Accelerants for the modification of non-natural amino acids and non-natural amino acid polypeptides

This application claims benefit of U.S. Provisional Application No. 60/734,589, entitled “Accelerants for the modification of non-natural amino acids and non-natural amino acid polypeptides” filed on Nov. 8, 2005.

Accelerants for the modification of molecules containing a carbonyl moiety, including non-natural amino acids and agents containing a non-natural amino acids.

The ability to incorporate non-genetically encoded amino acids (i.e., “non-natural amino acids”) into proteins permits the introduction of chemical functional groups that could provide valuable alternatives to the naturally-occurring functional groups, such as the epsilon —NH₂ of lysine, the sulfhydryl —SH of cysteine, the imino group of histidine, etc. Certain chemical functional groups are known to be inert to the functional groups found in the 20 common, genetically-encoded amino acids but react cleanly and efficiently to form stable linkages with functional groups that can be incorporated onto non-natural amino acids.

Methods are now available to selectively introduce chemical functional groups that are not found in proteins, that are chemically inert to all of the functional groups found in the 20 common, genetically-encoded amino acids and that may be used to react efficiently and selectively with reagents comprising certain functional groups to form stable covalent linkages.

Described herein are methods, compositions, techniques and strategies comprising accelerants for the reaction of hydroxylamine-containing compounds with carbonyl-containing compounds. The accelerants find use in the synthesis of oxime-containing compounds. The accelerants, in some embodiments, form bonds with the carbonyl-containing compounds, and as such, these new compounds are more reactive with hydroxylamine-containing compounds. Described herein are chemical compounds that can modulate the reaction of hydroxylamine-containing compounds with carbonyl-containing compounds. Also described herein are chemical compounds that can lower the activation barrier for the reaction of hydroxylamine-containing compounds with carbonyl-containing compounds. Also described herein are chemical compounds that, when included in a reaction comprising hydroxylamine-containing compounds and carbonyl-containing compounds, increase the rate at which oxime-containing compounds are formed. The hydroxylamine-, carbonyl-, and oxime-containing compounds can include non-natural amino acids, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides. The carbonyl-containing compounds include compounds comprising an aromatic ketone moiety. Such compounds comprising an aromatic ketone moiety include amino acids and polypeptides. By way of example only, para-acetylphenylalanine or pAcF, is an amino acid that comprises an aromatic ketone moiety.

In one aspect are compounds that accelerate (referred to herein as accelerants) the rate of reaction between hydroxylamine-containing compounds with carbonyl-containing compounds to form oxime-containing compounds.

In one embodiment, the hydroxylamine-containing compound is a non-natural amino acid, non-natural amino acid polypeptide or a modified non-natural amino acid polypeptide, and the carbonyl-containing compound comprises a desired functionality. In a further embodiment, the resulting oxime-containing compound comprises one of the aforementioned desired groups (i.e., a desired functionality). In a related aspect are the use of such compounds to accelerate the rate of reaction between a hydroxylamine-containing moiety on a non-natural amino acid, non-natural amino acid polypeptide or a modified non-natural amino acid polypeptide with a carbonyl-containing compound comprising a desired group (i.e., a desired functionality) to form an oxime-containing non-natural amino acid, non-natural amino acid polypeptide or modified non-natural amino acid polypeptide comprising a desired group. In another related aspect are reaction mixtures containing an accelerant, a hydroxylamine-containing non-natural amino acid, non-natural amino acid polypeptide or modified non-natural amino acid polypeptide, and a carbonyl-containing compound comprising a desired group. In another related aspect are oxime-containing non-natural amino acids, non-natural amino acid polypeptides or modified non-natural amino acid polypeptides comprising a desired group, wherein such oxime-containing compounds are formed from the reaction of a hydroxylamine-containing non-natural amino acid, non-natural amino acid polypeptide or modified non-natural amino acid polypeptide with a carbonyl-containing compound comprising a desired group in the presence of an accelerant. In one embodiment, the carbonyl group is not an aldehyde. In another embodiment, the carbonyl group is an aromatic ketone.

