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

FIELD OF THE INVENTION Accelerators for modifying carbonyl-containing molecules, including non-natural amino acids and non-natural amino acid-containing agents.

BACKGROUND OF THE INVENTION The ability to incorporate non-genetically encoded amino acids ("natural amino acids") into proteins allows the introduction of chemical functional sets that could provide useful alternatives to naturally occurring functional sets, such as ipsilon-NH2 lysine, sulfhydryl. -SH cysteine, imino histidine set, etc. Certain chemically functional assemblies are known to be inert to functional assemblies found in 20 common, genetically encoded amino acids, but react purely and efficiently to form stable bonds with functional assemblies that may be included in non-natural amino acids.

Methods are now available for the selective introduction of non-protein chemical functional assemblies, which are chemically inert to all functional assemblies contained in 20 common, genetically encoded amino acids, and which can be used for effective and selective reactions. with reagents containing certain functional sets to form stable covalent bonds.

SUMMARY OF THE INVENTION Methods, compositions, techniques and strategies comprising accelerators for reacting hydroxylamine-containing compounds with carbonyl-containing compounds are described herein. Accelerators are used in the synthesis of oxime-containing compounds. Accelerators, in some embodiments of the invention, form bonds with carbonyl-containing compounds, and

51998 Β as such, these new compounds are more reactive with hydroxylamine-containing compounds. Described herein are compounds that can modulate hydroxylamine-containing reaction compounds with carbonyl-containing compounds. Also described herein are chemical compounds that can lower the activation limit for the reaction of hydroxylamine-containing compounds with carbonyl-containing compounds. Also described herein are chemical compounds which, when involved in a reaction comprising hydroxylamine-containing compounds and carbonyl-containing compounds, increase the rate at which oxime-containing compounds are formed. Compounds containing hydroxylamine, carbonyl, and oxime may include, non-natural amino acids, non-natural amino acid polypeptides, and modified non-natural amino acid polypeptides. Carbonyl-containing compounds include compounds containing an aromatic ketone moiety. Such compounds containing an aromatic ketone moiety include amino acids and polypeptides. For example, paraacetylphenylalanine, or pAcF, is an amino acid that contains an aromatic ketone moiety.

In one aspect of the invention, the accelerating compounds (referred to herein as accelerators) are the rate of reaction between hydroxylamine-containing compounds with carbonyl-containing compounds to give oxime-containing compounds. In one embodiment, the hydroxylamine-containing compound is a non-natural amino acid, a non-natural amino acid polypeptide or a modified non-natural amino acid polypeptide, and the carbonyl-containing compound possesses the desired functionality. In a further embodiment, the resulting oxime-containing compound comprises one of said preferred sets (i.e., the desired functionality). An additional aspect is the use of such compounds to accelerate the reaction rate between a portion containing a hydroxylamine to a non-natural amino acid, a non-natural amino acid polypeptide or a modified non-natural amino acid polypeptide with a carbonyl-containing compound containing the desired set (i.e., the desired functionality) to form a non-natural amino acid containing an oxime, a non-natural amino acid polypeptide or a modified non-natural amino acid polypeptide containing the desired set. In another related aspect are reaction mixtures comprising an accelerator, a non-natural amino acid comprising a hydroxylamine, a non-natural amino acid polypeptide or a modified non-natural amino acid polypeptide, and a carbonyl-containing compound containing the desired set. In another related aspect are oxime-containing non-natural amino acids, a non-natural amino acid polypeptide, or a modified non-natural amino acid polypeptide comprising a desired set, wherein such compounds comprising

51998 The oxime is formed from the reaction of a hydroxylamine-containing non-natural amino acid, a non-natural amino acid polypeptide or a modified non-natural amino acid polypeptide with a carbonyl-containing compound containing the desired set in the presence of an accelerator. The carbonyl set is not an aldehyde. In particular, the carbonyl set is an aromatic ketone.

