mutant hGH was assessed by three assays prior to entry into animal experiments. The purity of PEG-hGH was detected by electrophoresis with 4% -12% acrylamide NuPAGE Bis-Tris gel under non-reducing conditions in MES SDS running buffer (Invitrogen, carlsbad, calif.). The gel was stained with Coomassie blue (Coomassie blue). According to densitometry scan, the purity of the PEG-hGH band was greater than 95%. By using KTA from Charles River Laboratories (Wilmington, mass.) 2 The kinetic LAL assay of the kit was used to test endotoxin content in each PEG-hGH and less than 5EU per dose. Biological activity of PEG-hGH was assessed by the IM-9pSTAT5 bioassay and EC was confirmed 50 The value was less than 15nM.
The pharmacokinetic properties of PEG-modified growth hormone compounds were compared to each other and to non-pegylated growth hormone in male Sprague-Dawley rats (261 g-425 g) obtained from Charles River Laboratories. Catheters are surgically installed in the carotid artery for blood collection. After successful catheter installation, animals were assigned to treatment groups (three to six per group) prior to dosing. 1mg/kg of the compound is subcutaneously administered to the animal in a dosage volume of 0.41-0.55 ml/kg. Blood samples were collected at various time points through an indwelling catheter and into EDTA-coated microcentrifuge tubes. Plasma was collected after centrifugation and stored at-80 ℃ until analysis. Compound concentrations were measured using antibody sandwich growth hormone ELISA kits from BioSource International (Camarillo, CA) or Diagnostic Systems Laboratories (Webster, TX). The concentration was calculated using the criteria corresponding to the given analogues. Model program WinNonlin (Pharsight, version 4.1) was used to estimate pharmacokinetic parameters. Non-compartmental model analysis (Noncompartmental analysis) with linear up/log down trapezoidal integration was used and the concentration data was uniformly weighted.
Plasma concentrations were obtained at regular time intervals following a single subcutaneous administration in rats. Rats (n=3-6 per group) were given a single bolus dose of 1mg per kg of protein. Wild-type hGH protein (WHO hGH), his-tagged hGH polypeptideThe (His-hGH) or His-tagged hGH polypeptides comprising an unnatural amino acid, para-acetyl-phenylalanine, covalently linked to 30kDa PEG at each of six different positions were compared to WHO hGH and (His) -hGH. Plasma samples are taken over a prescribed time interval and the compounds are injected therein in accordance with the assay. The following table shows the pharmacokinetic parameter values for single dose administration of various hGH polypeptides. Concentration versus time curves were evaluated by non-compartmental model analysis (Pharsight, version 4.1). The values shown are the mean (+/-standard deviation). Cmax is as follows: maximum concentration; end stage t1/2 : terminal half-life; AUC (AUC) 0->inf : area under the concentration-time curve extrapolated to infinity; MRT: average residence time; cl/f: apparent total plasma clearance; vz/f: apparent distribution volume during the end phase. A significant extension of 30KPEG-pAF92 (his) hGH circulation, increased serum half-life and increased bioavailability was observed compared to control hGH.
Table: pharmacokinetic parameter values for 1mg/kg bolus for a single dose were administered subcutaneously in normal male Sprague-Dawley rats.
Figure BDA0001546710350002781
Example 91:pharmacodynamic study
Male Sprague-Dawley rats with pituitary excised were obtained from Charles River Laboratories. Pituitary was surgically removed at 3-4 weeks of age. Animals were acclimatized to the new environment for a period of 3 weeks during which body weight was monitored. Animals that gained weight by 0g-8g over a period of 7 days prior to the start of the study were placed and randomized into treatment groups. Rats are administered bolus doses or subcutaneously daily. Throughout the study, rats were weighed, anesthetized, exsanguinated, and dosed daily and sequentially (as appropriate). Blood was collected from the orbital sinus using heparinized capillaries and placed into EDTA-coated microcentrifuge tubes. Plasma was separated by centrifugation and stored at-80 ℃ until analysis. Average (+/-s.d.) plasma concentration versus time interval was plotted.
The peptide IGF-1 is a member of the family of growth regulators or insulin-like growth factors. IGF-1 mediates many of the growth-promoting effects of growth hormone. IGF-1 concentration was measured using a competitive binding enzyme immunoassay kit (Diagnosic Systems Laboratories) against the rat/mouse IGF-1 standard provided. The pituitary glands of the rats were excised. Rats (n=5-7 per group) were given a single dose or daily dose subcutaneously. Animals were weighed, anesthetized, exsanguinated and dosed daily in sequence (as appropriate). Weight results were obtained for placebo treatment, wild-type hGH (hGH), his-tagged hGH ((His) hGH) and hGH polypeptides comprising a para-acetyl-phenylalanine covalently linked to 30kDa PEG at positions 35 and 92. The weight gain at day 9 was observed for the 30KPEG-pAF35 (his) hGH compound to be statistically different from the 30KPEG-pAF92 (his) hGH compound (p < 0.0005) in that a greater weight gain was observed. There was a significant difference in the effect on circulating plasma IGF-1 levels as determined by t-test using a two-tail distribution, unpaired, equal variance following administration of a single dose of hGH polypeptide comprising pegylated non-naturally encoded amino acid.
Example 92: human clinical trials comprising the safety and/or efficacy of non-naturally encoded amino acid PEGylated hGH.
Purpose(s)To compare a PEGylated recombinant human hGH including non-naturally encoded amino acids with one or more commercially available hGH products (including but not limited to Humatrope) for subcutaneous administration TM (Eli Lilly&Co.)、Nutropin TM (Genentech)、Norditropin TM (Novo-Nordisk)、Genotropin TM (Pfizer) and Saizer/Serostim TM (Serono)) safety and pharmacokinetics.
Patient(s)18 healthy volunteers with an age in the range of 20-40 years and a weight between 60-90 kg were enrolled in the study. The subject should not have clinically significant abnormal hematology or serum chemistry, negative urine toxicity screening, HIV screening, and hepatitis B surface antigen experimental values. It should have no sign of: hypertension; history of any primary blood disease; a history of major liver disease, kidney disease, cardiovascular disease, gastrointestinal disease, genitourinary disease, metabolic disease, and neuropathy; history of anemia or epilepsy; for a pair ofKnown sensitivity of products of bacterial or mammalian origin, PEG or human serum albumin; habitual and heavy consumers of caffeine-containing beverages; reference to any other clinical trial or transfusion or donation within 30 days of entry into the study; hGH had been contacted within 3 months of entering the study; disease within 7 days of entry into the study; and significant abnormalities for pre-study physical examination or clinical laboratory assessment within 14 days of study entry. All subjects were assessed for safety and all blood collections for pharmacokinetic analysis were collected at regular time. All studies were approved by the institutional ethics committee and patients were consented.
Study designIt should be a phase I, single center, open label, random, two cycle crossover study in healthy male volunteers. 18 subjects were randomly assigned to one of two treatment sequence groups (9 subjects per group). GH was administered in rapid subcutaneous injection at the upper thigh over two separate dosing periods using equal doses of PEGylated hGH, including non-naturally encoded amino acids, and selected commercial products. The dosage and frequency of administration of the commercial product is as specified in the package label. Other dosages, dosing frequencies, or other parameters using commercially available products may be added to the study by including other subject groups, as desired. Each dosing period was separated by a 14 day washout period. For each of the two dosing periods, the subject was confined to the study center at least 12 hours prior to dosing and 72 hours after dosing, but this was not required between dosing periods. Additional subject groups may also be added to test for pegylated hGH if other dosages, frequencies, or other parameters are present. A variety of GH formulations approved for human use can be used in this study. Humatrope TM (Eli Lilly&Co.)、Nutropin TM (Genentech)、Norditropin TM (Novo-Nordisk)、Genotropin TM (Pfizer) and Saizer/Serostim TM (Serono) is a commercially available GH product approved for human use. The experimental formulation for hGH was PEGylated hGH, which includes non-naturally encoded amino acids.