In another embodiment, the carbonyl-containing compound is a non-natural amino acid, non-natural amino acid polypeptide or a modified non-natural amino acid polypeptide, and the hydroxylamine-containing compound comprises a desired functionality. In a further embodiment, the oxime-containing compound comprises one of the aforementioned groups. In a related aspect are the use of such compounds to accelerate the rate of reaction between a carbonyl-containing moiety on a non-natural amino acid, non-natural amino acid polypeptide or a modified non-natural amino acid polypeptide with a hydroxylamine-containing compound comprising a desired group to form an oxime-containing non-natural amino acid, non-natural amino acid polypeptide or modified non-natural amino acid polypeptide comprising a desired group. In another related aspect are reaction mixtures containing an accelerant, a carbonyl-containing non-natural amino acid, non-natural amino acid polypeptide or modified non-natural amino acid polypeptide, and a hydroxylamine-containing compound comprising a desired group. In another related aspect are oxime-containing non-natural amino acids, non-natural amino acid polypeptides or modified non-natural amino acid polypeptides comprising a desired group, wherein such oxime-containing compounds are formed from the reaction of a carbonyl-containing non-natural amino acid, non-natural amino acid polypeptide or modified non-natural amino acid polypeptide with a hydroxylamine-containing compound comprising a desired group in the presence of an accelerant. In one embodiment, the carbonyl group is not an aldehyde. In another embodiment, the carbonyl group is an aromatic ketone.

In a further aspect are methods for optimizing the reaction of a carbonyl-containing compound and a hydroxylamine-containing compound to form an oxime-containing compound by selection of at least one appropriate accelerant. In one embodiment, such optimization comprises comparing the yield of the oxime-containing compound in the presence of different accelerants, different molar ratios of accelerants, or a combination of the foregoing. In a further embodiment the yield of the oxime-containing compound is monitored by chromatography. In another embodiment, such optimization comprises comparing the amount of side-products that result in the presence of different accelerants, different molar ratios of accelerants, or a combination of the foregoing. In a further embodiment the quantity of side products is monitored by chromatography. In further embodiments, such optimization includes changing additional reaction conditions, including by way of example only pH and temperature. In one embodiment, the carbonyl group is not an aldehyde. In another embodiment, the carbonyl group is an aromatic ketone.

In one aspect are non-natural amino acids based upon an oxime bond in which the oxime bond was formed in the presence of an accelerant described herein. In further or additional embodiments, the non-natural amino acid is incorporated into a polypeptide, that is, such embodiments are non-natural amino acid polypeptides. In further or additional embodiments, the non-natural amino acids are functionalized on their sidechains such that their reaction with a derivatizing molecule generates an oxime bond formed in the presence of an accelerant described herein. In further or additional embodiments are non-natural amino acid polypeptides that can react with a derivatizing molecule, formed in the presence of an accelerant described herein (although such a reaction may be less efficient in the absence of an accelerant described herein), to generate an oxime-containing non-natural amino acid polypeptide. In further or additional embodiments, the non-natural amino acids are selected from amino acids having carbonyl, dicarbonyl or hydroxylamine sidechains. In further or additional embodiments, the non-natural amino acids comprise carbonyl or dicarbonyl sidechains where the carbonyl or dicarbonyl is selected from a ketone or an aldehyde. In another embodiment are non-natural amino acids containing a functional group that is capable of forming an oxime upon treatment with an appropriately functionalized co-reactant in the presence of an accelerant described herein (although such a reaction may be less efficient in the absence of an accelerant described herein). In a further or additional embodiment, the non-natural amino acids resemble a natural amino acid in structure but contain one of the aforementioned functional groups. In another or further embodiment the non-natural amino acids resemble phenylalanine or tyrosine (aromatic amino acids); while in a separate embodiment, the non-natural amino acids resemble alanine and leucine (hydrophobic amino acids). In one embodiment, the non-natural amino acids have properties that are distinct from those of the natural amino acids. In one embodiment, such distinct properties are the chemical reactivity of the sidechain, in a further embodiment this distinct chemical reactivity permits the sidechain of the non-natural amino acid to undergo a reaction while being a unit of a polypeptide even though the sidechains of the naturally-occurring amino acid units in the same polypeptide do not undergo the aforementioned reaction. In a further embodiment, the sidechain of the non-natural amino acid has a chemistry orthogonal to those of the naturally-occurring amino acids. In a further embodiment, the sidechain of the non-natural amino acid comprises an electrophile-containing moiety; in a further embodiment, the electrophile-containing moiety on the sidechain of the non-natural amino acid can undergo nucleophilic attack to generate an oxime-derivatized protein in the presence of an accelerant described herein (although such a reaction may be less efficient in the absence of an accelerant described herein). In any of the aforementioned embodiments in this paragraph, the non-natural amino acid may exist as a separate molecule or may be incorporated into a polypeptide of any length; if the latter, then the polypeptide may further incorporate naturally-occurring or non-natural amino acids. In one embodiment, the carbonyl group is not an aldehyde. In another embodiment, the carbonyl group is an aromatic ketone.