In another embodiment, the carbonyl-containing compound is a non-natural amino acid, a non-natural amino acid polypeptide or a modified non-natural amino acid polypeptide, and the hydroxylamine-containing compound possesses the desired functionality. In a further embodiment, the oxime-containing compound comprises one of the above sets. In a related aspect, the use of such compounds is to accelerate the rate of reaction between a carbonyl-containing portion with a non-natural amino acid, a non-natural amino acid polypeptide or a modified non-natural amino acid polypeptide with a hydroxylamine-containing compound to form the desired moiety. a natural oxime-containing amino acid, a non-natural amino acid polypeptide or a modified non-natural amino acid polypeptide containing the desired set. In another related aspect are reaction mixtures comprising an accelerator, a carbonyl-containing non-natural amino acid, a non-natural amino acid polypeptide or a modified non-natural amino acid polypeptide, and a hydroxylamine-containing compound containing the desired set. In another related aspect, the oxime-containing non-natural amino acids, the non-natural amino acid polypeptide, or the modified non-natural amino acid polypeptide containing the desired set, such oxime-containing compounds are formed from a non-natural amino acid reaction that comprising a carbonyl, a non-natural amino acid polypeptide or a modified non-natural amino acid polypeptide with a hydroxylamine-containing compound containing the desired assembly in the presence of an accelerator. The carbonyl set is not an aldehyde. In particular, the carbonyl set is an aromatic ketone.

Further aspects of the invention are methods for optimizing the reaction of a carbonyl-containing compound and a hydroxylamine-containing compound while forming an oxime-containing compound with the selection of at least one suitable accelerator. In one embodiment, such optimization comprises comparing the yield of an oxime-containing compound in the presence of different accelerators, different molar ratios of accelerators, or said combinations. 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 by-products resulting in

51998 Β the presence of different accelerators, different molar ratios of accelerators, or the above combinations. In another embodiment, the amount of by-products is monitored by chromatography. In further embodiments, such optimization involves changing additional reaction conditions, including, by way of example only, pH and temperature. The carbonyl set is not an aldehyde. In particular, the carbonyl set is an aromatic ketone.

One aspect is non-natural amino acids based on an oxime bond in which the oxime bond is formed in the presence of the accelerator described herein. In further or additional embodiments, the non-natural amino acid is included in the polypeptide, i.e., such embodiments are non-natural amino acid polypeptides. In further or additional embodiments, the non-natural amino acids are functional on their side chains such that their reaction with the derived molecule generates an oxime bond formed in the presence of the accelerator described herein. In further or additional embodiments are non-natural amino acid polypeptides that can react with a derived molecule, which is formed in the presence of the accelerator described herein (although such a reaction may be less effective in the absence of the accelerator described herein), to obtain a non-natural amino polypeptide. acid containing oxime. In further or additional embodiments, non-natural amino acids are selected from amino acids having carbonyl, dicarbonyl or hydroxylamine side chains. In further or additional embodiments, the non-natural amino acids comprise carbonyl or dicarbonyl side chains wherein the carbonyl or dicarbonyl is selected from a ketone or aldehyde. In another embodiment are non-natural amino acids comprising a functional assembly capable of forming an oxime after treatment with a suitably functionalized co-reactant in the presence of the accelerator described herein (although such a reaction may be less effective in the absence of the accelerator described herein). In a further or additional embodiment, the non-natural amino acids are similar in structure to natural amino acids but contain one of the above functional sets. In another or further embodiment, the non-natural amino acids are similar to phenylalanine or tyrosine (aromatic amino acids); while in a separate embodiment, the non-natural amino acids are similar to alanine and leukin (hydrophobic amino acids). In one embodiment, the non-natural amino acids have properties that are different from those for natural amino acids. In one embodiment, such different properties are the chemical reactivity of the side chain, in a further embodiment this different chemical reactivity allows the side chain not to