Blood samplingBlood was continuously withdrawn by direct venipuncture before and after administration of hGH. In the administration of drugsAbout 30 minutes, 20 minutes, and 10 minutes (3 baseline samples) before and about the following times after dosing: venous blood samples (5 mL) for determining serum GH concentration were obtained for 30 minutes and 1 hour, 2 hours, 5 hours, 8 hours, 12 hours, 15 hours, 18 hours, 24 hours, 30 hours, 36 hours, 48 hours, 60 hours and 72 hours. Each serum sample was split into two aliquots. All serum samples were stored at-20 ℃. Serum samples were transported on dry ice. Fasted clinical trial tests (hematology, serum chemistry, and urine tests) were performed immediately prior to initial dosing on day 1, immediately prior to dosing on day 4, immediately prior to dosing on day 16, and on day 19.
Biological analysis methodUsing an ELISA kit procedure (Diagnostic Systems Laboratory [ DSL)]Webster TX) to determine serum GH concentration.
Safety determinationVital signs were recorded immediately before each dose (day 1 and day 16) and 6 hours, 24 hours, 48 hours and 72 hours after each dose. Safety assays are based on the incidence and type of adverse events and the change from baseline in clinical trial tests. In addition, changes from pre-study in vital sign measurements (including blood pressure) and physical examination results were assessed.
Data analysisPost-dose serum concentration values were corrected for pre-dose baseline GH concentration by subtracting from each of the post-dose values the average baseline GH concentration determined by averaging GH levels of three samples taken at 30, 20 and 10 minutes prior to dose. If the serum GH concentration is below the quantitative level of the assay before administration, it is not included in the calculation of the mean. Pharmacokinetic parameters were determined from corrected serum concentration data for baseline GH concentration. Pharmacokinetic parameters were calculated by a model independent method on a Digital Equipment Corporation VAX 8600 computer system using the latest version of the BIOAVL software. The following pharmacokinetic parameters were determined: peak serum concentration (C) max ) The method comprises the steps of carrying out a first treatment on the surface of the Time to peak serum concentration (t max ) The method comprises the steps of carrying out a first treatment on the surface of the From time zero to last blood sampling time (AUC) calculated using linear trapezoidal rule 0-72 ) Area under concentration-time curve (AUC); and is composed ofTerminal elimination half-life (t) calculated by elimination rate constant 1/2 ). The elimination rate constant is estimated by linear regression of successive data points in the end linear region of the log-linear concentration-time curve. For each treatment, the mean Standard Deviation (SD) and Coefficient of Variation (CV) of the pharmacokinetic parameters were calculated. The ratio of the parameter averages (hold recipe/non-hold recipe) was calculated.
Security resultsThe incidence distribution of adverse events was the same in the treatment group. There were no clinically significant changes from baseline or pre-study clinical laboratory tests or blood pressure, and no significant changes from pre-study physical examination results and vital sign measurements. The safety profiles of the two treatment groups should appear similar.
Pharmacokinetic resultsAt each time point measured, a single dose of one or more commercial hGH products (including but not limited to Humatrope) will be received TM (Eli Lilly&Co.)、Nutropin TM (Genentech)、Norditropin TM (Novo-Nordisk)、Genotropin TM (Pfizer) and Saizer/Serostim TM (Serono)) and the average serum GH concentration-time profile (not corrected for baseline GH content) of all 18 subjects was compared to pegylated hGH comprising non-naturally encoded amino acids. All subjects should have a pre-dose baseline GH concentration within the normal physiological range. Determination of pharmacokinetic parameters and determination of C from serum data corrected for pre-dosing mean baseline GH concentration max And t max . Selected clinical comparisons (humatopcope) TM (Eli Lilly&Co.)、Nutropin TM (Genentech)、Norditropin TM (Novo-Nordisk)、Genotropin TM (Pfizer)、Saizen/Serostim TM (Serono)) average t max Compared to t comprising a PEGylated hGH which does not naturally encode an amino acid max Significantly shorter. The terminal half-life values of the commercial hGH products tested were significantly shorter compared to the terminal half-life of the pegylated hGH comprising the non-naturally encoded amino acid.
Although the study of the present invention was conducted in healthy male subjects, it is contemplated that similar absorption characteristics and safety profiles may be provided in other patient populations, such as male or female patients with cancer or chronic kidney failure, pediatric kidney failure patients, patients in autologous pre-stored procedures, or patients scheduled for selective surgery.
In general, a single dose of pegylated hGH comprising an unnatural encoded amino acid administered subcutaneously should be safe and well accepted by healthy male subjects. Depending on the relative incidence of adverse events, the clinical laboratory values, vital signs, and physical examination results and safety profiles of commercial forms of hGH and pegylated hGH including non-naturally encoded amino acids should be comparable. PEGylated hGH, including non-naturally encoded amino acids, potentially provides great clinical utility to patients and healthcare providers.
Example 93: comparison of Water solubility of PEGylated hGH and non-PEGylated hGH
The water solubility of hGH wild-type protein (WHO hGH), his-tagged hGH polypeptide (His-hGH) or His-tagged hGH polypeptide comprising the unnatural amino acid para-acetyl-phenylalanine covalently linked to 30kDa PEG at position 92 was obtained by determining the amount of the respective polypeptide soluble in 100. Mu.L of water. The amount of PEGylated hGH is greater than the amounts of WHO hGH and hGH, which demonstrates that PEGylation of the unnatural amino acid polypeptide increases water solubility.
Example 94: in vivo studies of modified therapeutically active unnatural amino acid polypeptides
Prostate cancer tumor xenografts were implanted into mice, which were then divided into two groups. One group is treated daily with a modified therapeutically active non-natural amino acid polypeptide and the other group is treated daily with a therapeutically active natural amino acid polypeptide. Tumor size was measured daily and modified therapeutically active unnatural amino acid polypeptides have improved therapeutic efficacy compared to the therapeutically active natural amino acid polypeptides as indicated by a decrease in tumor size in the group treated with the modified therapeutically active unnatural amino acid polypeptides.
Example 95: in vivo studies of modified therapeutically active unnatural amino acid polypeptides
Prostate cancer tumor xenografts were implanted into mice, which were then divided into two groups. One group is treated daily with a modified therapeutically active non-natural amino acid polypeptide and the other group is treated daily with a therapeutically active natural amino acid polypeptide. Tumor size was measured daily and modified therapeutically active unnatural amino acid polypeptides have improved therapeutic efficacy compared to the therapeutically active natural amino acid polypeptides as indicated by a decrease in tumor size in the group treated with the modified therapeutically active unnatural amino acid polypeptides.
It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof should be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.