In another aspect are hydroxylamine-substituted molecules for the production of derivatized non-natural amino acid polypeptides based upon an oxime bond in the presence of an accelerant described herein (although such a reaction may be less efficient in the absence of an accelerant described herein). In a further embodiment are hydroxylamine-substituted molecules used to derivatize carbonyl- or dicarbonyl-containing non-natural amino acid polypeptides via the formation of an oxime bond between the derivatizing molecule and the carbonyl- or dicarbonyl-containing non-natural amino acid polypeptide in the presence of an accelerant described herein (although such a reaction may be less efficient in the absence of an accelerant described herein). In further embodiments the aforementioned carbonyl- or dicarbonyl-containing non-natural amino acid polypeptides are keto-containing non-natural amino acid polypeptides. In further or additional embodiments, the carbonyl- or dicarbonyl-containing non-natural amino acids comprise sidechains selected from a ketone or an aldehyde. In further or additional embodiments, the hydroxylamine-substituted molecules comprise a desired functionality. In further or additional embodiments, the hydroxylamine-substituted molecules are hydroxylamine-substituted polyethylene glycol (PEG) molecules. In a further embodiment, the sidechain of the non-natural amino acid has a chemistry orthogonal to those of the naturally-occurring amino acids that allows the non-natural amino acid to react selectively with the hydroxylamine-substituted molecules in the presence of an accelerant described herein (although such a reaction may be less efficient in the absence of an accelerant described herein). In a further embodiment, the sidechain of the non-natural amino acid comprises an electrophile-containing moiety that reacts selectively with the hydroxylamine-containing molecule in the presence of an accelerant described herein (although such a reaction may be less efficient in the absence of an accelerant described herein); in a further embodiment, the electrophile-containing moiety on the sidechain of the non-natural amino acid can undergo nucleophilic attack to generate an oxime-derivatized protein in the presence of an accelerant described herein (although such a reaction may be less efficient in the absence of an accelerant described herein). In a further aspect related to the embodiments described in this paragraph are the modified non-natural amino acid polypeptides that result from the reaction of the derivatizing molecule with the non-natural amino acid polypeptides in the presence of an accelerant described herein (although such a reaction may be less efficient in the absence of an accelerant described herein). Further embodiments include any further modifications of the already modified non-natural amino acid polypeptides. In one embodiment, the carbonyl group is not an aldehyde. In another embodiment, the carbonyl group is an aromatic ketone.

In another aspect are carbonyl- or dicarbonyl-substituted molecules for the production of derivatized non-natural amino acid polypeptides based upon an oxime bond, wherein the oxime bond is formed in the presence of an accelerant described herein (although such a reaction may be less efficient in the absence of an accelerant described herein). In a further embodiment are carbonyl- or dicarbonyl-substituted molecules used to derivatize hydroxylamine-containing non-natural amino acid polypeptides via the formation of an oxime bond in the presence of an accelerant described herein (although such a reaction may be less efficient in the absence of an accelerant described herein). In a further embodiment the carbonyl- or dicarbonyl-substituted molecules are aldehyde-substituted molecules or ketone-substituted moieties. In further embodiments, the carbonyl- or dicarbonyl-substituted molecules comprise a desired functionality. In further or additional embodiments, the aldehyde-substituted molecules are aldehyde-substituted polyethylene glycol (PEG) molecules. In further or additional embodiments, the ketone-substituted molecules are ketone-substituted polyethylene glycol (PEG) molecules. In a further embodiment, the sidechain of the non-natural amino acid has a chemistry orthogonal to those of the naturally-occurring amino acids that allows the non-natural amino acid to react selectively with the carbonyl- or dicarbonyl-substituted molecules in the presence of an accelerant described herein (although such a reaction may be less efficient in the absence of an accelerant described herein). In a further embodiment, the sidechain of the non-natural amino acid comprises a moiety (e.g., hydroxylamine group) that reacts selectively with the carbonyl- or dicarbonyl-containing molecule in the presence of an accelerant described herein (although such a reaction may be less efficient in the absence of an accelerant described herein); in a further embodiment, the nucleophilic moiety on the sidechain of the non-natural amino acid can undergo electrophilic attack to generate an oxime-derivatized protein in the presence of an accelerant described herein (although such a reaction may be less efficient in the absence of an accelerant described herein). In a further aspect related to the embodiments described in this paragraph are the modified non-natural amino acid polypeptides that result from the reaction of the derivatizing molecule with the non-natural amino acid polypeptides in the presence of an accelerant described herein (although such a reaction may be less efficient in the absence of an accelerant described herein). Further embodiments include any further modifications of the already modified non-natural amino acid polypeptides. In one embodiment, the carbonyl group is not an aldehyde. In another embodiment, the carbonyl group is an aromatic ketone.

In another aspect are mono-, bi- and multi-functional linkers for the generation of derivatized non-natural amino acid polypeptides based upon an oxime bond, wherein the oxime bond is formed in the presence of an accelerant described herein (although such a reaction may be less efficient in the absence of an accelerant described herein). In one embodiment are molecular linkers (bi- and multi-functional) that can be used to connect carbonyl- or dicarbonyl-containing non-natural amino acid polypeptides to other molecules in the presence of an accelerant described herein (although such a reaction may be less efficient in the absence of an accelerant described herein). In another embodiment are molecular linkers (bi- and multi-functional) that can be used to connect hydroxylamine-containing non-natural amino acid polypeptides to other molecules in the presence of an accelerant described herein (although such a reaction may be less efficient in the absence of an accelerant described herein). In another embodiment the carbonyl- or dicarbonyl-containing non-natural amino acid polypeptides comprise a ketone and/or an aldehyde sidechain. In an embodiment utilizing a hydroxylamine-containing non-natural amino acid polypeptide, the molecular linker contains a carbonyl or dicarbonyl group at one of its termini; in further embodiments, the carbonyl or dicarbonyl group is selected from an aldehyde group or a ketone group. In further or additional embodiments, the hydroxylamine-substituted linker molecules are hydroxylamine-substituted polyethylene glycol (PEG) linker molecules. In further or additional embodiments, the carbonyl- or dicarbonyl-substituted linker molecules are carbonyl- or dicarbonyl-substituted polyethylene glycol (PEG) linker molecules. Throughout, the phrase “other molecules” includes, by way of example only, proteins, other polymers (branched and unbranched), small molecules, and groups also identified as a “desired functionality.” In further or additional embodiments, the hydroxylamine-containing molecular linkers comprise the same or equivalent groups on all termini so that upon reaction with a carbonyl- or dicarbonyl-containing non-natural amino acid polypeptide in the presence of an accelerant described herein (although such a reaction may be less efficient in the absence of an accelerant described herein), the resulting product is the homo-multimerization of the carbonyl- or dicarbonyl-containing non-natural amino acid polypeptide. In further embodiments, the homo-multimerization is a homo-dimerization. In further or additional embodiments, the carbonyl- or dicarbonyl-containing molecular linkers comprise the same or equivalent groups on all termini so that upon reaction with a hydroxylamine-containing non-natural amino acid polypeptide in the presence of an accelerant described herein (although such a reaction may be less efficient in the absence of an accelerant described herein), the resulting product is the homo-multimerization of the hydroxylamine-containing non-natural amino acid polypeptide. In further embodiments, the homo-multimerization is a homo-dimerization. In a further embodiment, the sidechain of the non-natural amino acid has a chemistry orthogonal to those of the naturally-occurring amino acids that allows the non-natural amino acid to react selectively with the hydroxylamine-substituted linker molecules in the presence of an accelerant described herein (although such a reaction may be less efficient in the absence of an accelerant described herein). In a further embodiment, the sidechain of the non-natural amino acid has a chemistry orthogonal to those of the naturally-occurring amino acids that allows the non-natural amino acid to react selectively with the carbonyl- or dicarbonyl-substituted linker molecules in the presence of an accelerant described herein (although such a reaction may be less efficient in the absence of an accelerant described herein). In a further embodiment, the sidechain of the non-natural amino acid comprises an electrophile-containing moiety that reacts selectively with the hydroxylamine-containing linker molecule in the presence of an accelerant described herein (although such a reaction may be less efficient in the absence of an accelerant described herein); in a further embodiment, the electrophile-containing moiety on the sidechain of the non-natural amino acid can undergo nucleophilic attack by the hydroxylamine-containing linker molecule to generate an oxime-derivatized protein in the presence of an accelerant described herein (although such a reaction may be less efficient in the absence of an accelerant described herein). In a further aspect related to the embodiments described in this paragraph are the linked (modified) non-natural amino acid polypeptides that result from the reaction of the linker molecule with the non-natural amino acid polypeptides. Further embodiments include any further modifications of the already linked (modified) non-natural amino acid polypeptides. In one embodiment, the carbonyl group is not an aldehyde. In another embodiment, the carbonyl group is an aromatic ketone.