Sequence listing
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<140> PCT/US05/046618
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Met Asp Glu Phe Glu Met Ile Lys Arg Asn Thr Ser Glu Ile Ile Ser
1 5 10 15
Glu Glu Glu Leu Arg Glu Val Leu Lys Lys Asp Glu Lys Ser Ala Gly
20 25 30
Ile Gly Phe Glu Pro Ser Gly Lys Ile His Leu Gly His Tyr Leu Gln
35 40 45
Ile Lys Lys Met Ile Asp Leu Gln Asn Ala Gly Phe Asp Ile Ile Ile
50 55 60
Leu Leu Ala Asp Leu His Ala Tyr Leu Asn Gln Lys Gly Glu Leu Asp
65 70 75 80
Glu Ile Arg Lys Ile Gly Asp Tyr Asn Lys Lys Val Phe Glu Ala Met
85 90 95
Gly Leu Lys Ala Lys Tyr Val Tyr Gly Ser Thr Phe Gln Leu Asp Lys
100 105 110
Asp Tyr Thr Leu Asn Val Tyr Arg Leu Ala Leu Lys Thr Thr Leu Lys
115 120 125
Arg Ala Arg Arg Ser Met Glu Leu Ile Ala Arg Glu Asp Glu Asn Pro
130 135 140
Lys Val Ala Glu Val Ile Tyr Pro Ile Met Gln Val Asn Thr Tyr Tyr
145 150 155 160
Tyr Leu Gly Val Asp Val Ala Val Gly Gly Met Glu Gln Arg Lys Ile
165 170 175
His Met Leu Ala Arg Glu Leu Leu Pro Lys Lys Val Val Cys Ile His
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Asn Pro Val Leu Thr Gly Leu Asp Gly Glu Gly Lys Met Ser Ser Ser
195 200 205
Lys Gly Asn Phe Ile Ala Val Asp Asp Ser Pro Glu Glu Ile Arg Ala
210 215 220
Lys Ile Lys Lys Ala Tyr Cys Pro Ala Gly Val Val Glu Gly Asn Pro
225 230 235 240
Ile Met Glu Ile Ala Lys Tyr Phe Leu Glu Tyr Pro Leu Thr Ile Lys
245 250 255
Arg Pro Glu Lys Phe Gly Gly Asp Leu Thr Val Asn Ser Tyr Glu Glu
260 265 270
Leu Glu Ser Leu Phe Lys Asn Lys Glu Leu His Pro Met Asp Leu Lys
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Asn Ala Val Ala Glu Glu Leu Ile Lys Ile Leu Glu Pro Ile Arg Lys
290 295 300
Arg Leu
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Met Asp Glu Phe Glu Met Ile Lys Arg Asn Thr Ser Glu Ile Ile Ser
1 5 10 15
Glu Glu Glu Leu Arg Glu Val Leu Lys Lys Asp Glu Lys Ser Ala Gly
20 25 30
Ile Gly Phe Glu Pro Ser Gly Lys Ile His Leu Gly His Tyr Leu Gln
35 40 45
Ile Lys Lys Met Ile Asp Leu Gln Asn Ala Gly Phe Asp Ile Ile Ile
50 55 60
Leu Leu Ala Asp Leu His Ala Tyr Leu Asn Gln Lys Gly Glu Leu Asp
65 70 75 80
Glu Ile Arg Lys Ile Gly Asp Tyr Asn Lys Lys Val Phe Glu Ala Met
85 90 95
Gly Leu Lys Ala Lys Tyr Val Tyr Gly Ser Ser Phe Gln Leu Asp Lys
100 105 110
Asp Tyr Thr Leu Asn Val Tyr Arg Leu Ala Leu Lys Thr Thr Leu Lys
115 120 125
Arg Ala Arg Arg Ser Met Glu Leu Ile Ala Arg Glu Asp Glu Asn Pro
130 135 140
Lys Val Ala Glu Val Ile Tyr Pro Ile Met Gln Val Asn Thr Ser His
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Tyr Leu Gly Val Asp Val Ala Val Gly Gly Met Glu Gln Arg Lys Ile
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His Met Leu Ala Arg Glu Leu Leu Pro Lys Lys Val Val Cys Ile His
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Asn Pro Val Leu Thr Gly Leu Asp Gly Glu Gly Lys Met Ser Ser Ser
195 200 205
Lys Gly Asn Phe Ile Ala Val Asp Asp Ser Pro Glu Glu Ile Arg Ala
210 215 220
Lys Ile Lys Lys Ala Tyr Cys Pro Ala Gly Val Val Glu Gly Asn Pro
225 230 235 240
Ile Met Glu Ile Ala Lys Tyr Phe Leu Glu Tyr Pro Leu Thr Ile Lys
245 250 255
Arg Pro Glu Lys Phe Gly Gly Asp Leu Thr Val Asn Ser Tyr Glu Glu
260 265 270
Leu Glu Ser Leu Phe Lys Asn Lys Glu Leu His Pro Met Asp Leu Lys
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Asn Ala Val Ala Glu Glu Leu Ile Lys Ile Leu Glu Pro Ile Arg Lys
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Arg Leu
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Met Asp Glu Phe Glu Met Ile Lys Arg Asn Thr Ser Glu Ile Ile Ser
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Glu Glu Glu Leu Arg Glu Val Leu Lys Lys Asp Glu Lys Ala Ala Ile
20 25 30
Gly Phe Glu Pro Ser Gly Lys Ile His Leu Gly His Tyr Leu Gln Ile
35 40 45
Lys Lys Met Ile Asp Leu Gln Asn Ala Gly Phe Asp Ile Ile Ile Leu
50 55 60
Leu Ala Asp Leu His Ala Tyr Leu Asn Gln Lys Gly Glu Leu Asp Glu
65 70 75 80
Ile Arg Lys Ile Gly Asp Tyr Asn Lys Lys Val Phe Glu Ala Met Gly
85 90 95
Leu Lys Ala Lys Tyr Val Tyr Gly Ser Pro Phe Gln Leu Asp Lys Asp
100 105 110
Tyr Thr Leu Asn Val Tyr Arg Leu Ala Leu Lys Thr Thr Leu Lys Arg
115 120 125
Ala Arg Arg Ser Met Glu Leu Ile Ala Arg Glu Asp Glu Asn Pro Lys
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Val Ala Glu Val Ile Tyr Pro Ile Met Gln Val Asn Ala Ile Tyr Leu
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Ala Val Asp Val Ala Val Gly Gly Met Glu Gln Arg Lys Ile His Met
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Leu Ala Arg Glu Leu Leu Pro Lys Lys Val Val Cys Ile His Asn Pro
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Val Leu Thr Gly Leu Asp Gly Glu Gly Lys Met Ser Ser Ser Lys Gly
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Asn Phe Ile Ala Val Asp Asp Ser Pro Glu Glu Ile Arg Ala Lys Ile
210 215 220
Lys Lys Ala Tyr Cys Pro Ala Gly Val Val Glu Gly Asn Pro Ile Met
225 230 235 240
Glu Ile Ala Lys Tyr Phe Leu Glu Tyr Pro Leu Thr Ile Lys Arg Pro
245 250 255
Glu Lys Phe Gly Gly Asp Leu Thr Val Asn Ser Tyr Glu Glu Leu Glu