In one aspect are methods to derivatize proteins via the condensation of carbonyl or dicarbonyl and hydroxylamine reactants in the presence of an accelerant described herein (although such a reaction may be less efficient in the absence of an accelerant described herein) to generate an oxime-based product. Included within this aspect are methods for the derivatization of proteins based upon the condensation of carbonyl- or dicarbonyl- and hydroxylamine-containing reactants to generate an oxime-derivatized protein adduct. In additional or further embodiments are methods to derivatize keto-containing proteins with hydroxylamine-functionalized polyethylene glycol (PEG) molecules. In yet additional or further aspects, the hydroxylamine-substituted molecule can include proteins, other polymers (branched and unbranched), small molecules and groups also identified as a “desired functionality.” In one embodiment, the carbonyl group is not an aldehyde. In another embodiment, the carbonyl group is an aromatic ketone.

In another aspect are methods for the chemical synthesis of hydroxylamine-substituted molecules for the derivatization of keto-substituted proteins in the presence of an accelerant described herein (although such a reaction may be less efficient in the absence of an accelerant described herein). In one embodiment, the hydroxylamine-substituted molecule can comprise peptides, other polymers (non-branched and branched) and small molecules. In one embodiment are methods for the preparation of hydroxylamine-substituted molecules suitable for the derivatization of carbonyl- or dicarbonyl-containing non-natural amino acid polypeptides in the presence of an accelerant described herein (although such a reaction may be less efficient in the absence of an accelerant described herein), including by way of example only, keto-containing non-natural amino acid polypeptides. In a further or additional embodiment, the non-natural amino acids are incorporated site-specifically during the in vivo translation of proteins. In a further or additional embodiment, the hydroxylamine-substituted molecules allow for the site-specific derivatization of this carbonyl- or dicarbonyl-containing non-natural amino acid via nucleophilic attack of the carbonyl or dicarbonyl group to form an oxime-derivatized polypeptide in a site-specific fashion, wherein the oxime-derivatized polypeptide is formed in the presence of an accelerant described herein (although such a reaction may be less efficient in the absence of an accelerant described herein). In a further or additional embodiment, the method for the preparation of hydroxylamine-substituted molecules provides access to a wide variety of site-specifically derivatized polypeptides. In a further or additional embodiment are methods for synthesizing hydroxylamine-functionalized polyethylene glycol (PEG) molecules.

In another aspect are methods for the chemical derivatization of carbonyl- or dicarbonyl-substituted non-natural amino acid polypeptides, in the presence of an accelerant described herein (although such a reaction may be less efficient in the absence of an accelerant described herein), using a hydroxylamine-containing bi-functional linker. In one embodiment are methods for attaching a hydroxylamine-substituted linker to a carbonyl- or dicarbonyl-substituted protein via a condensation reaction to generate an oxime bond in the presence of an accelerant described herein (although such a reaction may be less efficient in the absence of an accelerant described herein). In further or additional embodiments, the carbonyl- or dicarbonyl-substituted non-natural amino acid is a keto-substituted non-natural amino acid. In further or additional embodiments, the non-natural amino acid polypeptides are derivatized site-specifically and/or with precise control of three-dimensional structure, using a hydroxylamine-containing bi-functional linker. In one embodiment, such methods are used to attach molecular linkers (mono- bi- and multi-functional) to carbonyl- or dicarbonyl-containing (including by way of example keto-containing) non-natural amino acid polypeptides, wherein at least one of the linker termini contains a hydroxylamine group which can link to the carbonyl- or dicarbonyl-containing non-natural amino acid polypeptides via an oxime bond in the presence of an accelerant described herein (although such a reaction may be less efficient in the absence of an accelerant described herein). In a further or additional embodiment, these linkers are used to connect the carbonyl- or dicarbonyl-containing non-natural amino acid polypeptides to other molecules, including by way of example, proteins, other polymers (branched and non-branched), small molecules and groups also identified as a “desired functionality.” In one embodiment, the carbonyl group is not an aldehyde. In another embodiment, the carbonyl group is an aromatic ketone.

In some embodiments, the non-natural amino acid polypeptide is linked to a water soluble polymer. In some embodiments, the water soluble polymer comprises a poly(ethylene glycol) moiety. In some embodiments, the poly(ethylene glycol) molecule is a bifunctional polymer. In some embodiments, the bifunctional polymer is linked to a second polypeptide. In some embodiments, the second polypeptide is identical to the first polypeptide, in other embodiments, the second polypeptide is a different polypeptide. In some embodiments, the non-natural amino acid polypeptide comprises at least two amino acids linked to a water soluble polymer comprising a poly(ethylene glycol) moiety.