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Ser Leu Phe Lys Asn Lys Glu Leu His Pro Met Asp Leu Lys Asn Ala
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Val Ala Glu Glu Leu Ile Lys Ile Leu Glu Pro Ile Arg Lys Arg Leu
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Met Asp Glu Phe Glu Met Ile Lys Arg Asn Thr Ser Glu Ile Ile Ser
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Glu Glu Glu Leu Arg Glu Val Leu Lys Lys Asp Glu Lys Ser Ala Ala
20 25 30
Ile Gly Phe Glu Pro Ser Gly Lys Ile His Leu Gly His Tyr Leu Gln
35 40 45
Ile Lys Lys Met Ile Asp Leu Gln Asn Ala Gly Phe Asp Ile Ile Ile
50 55 60
Leu Leu Ala Asp Leu His Ala Tyr Leu Asn Gln Lys Gly Glu Leu Asp
65 70 75 80
Glu Ile Arg Lys Ile Gly Asp Tyr Asn Lys Lys Val Phe Glu Ala Met
85 90 95
Gly Leu Lys Ala Lys Tyr Val Tyr Gly Ser Pro Phe Gln Leu Asp Lys
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Asp Tyr Thr Leu Asn Val Tyr Arg Leu Ala Leu Lys Thr Thr Leu Lys
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Arg Ala Arg Arg Ser Met Glu Leu Ile Ala Arg Glu Asp Glu Asn Pro
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Lys Val Ala Glu Val Ile Tyr Pro Ile Met Gln Val Asn Ile Pro Tyr
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Leu Pro Val Asp Val Ala Val Gly Gly Met Glu Gln Arg Lys Ile His
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Met Leu Ala Arg Glu Leu Leu Pro Lys Lys Val Val Cys Ile His Asn
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Pro Val Leu Thr Gly Leu Asp Gly Glu Gly Lys Met Ser Ser Ser Lys
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Gly Asn Phe Ile Ala Val Asp Asp Ser Pro Glu Glu Ile Arg Ala Lys
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Ile Lys Lys Ala Tyr Cys Pro Ala Gly Val Val Glu Gly Asn Pro Ile
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Met Glu Ile Ala Lys Tyr Phe Leu Glu Tyr Pro Leu Thr Ile Lys Arg
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Pro Glu Lys Phe Gly Gly Asp Leu Thr Val Asn Ser Tyr Glu Glu Leu
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Glu Ser Leu Phe Lys Asn Lys Glu Leu His Pro Met Asp Leu Lys Asn
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Ala Val Ala Glu Glu Leu Ile Lys Ile Leu Glu Pro Ile Arg Lys Arg
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Leu
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Met Asp Glu Phe Glu Met Ile Lys Arg Asn Thr Ser Glu Ile Ile Ser
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Glu Glu Glu Leu Arg Glu Val Leu Lys Lys Asp Glu Lys Ser Ala Ala
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Ile Gly Phe Glu Pro Ser Gly Lys Ile His Leu Gly His Tyr Leu Gln
35 40 45
Ile Lys Lys Met Ile Asp Leu Gln Asn Ala Gly Phe Asp Ile Ile Ile
50 55 60
Leu Leu Ala Asp Leu His Ala Tyr Leu Asn Gln Lys Gly Glu Leu Asp
65 70 75 80
Glu Ile Arg Lys Ile Gly Asp Tyr Asn Lys Lys Val Phe Glu Ala Met
85 90 95
Gly Leu Lys Ala Lys Tyr Val Tyr Gly Ser Lys Phe Gln Leu Asp Lys
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Asp Tyr Thr Leu Asn Val Tyr Arg Leu Ala Leu Lys Thr Thr Leu Lys
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Arg Ala Arg Arg Ser Met Glu Leu Ile Ala Arg Glu Asp Glu Asn Pro
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Lys Val Ala Glu Val Ile Tyr Pro Ile Met Gln Val Asn Ala Ile Tyr
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Leu Ala Val Asp Val Ala Val Gly Gly Met Glu Gln Arg Lys Ile His
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Met Leu Ala Arg Glu Leu Leu Pro Lys Lys Val Val Cys Ile His Asn
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Pro Val Leu Thr Gly Leu Asp Gly Glu Gly Lys Met Ser Ser Ser Lys
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Gly Asn Phe Ile Ala Val Asp Asp Ser Pro Glu Glu Ile Arg Ala Lys
210 215 220
Ile Lys Lys Ala Tyr Cys Pro Ala Gly Val Val Glu Gly Asn Pro Ile
225 230 235 240
Met Glu Ile Ala Lys Tyr Phe Leu Glu Tyr Pro Leu Thr Ile Lys Arg
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Pro Glu Lys Phe Gly Gly Asp Leu Thr Val Asn Ser Tyr Glu Glu Leu
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Glu Ser Leu Phe Lys Asn Lys Glu Leu His Pro Met Asp Leu Lys Asn
275 280 285
Ala Val Ala Glu Glu Leu Ile Lys Ile Leu Glu Pro Ile Arg Lys Arg
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Leu
305
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Met Asp Glu Phe Glu Met Ile Lys Arg Asn Thr Ser Glu Ile Ile Ser
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Glu Glu Glu Leu Arg Glu Val Leu Lys Lys Asp Glu Lys Ser Ala Thr
20 25 30
Ile Gly Phe Glu Pro Ser Gly Lys Ile His Leu Gly His Tyr Leu Gln
35 40 45
Ile Lys Lys Met Ile Asp Leu Gln Asn Ala Gly Phe Asp Ile Ile Ile
50 55 60
Leu Leu Ala Asp Leu His Ala Tyr Leu Asn Gln Lys Gly Glu Leu Asp
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Glu Ile Arg Lys Ile Gly Asp Tyr Asn Lys Lys Val Phe Glu Ala Met
85 90 95
Gly Leu Lys Ala Lys Tyr Val Tyr Gly Ser Asn Phe Gln Leu Asp Lys
100 105 110
Asp Tyr Thr Leu Asn Val Tyr Arg Leu Ala Leu Lys Thr Thr Leu Lys
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Arg Ala Arg Arg Ser Met Glu Leu Ile Ala Arg Glu Asp Glu Asn Pro
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Lys Val Ala Glu Val Ile Tyr Pro Ile Met Gln Val Asn Pro Leu His
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Tyr Gln Gly Val Asp Val Ala Val Gly Gly Met Glu Gln Arg Lys Ile
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His Met Leu Ala Arg Glu Leu Leu Pro Lys Lys Val Val Cys Ile His
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Asn Pro Val Leu Thr Gly Leu Asp Gly Glu Gly Lys Met Ser Ser Ser
195 200 205
Lys Gly Asn Phe Ile Ala Val Asp Asp Ser Pro Glu Glu Ile Arg Ala
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Lys Ile Lys Lys Ala Tyr Cys Pro Ala Gly Val Val Glu Gly Asn Pro
225 230 235 240
Ile Met Glu Ile Ala Lys Tyr Phe Leu Glu Tyr Pro Leu Thr Ile Lys
245 250 255
Arg Pro Glu Lys Phe Gly Gly Asp Leu Thr Val Asn Ser Tyr Glu Glu
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Leu Glu Ser Leu Phe Lys Asn Lys Glu Leu His Pro Met Asp Leu Lys
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Asn Ala Val Ala Glu Glu Leu Ile Lys Ile Leu Glu Pro Ile Arg Lys
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Arg Leu
305
<210> 10
<211> 306
<212> PRT
<213> Methanocaldococcus jannaschii
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Met Asp Glu Phe Glu Met Ile Lys Arg Asn Thr Ser Glu Ile Ile Ser
1 5 10 15
Glu Glu Glu Leu Arg Glu Val Leu Lys Lys Asp Glu Lys Ser Ala Thr
20 25 30
Ile Gly Phe Glu Pro Ser Gly Lys Ile His Leu Gly His Tyr Leu Gln
35 40 45
Ile Lys Lys Met Ile Asp Leu Gln Asn Ala Gly Phe Asp Ile Ile Ile
50 55 60
Leu Leu Ala Asp Leu His Ala Tyr Leu Asn Gln Lys Gly Glu Leu Asp
65 70 75 80
Glu Ile Arg Lys Ile Gly Asp Tyr Asn Lys Lys Val Phe Glu Ala Met
85 90 95
Gly Leu Lys Ala Lys Tyr Val Tyr Gly Ser Ser Phe Gln Leu Asp Lys
100 105 110
Asp Tyr Thr Leu Asn Val Tyr Arg Leu Ala Leu Lys Thr Thr Leu Lys
115 120 125
Arg Ala Arg Arg Ser Met Glu Leu Ile Ala Arg Glu Asp Glu Asn Pro
130 135 140
Lys Val Ala Glu Val Ile Tyr Pro Ile Met Gln Val Asn Pro Leu His
145 150 155 160
Tyr Gln Gly Val Asp Val Ala Val Gly Gly Met Glu Gln Arg Lys Ile
165 170 175
His Met Leu Ala Arg Glu Leu Leu Pro Lys Lys Val Val Cys Ile His
180 185 190
Asn Pro Val Leu Thr Gly Leu Asp Gly Glu Gly Lys Met Ser Ser Ser
195 200 205
Lys Gly Asn Phe Ile Ala Val Asp Asp Ser Pro Glu Glu Ile Arg Ala
210 215 220
Lys Ile Lys Lys Ala Tyr Cys Pro Ala Gly Val Val Glu Gly Asn Pro
225 230 235 240
Ile Met Glu Ile Ala Lys Tyr Phe Leu Glu Tyr Pro Leu Thr Ile Lys
245 250 255
Arg Pro Glu Lys Phe Gly Gly Asp Leu Thr Val Asn Ser Tyr Glu Glu
260 265 270
Leu Glu Ser Leu Phe Lys Asn Lys Glu Leu His Pro Met Asp Leu Lys
275 280 285
Asn Ala Val Ala Glu Glu Leu Ile Lys Ile Leu Glu Pro Ile Arg Lys
290 295 300
Arg Leu
305
<210> 11
<211> 306
<212> PRT
<213> Methanocaldococcus jannaschii
<400> 11
Met Asp Glu Phe Glu Met Ile Lys Arg Asn Thr Ser Glu Ile Ile Ser
1 5 10 15
Glu Glu Glu Leu Arg Glu Val Leu Lys Lys Asp Glu Lys Ser Ala Leu
20 25 30
Ile Gly Phe Glu Pro Ser Gly Lys Ile His Leu Gly His Tyr Leu Gln
35 40 45
Ile Lys Lys Met Ile Asp Leu Gln Asn Ala Gly Phe Asp Ile Ile Ile
50 55 60
Leu Leu Ala Asp Leu His Ala Tyr Leu Asn Gln Lys Gly Glu Leu Asp
65 70 75 80
Glu Ile Arg Lys Ile Gly Asp Tyr Asn Lys Lys Val Phe Glu Ala Met
85 90 95
Gly Leu Lys Ala Lys Tyr Val Tyr Gly Ser Thr Phe Gln Leu Asp Lys
100 105 110
Asp Tyr Thr Leu Asn Val Tyr Arg Leu Ala Leu Lys Thr Thr Leu Lys
115 120 125
Arg Ala Arg Arg Ser Met Glu Leu Ile Ala Arg Glu Asp Glu Asn Pro
130 135 140
Lys Val Ala Glu Val Ile Tyr Pro Ile Met Gln Val Asn Pro Val His
145 150 155 160
Tyr Gln Gly Val Asp Val Ala Val Gly Gly Met Glu Gln Arg Lys Ile
165 170 175
His Met Leu Ala Arg Glu Leu Leu Pro Lys Lys Val Val Cys Ile His
180 185 190
Asn Pro Val Leu Thr Gly Leu Asp Gly Glu Gly Lys Met Ser Ser Ser
195 200 205
Lys Gly Asn Phe Ile Ala Val Asp Asp Ser Pro Glu Glu Ile Arg Ala
210 215 220
Lys Ile Lys Lys Ala Tyr Cys Pro Ala Gly Val Val Glu Gly Asn Pro
225 230 235 240
Ile Met Glu Ile Ala Lys Tyr Phe Leu Glu Tyr Pro Leu Thr Ile Lys
245 250 255
Arg Pro Glu Lys Phe Gly Gly Asp Leu Thr Val Asn Ser Tyr Glu Glu
260 265 270
Leu Glu Ser Leu Phe Lys Asn Lys Glu Leu His Pro Met Asp Leu Lys
275 280 285
Asn Ala Val Ala Glu Glu Leu Ile Lys Ile Leu Glu Pro Ile Arg Lys
290 295 300
Arg Leu
305
<210> 12
<211> 306
<212> PRT
<213> Methanocaldococcus jannaschii
<400> 12
Met Asp Glu Phe Glu Met Ile Lys Arg Asn Thr Ser Glu Ile Ile Ser
1 5 10 15
Glu Glu Glu Leu Arg Glu Val Leu Lys Lys Asp Glu Lys Ser Ala Thr
20 25 30
Ile Gly Phe Glu Pro Ser Gly Lys Ile His Leu Gly His Tyr Leu Gln
35 40 45
Ile Lys Lys Met Ile Asp Leu Gln Asn Ala Gly Phe Asp Ile Ile Ile
50 55 60
Leu Leu Ala Asp Leu His Ala Tyr Leu Asn Gln Lys Gly Glu Leu Asp
65 70 75 80
Glu Ile Arg Lys Ile Gly Asp Tyr Asn Lys Lys Val Phe Glu Ala Met
85 90 95
Gly Leu Lys Ala Lys Tyr Val Tyr Gly Ser Ser Phe Gln Leu Asp Lys
100 105 110
Asp Tyr Thr Leu Asn Val Tyr Arg Leu Ala Leu Lys Thr Thr Leu Lys
115 120 125
Arg Ala Arg Arg Ser Met Glu Leu Ile Ala Arg Glu Asp Glu Asn Pro
130 135 140
Lys Val Ala Glu Val Ile Tyr Pro Ile Met Gln Val Asn Pro Ser His
145 150 155 160
Tyr Gln Gly Val Asp Val Ala Val Gly Gly Met Glu Gln Arg Lys Ile
165 170 175
His Met Leu Ala Arg Glu Leu Leu Pro Lys Lys Val Val Cys Ile His
180 185 190
Asn Pro Val Leu Thr Gly Leu Asp Gly Glu Gly Lys Met Ser Ser Ser
195 200 205
Lys Gly Asn Phe Ile Ala Val Asp Asp Ser Pro Glu Glu Ile Arg Ala
210 215 220
Lys Ile Lys Lys Ala Tyr Cys Pro Ala Gly Val Val Glu Gly Asn Pro
225 230 235 240
Ile Met Glu Ile Ala Lys Tyr Phe Leu Glu Tyr Pro Leu Thr Ile Lys
245 250 255
Arg Pro Glu Lys Phe Gly Gly Asp Leu Thr Val Asn Ser Tyr Glu Glu
260 265 270
Leu Glu Ser Leu Phe Lys Asn Lys Glu Leu His Pro Met Asp Leu Lys
275 280 285
Asn Ala Val Ala Glu Glu Leu Ile Lys Ile Leu Glu Pro Ile Arg Lys
290 295 300
Arg Leu
305
<210> 13
<211> 306
<212> PRT
<213> Methanocaldococcus jannaschii
<400> 13
Met Asp Glu Phe Glu Met Ile Lys Arg Asn Thr Ser Glu Ile Ile Ser
1 5 10 15
Glu Glu Glu Leu Arg Glu Val Leu Lys Lys Asp Glu Lys Ser Ala Leu
20 25 30
Ile Gly Phe Glu Pro Ser Gly Lys Ile His Leu Gly His Tyr Leu Gln
35 40 45
Ile Lys Lys Met Ile Asp Leu Gln Asn Ala Gly Phe Asp Ile Ile Ile
50 55 60
Leu Leu Ala Asp Leu His Ala Tyr Leu Asn Gln Lys Gly Glu Leu Asp
65 70 75 80
Glu Ile Arg Lys Ile Gly Asp Tyr Asn Lys Lys Val Phe Glu Ala Met
85 90 95
Gly Leu Lys Ala Lys Tyr Val Tyr Gly Ser Glu Phe Gln Leu Asp Lys
100 105 110
Asp Tyr Thr Leu Asn Val Tyr Arg Leu Ala Leu Lys Thr Thr Leu Lys
115 120 125
Arg Ala Arg Arg Ser Met Glu Leu Ile Ala Arg Glu Asp Glu Asn Pro
130 135 140
Lys Val Ala Glu Val Ile Tyr Pro Ile Met Gln Val Asn Gly Cys His
145 150 155 160
Tyr Arg Gly Val Asp Val Ala Val Gly Gly Met Glu Gln Arg Lys Ile
165 170 175
His Met Leu Ala Arg Glu Leu Leu Pro Lys Lys Val Val Cys Ile His
180 185 190
Asn Pro Val Leu Thr Gly Leu Asp Gly Glu Gly Lys Met Ser Ser Ser
195 200 205
Lys Gly Asn Phe Ile Ala Val Asp Asp Ser Pro Glu Glu Ile Arg Ala
210 215 220
Lys Ile Lys Lys Ala Tyr Cys Pro Ala Gly Val Val Glu Gly Asn Pro
225 230 235 240
Ile Met Glu Ile Ala Lys Tyr Phe Leu Glu Tyr Pro Leu Thr Ile Lys
245 250 255
Arg Pro Glu Lys Phe Gly Gly Asp Leu Thr Val Asn Ser Tyr Glu Glu
260 265 270
Leu Glu Ser Leu Phe Lys Asn Lys Glu Leu His Pro Met Asp Leu Lys
275 280 285
Asn Ala Val Ala Glu Glu Leu Ile Lys Ile Leu Glu Pro Ile Arg Lys
290 295 300
Arg Leu
305
<210> 14
<211> 306
<212> PRT
<213> Methanocaldococcus jannaschii
<400> 14
Met Asp Glu Phe Glu Met Ile Lys Arg Asn Thr Ser Glu Ile Ile Ser
1 5 10 15
Glu Glu Glu Leu Arg Glu Val Leu Lys Lys Asp Glu Lys Ser Ala Leu
20 25 30
Ile Gly Phe Glu Pro Ser Gly Lys Ile His Leu Gly His Tyr Leu Gln
35 40 45
Ile Lys Lys Met Ile Asp Leu Gln Asn Ala Gly Phe Asp Ile Ile Ile
50 55 60
Leu Leu Ala Asp Leu His Ala Tyr Leu Asn Gln Lys Gly Glu Leu Asp
65 70 75 80
Glu Ile Arg Lys Ile Gly Asp Tyr Asn Lys Lys Val Phe Glu Ala Met
85 90 95
Gly Leu Lys Ala Lys Tyr Val Tyr Gly Ser Glu Phe Gln Leu Asp Lys
100 105 110
Asp Tyr Thr Leu Asn Val Tyr Arg Leu Ala Leu Lys Thr Thr Leu Lys
115 120 125
Arg Ala Arg Arg Ser Met Glu Leu Ile Ala Arg Glu Asp Glu Asn Pro
130 135 140
Lys Val Ala Glu Val Ile Tyr Pro Ile Met Gln Val Asn Gly Thr His
145 150 155 160
Tyr Arg Gly Val Asp Val Ala Val Gly Gly Met Glu Gln Arg Lys Ile
165 170 175
His Met Leu Ala Arg Glu Leu Leu Pro Lys Lys Val Val Cys Ile His
180 185 190
Asn Pro Val Leu Thr Gly Leu Asp Gly Glu Gly Lys Met Ser Ser Ser
195 200 205
Lys Gly Asn Phe Ile Ala Val Asp Asp Ser Pro Glu Glu Ile Arg Ala
210 215 220
Lys Ile Lys Lys Ala Tyr Cys Pro Ala Gly Val Val Glu Gly Asn Pro
225 230 235 240
Ile Met Glu Ile Ala Lys Tyr Phe Leu Glu Tyr Pro Leu Thr Ile Lys
245 250 255
Arg Pro Glu Lys Phe Gly Gly Asp Leu Thr Val Asn Ser Tyr Glu Glu
260 265 270
Leu Glu Ser Leu Phe Lys Asn Lys Glu Leu His Pro Met Asp Leu Lys
275 280 285
Asn Ala Val Ala Glu Glu Leu Ile Lys Ile Leu Glu Pro Ile Arg Lys
290 295 300
Arg Leu
305
<210> 15
<211> 306
<212> PRT
<213> Methanocaldococcus jannaschii
<400> 15
Met Asp Glu Phe Glu Met Ile Lys Arg Asn Thr Ser Glu Ile Ile Ser
1 5 10 15
Glu Glu Glu Leu Arg Glu Val Leu Lys Lys Asp Glu Lys Ser Ala Ala
20 25 30
Ile Gly Phe Glu Pro Ser Gly Lys Ile His Leu Gly His Tyr Leu Gln
35 40 45
Ile Lys Lys Met Ile Asp Leu Gln Asn Ala Gly Phe Asp Ile Ile Ile
50 55 60
Leu Leu Ala Asp Leu His Ala Tyr Leu Asn Gln Lys Gly Glu Leu Asp
65 70 75 80
Glu Ile Arg Lys Ile Gly Asp Tyr Asn Lys Lys Val Phe Glu Ala Met
85 90 95
Gly Leu Lys Ala Lys Tyr Val Tyr Gly Ser Glu Phe Gln Leu Asp Lys
100 105 110
Asp Tyr Thr Leu Asn Val Tyr Arg Leu Ala Leu Lys Thr Thr Leu Lys
115 120 125
Arg Ala Arg Arg Ser Met Glu Leu Ile Ala Arg Glu Asp Glu Asn Pro
130 135 140
Lys Val Ala Glu Val Ile Tyr Pro Ile Met Gln Val Asn Gly Gly His
145 150 155 160
Tyr Leu Gly Val Asp Val Ile Val Gly Gly Met Glu Gln Arg Lys Ile
165 170 175
His Met Leu Ala Arg Glu Leu Leu Pro Lys Lys Val Val Cys Ile His
180 185 190
Asn Pro Val Leu Thr Gly Leu Asp Gly Glu Gly Lys Met Ser Ser Ser
195 200 205
Lys Gly Asn Phe Ile Ala Val Asp Asp Ser Pro Glu Glu Ile Arg Ala
210 215 220
Lys Ile Lys Lys Ala Tyr Cys Pro Ala Gly Val Val Glu Gly Asn Pro
225 230 235 240
Ile Met Glu Ile Ala Lys Tyr Phe Leu Glu Tyr Pro Leu Thr Ile Lys
245 250 255
Arg Pro Glu Lys Phe Gly Gly Asp Leu Thr Val Asn Ser Tyr Glu Glu
260 265 270
Leu Glu Ser Leu Phe Lys Asn Lys Glu Leu His Pro Met Asp Leu Lys
275 280 285
Asn Ala Val Ala Glu Glu Leu Ile Lys Ile Leu Glu Pro Ile Arg Lys
290 295 300
Arg Leu
305
<210> 16
<211> 306
<212> PRT
<213> Methanocaldococcus jannaschii
<400> 16
Met Asp Glu Phe Glu Met Ile Lys Arg Asn Thr Ser Glu Ile Ile Ser
1 5 10 15
Glu Glu Glu Leu Arg Glu Val Leu Lys Lys Asp Glu Lys Ser Ala Ala
20 25 30
Ile Gly Phe Glu Pro Ser Gly Lys Ile His Leu Gly His Tyr Leu Gln
35 40 45
Ile Lys Lys Met Ile Asp Leu Gln Asn Ala Gly Phe Asp Ile Ile Ile
50 55 60
Leu Leu Ala Asp Leu His Ala Tyr Leu Asn Gln Lys Gly Glu Leu Asp
65 70 75 80
Glu Ile Arg Lys Ile Gly Asp Tyr Asn Lys Lys Val Phe Glu Ala Met
85 90 95
Gly Leu Lys Ala Lys Tyr Val Tyr Gly Ser Arg Phe Gln Leu Asp Lys
100 105 110
Asp Tyr Thr Leu Asn Val Tyr Arg Leu Ala Leu Lys Thr Thr Leu Lys
115 120 125
Arg Ala Arg Arg Ser Met Glu Leu Ile Ala Arg Glu Asp Glu Asn Pro
130 135 140
Lys Val Ala Glu Val Ile Tyr Pro Ile Met Gln Val Asn Val Ile His
145 150 155 160
Tyr Asp Gly Val Asp Val Ala Val Gly Gly Met Glu Gln Arg Lys Ile
165 170 175
His Met Leu Ala Arg Glu Leu Leu Pro Lys Lys Val Val Cys Ile His
180 185 190
Asn Pro Val Leu Thr Gly Leu Asp Gly Glu Gly Lys Met Ser Ser Ser
195 200 205
Lys Gly Asn Phe Ile Ala Val Asp Asp Ser Pro Glu Glu Ile Arg Ala
210 215 220
Lys Ile Lys Lys Ala Tyr Cys Pro Ala Gly Val Val Glu Gly Asn Pro
225 230 235 240
Ile Met Glu Ile Ala Lys Tyr Phe Leu Glu Tyr Pro Leu Thr Ile Lys
245 250 255
Arg Pro Glu Lys Phe Gly Gly Asp Leu Thr Val Asn Ser Tyr Glu Glu
260 265 270
Leu Glu Ser Leu Phe Lys Asn Lys Glu Leu His Pro Met Asp Leu Lys
275 280 285
Asn Ala Val Ala Glu Glu Leu Ile Lys Ile Leu Glu Pro Ile Arg Lys
290 295 300
Arg Leu
305
<210> 17
<211> 306
<212> PRT
<213> Methanocaldococcus jannaschii
<400> 17
Met Asp Glu Phe Glu Met Ile Lys Arg Asn Thr Ser Glu Ile Ile Ser
1 5 10 15
Glu Glu Glu Leu Arg Glu Val Leu Lys Lys Asp Glu Lys Ser Ala Gly
20 25 30
Ile Gly Phe Glu Pro Ser Gly Lys Ile His Leu Gly His Tyr Leu Gln
35 40 45
Ile Lys Lys Met Ile Asp Leu Gln Asn Ala Gly Phe Asp Ile Ile Ile
50 55 60
Leu Leu Ala Asp Leu His Ala Tyr Leu Asn Gln Lys Gly Glu Leu Asp
65 70 75 80
Glu Ile Arg Lys Ile Gly Asp Tyr Asn Lys Lys Val Phe Glu Ala Met
85 90 95
Gly Leu Lys Ala Lys Tyr Val Tyr Gly Ser Thr Phe Gln Leu Asp Lys
100 105 110
Asp Tyr Thr Leu Asn Val Tyr Arg Leu Ala Leu Lys Thr Thr Leu Lys
115 120 125
Arg Ala Arg Arg Ser Met Glu Leu Ile Ala Arg Glu Asp Glu Asn Pro
130 135 140
Lys Val Ala Glu Val Ile Tyr Pro Ile Met Gln Val Asn Thr Tyr Tyr
145 150 155 160
Tyr Leu Gly Val Asp Val Ala Val Gly Gly Met Glu Gln Arg Lys Ile
165 170 175
His Met Leu Ala Arg Glu Leu Leu Pro Lys Lys Val Val Cys Ile His
180 185 190
Asn Pro Val Leu Thr Gly Leu Asp Gly Glu Gly Lys Met Ser Ser Ser
195 200 205
Lys Gly Asn Phe Ile Ala Val Asp Asp Ser Pro Glu Glu Ile Arg Ala
210 215 220
Lys Ile Lys Lys Ala Tyr Cys Pro Ala Gly Val Val Glu Gly Asn Pro
225 230 235 240
Ile Met Glu Ile Ala Lys Tyr Phe Leu Glu Tyr Pro Leu Thr Ile Lys
245 250 255
Arg Pro Glu Lys Phe Gly Gly Asp Leu Thr Val Asn Ser Tyr Glu Glu
260 265 270
Leu Glu Ser Leu Phe Lys Asn Lys Glu Leu His Pro Met Asp Leu Lys
275 280 285
Asn Ala Val Ala Glu Glu Leu Ile Lys Ile Leu Glu Pro Ile Arg Lys
290 295 300
Arg Leu
305

Claims (7)

1. A polypeptide incorporating at least one compound or salt thereof, wherein the compound or salt thereof comprises formula (XI), wherein the compound or salt thereof is an unnatural amino acid, and wherein formula (XI) is:
Figure FDA0004105855270000011
wherein:
a is phenylene;
b is absent;
r is methyl;
R 1 is H, an amino acid, a polypeptide or a polynucleotide;
R 2 is OH, an amino acid, a polypeptide or a polynucleotide; and is also provided with
Wherein R is 1 And/or R 2 Is a polypeptide;
R 3 and R is R 4 Each independently is H;
R 5 is L-X, wherein
X is selected from polyethylene glycol derivatives or drugs; and is also provided with
L is optional and when present is a linker selected from the group consisting of: alkylene, alkenylene, -O- (alkylene) -, -S- (alkylene) -, wherein k is 1 S (O) of 2 or 3 k -、-C(O)-、
-C (O) - (alkylene) -, -C (S) - (alkylene) -, -N (R ') -, NR ' - (alkylene) -, -C (O) N (R ') -,
-CON (R ') - (alkylene) -, -CSN (R ') - (alkylene) -, -N (R ') CO- (alkylene) -, and-,
-N (R ') C (O) O-, - (alkylene) -O-n=cr ' -, - (alkylene) -C (O) NR ' - (alkylene) -, - (alkylene) -S-
-N(R')C(O)N(R')-、-N(R')C(S)N(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 ') -, wherein each R' is independently H or alkyl.
2. The polypeptide of claim 1, wherein X is a drug selected from the group consisting of antibiotics, fungicides, antiviral agents, anti-inflammatory agents, antitumor agents, cardiovascular agents, anxiolytic agents, hormones, growth factors, and steroid agents.
3. A method of providing a derivative polypeptide comprising at least one oxime-containing amino acid having the structure of formula (XI),
the method comprises contacting a polypeptide comprising an unnatural amino acid of formula (I) with an agent of formula (XIX), thereby derivatizing the polypeptide, wherein formula (I) corresponds to:
Figure FDA0004105855270000021
wherein:
a is phenylene;
b is absent;
j is
Figure FDA0004105855270000022
R is methyl;
R 1 is H, an amino acid, a polypeptide or a polynucleotide;
R 2 is OH, an amino acid, a polypeptide or a polynucleotide; and is also provided with
Wherein R is 1 And/or R 2 Is a polypeptide;
R 3 And R is R 4 Each independently is H;
wherein formula (XIX) corresponds to:
Figure FDA0004105855270000023
wherein:
each X is independently a drug or a derivative of polyethylene glycol;
each L is a linker independently selected from the group consisting of: alkylene, alkenylene, -O- (alkylene) -, -S- (alkylene) -, S (O) wherein k is 1, 2 or 3 k -C (O) -, -C (O) - (alkylene) -, -C (S) - (alkylene) -, -N (R '), -NR ' - (alkylene) -, -C (O) N (R ') -, -CON (R ') - (alkylene) -, - (alkylene) NR ' C (O) O- (alkylene) -, -O-CON (R ') - (alkylene) -, -CSN (R ') -, and-CSN (R ') - (alkylene) -, -N (R ') CO- (alkylene) -, -N (R ') C (O) O- (alkylene) -, -N (R ') C (O) N (R ') -, -N (R ') C (O) N (R ') - (alkylene) -, -N (R ') C (S) N (R ') -, -N (R ') -n=, -C (R ')=n-N (R ') - -C (R ')=n-n=, -C (R') 2 -n=n-and-C (R') 2 -N (R ') -, wherein each R' is independently H or alkyl;
L 1 is optional and when present is-C (R') p -NR' -C (O) O- (alkylene) -, wherein p is 0, 1 or 2;
w is-ON (R) 1 ) 2 Wherein R is 1 Each independently is H or an amino protecting group; and is also provided with
n is a number from 1 to 3,
and wherein the derivatized polypeptide comprises at least one oxime-containing amino acid having the structure of formula (XI):
Figure FDA0004105855270000031
Wherein:
a is phenylene;
b is absent;
r is methyl;
R 1 is H, an amino acid, a polypeptide or a polynucleotide;
R 2 is OH, an amino acid, a polypeptide or a polynucleotide; and is also provided with
Wherein R is 1 And/or R 2 Is a polypeptide;
R 3 and R is R 4 Each independently is H;
R 5 is L-X, wherein
X is a drug or a derivative of polyethylene glycol; and is also provided with
L is optional and when present is a linker selected from the group consisting of: alkylene, alkenylene, -O- (alkylene) -, -S- (alkylene) -, S (O) wherein k is 1, 2 or 3 k -C (O) -, -C (O) - (alkylene) -, -C (S) - (alkylene) -, -N (R '), -NR ' - (alkylene) -, -C (O) N (R '), -CON (R ') - (alkylene) -, -CSN (R ') - (alkylene) -, -N (R ') CO- (alkylene) -, -N (R ') C (O) O-, -N (R ') C (O) N (R '), -N (R ') C (S) N (R '), -N =, -C (R ') = N-N (R '), -C (R ') = N-n=, -C (R ') = 2 -n=n-and-C (R') 2 -N (R ') -, wherein each R' is independently H or alkyl.
4. A method according to claim 3, wherein the reagent corresponds to formula (XXVII):
Figure FDA0004105855270000041
5. a method according to claim 3, wherein the polypeptide is contacted with the agent of formula (XIX) in aqueous solution under moderately acidic conditions.
6. The method of claim 5, wherein the conditions are pH 2 to 8.
7. A method according to claim 3, wherein the polypeptide is contacted with the agent of formula (XIX) in the presence of a promoter selected from the group consisting of:
Figure FDA0004105855270000042
and
Figure FDA0004105855270000043
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US10800856B2 (en) * 2012-06-07 2020-10-13 Ambrx, Inc. Prostate-specific membrane antigen antibody drug conjugates
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CN106248742B (en) * 2016-09-23 2019-03-29 郑州轻工业学院 Chitosan/gold complex system, preparation method and application
CN111479800A (en) * 2018-02-12 2020-07-31 四川科伦博泰生物医药股份有限公司 Intermediate compound, preparation method thereof and solid-phase synthesis method for preparing polypeptide by using intermediate compound
CN110423280B (en) * 2019-07-31 2021-02-02 北京泓恩生物科技有限公司 Preparation method of human papilloma virus and heat shock protein recombinant protein
KR20240024235A (en) * 2021-07-29 2024-02-23 노보코덱스 바이오파마슈티컬즈 컴퍼니 리미티드 Non-natural amino acids and uses thereof, recombinant proteins containing them, and recombinant protein conjugates
CN113979889A (en) * 2021-11-09 2022-01-28 西安康福诺生物科技有限公司 Synthesis method of bifunctional polyethylene glycol amine
CN115093487B (en) * 2021-12-30 2023-07-25 江苏超力建材科技有限公司 Hydration heat inhibitor and preparation method thereof
CN117542460B (en) * 2024-01-09 2024-03-22 江苏尤里卡生物科技有限公司 Adaptive parameter optimization method and system for urokinase separation

Family Cites Families (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3849576A (en) * 1964-01-29 1974-11-19 Oreal Process and cosmetic compositions for the treatment of skin and scalp
ZA783356B (en) * 1977-07-21 1979-06-27 Merrell Toraude & Co A-halomethyl amino acid derivatives
DE3043159C2 (en) * 1980-11-15 1982-12-09 Degussa Ag, 6000 Frankfurt Cyclic acetals of glutamic acid-γ-semialdehyde, process for their preparation and their use
US5876916A (en) * 1996-03-18 1999-03-02 Case Western Reserve University Pyruvate compounds and methods for use thereof
JPH11140076A (en) * 1997-11-04 1999-05-25 Dai Ichi Pure Chem Co Ltd N-acyl-alpha-aminoadipic acid-gamma-semialdehyde ethylene acetal

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
Optically active aromatic amino acids. Part II. Synthesis of 3(E)-(hydroxyiminomethyl) derivatives of L-tyrosine;Arnold, Zdzislaw;《Polish Journal of Chemistry》;19831231;1021-5 *
Optically active aromatic amino acids. Part IV. Synthesis and analgesic activity of four derivatives of D-tyrosine;Arnold, Zdzislaw S.;《Polish Journal of Chemistry》;19851231;837-43 *
Optically active aromatic amino acids. Part V: Some N-t-butyloxycarbonyl-O-methyl-L-tyrosine analogs with ring substitution at position 3;Arnold, Zdzislaw S.;《 Journal of Peptide Science》;19971231;354-360 *
Optically Active Aromatic Amino Acids. Part VI. Synthesis and Properties of [Leu5]-enkephalin Analogues Containing O-methyl-L-tyrosine1 with Ring Substitution at Position 3;ZDZISLAW S. ARNOLD etal;《J. Peptide Sci.》;20001231;Fig 1 *
SUPPORTING INFORMATION FOR: A STRATEGY FOR THE CHEMOSELECTIVE SYNTHESIS OF O-LINKED GLYCOPEPTIDES WITH NATIVE SUGAR-PEPTIDE LINKAGES;Elena C. Rodriguez etal;《J. Am. Chem. Soc.》;19971231;9905 *
The effects of D-phenylalanine and its derivatives on enkephalin degradation in vitro: relation to analgesia and attenuation of the morphine withdrawal syndrome;Janicki, Piotr K.etal;《Polish Journal of Pharmacology and Pharmacy》;19861231;41-9 *

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