51998 Β natural amino acids to be subjected to a reaction, while the polypeptide unit is even though the side chains of naturally occurring amino acid units in the same polypeptide are not subjected to said reaction. In a further embodiment, the non-natural amino acid side chain has orthogonal chemistry to naturally occurring amino acids. In a further embodiment, the non-natural amino acid side chain comprises an electrophilic moiety; in a further embodiment, the electrophilic moiety on the side chain of a non-natural amino acid may be subjected to a nucleophilic attack to generate an oxime-derived protein in the presence of the accelerator described herein (although such a reaction may be less effective in the absence of the accelerator described herein). In any of the foregoing embodiments in this paragraph, a non-natural amino acid may exist as a separate molecule or may be included in a polypeptide of any length; in the case of the latter, then the polypeptide may further include naturally occurring amino acids or unnatural amino acids. In particular, the carbonyl set is an aromatic ketone.

In another aspect, the substituted hydroxylamine molecules for producing non-natural amino acid derived polypeptides are based on an oxime bond in the presence of the accelerator described herein (although such a reaction may be less effective in the absence of the accelerator described herein). In a further embodiment, the substituted hydroxylamine molecules are used to derive a carbonyl or dicarbonyl-containing non-natural amino acid polypeptide by forming an oxime bond between the derivative molecule and a carbonyl or dicarbonyl-containing non-natural amino acid polypeptide in the presence of such an accelerator. be less effective in the absence of the accelerator described here). In further embodiments, the aforementioned carbonyl-containing or dicarbonyl-containing non-natural amino acid polypeptides are keto-containing non-natural amino acid polypeptides. In further or additional embodiments, the non-natural amino acids comprising carbonyl- or dicarbonyl- consist of side chains selected from ketones or aldehydes. In further or additional embodiments, the substituted hydroxylamine molecule contains the desired functionality. In a further or additional embodiment, the substituted hydroxylamine molecules are hydroxylamine-substituted polyethylene glycol (PEG) molecules. In a further embodiment, the side chain of the non-natural amino acid has orthogonal chemistry to naturally occurring amino acids, allowing the non-natural amino acid to react selectively with the substituted hydroxylamine molecules in the presence described herein.

51998 Β accelerator (although such a reaction may be less effective in the absence of the accelerator described here). In a further embodiment, the non-natural amino acid side chain comprises an electrophilic moiety that reacts selectively with a hydroxylamine-containing molecule in the presence of the accelerator described herein (although such a reaction may be less effective in the absence of the accelerator described herein); in a further embodiment, the electrophilic moiety on the side chain of a non-natural amino acid may be subjected to a nucleophilic attack to generate an oxime-derived protein in the presence of the accelerator described herein (although such a reaction may be less effective in the absence of the accelerator described herein), In a further aspect relating to the embodiments described in this paragraph, modified non-natural amino acid polypeptides resulting from the reaction of a derivative molecule with non-natural amino acid polypeptides in the presence of the accelerator described herein (although such a reaction may be less effective in the absence described herein). accelerator). Further embodiments include any further modification of already modified non-natural amino acid polypeptides. The carbonyl set is not an aldehyde. In particular, the carbonyl set is an aromatic ketone.

In another aspect, the carbonyl- or dicarbonyl-substituted molecules are for the production of derivative polypeptides of non-natural amino acids based on an oxime bond, wherein the oxime bond is formed in the presence of the accelerator described herein (although such a reaction may be less effective in the absence of described accelerator). In a further embodiment, the carbonyl- or dicarbonyl-substituted molecules are used to derive hydroxylamine-containing non-natural amino acid polypeptides by forming an oxime bond in the presence of the accelerator described herein (although such a reaction may be less effective in the absence of the accelerator 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 contain the 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 side chain of the non-natural amino acid has orthogonal chemistry to naturally occurring amino acids, allowing the non-natural amino acid to react selectively with carbonyl- or dicarbonyl-substituted

51998 Β molecules in the presence of the accelerator described herein (although such a reaction may be less effective in the absence of the accelerator described herein). In a further embodiment, the non-natural amino acid side chain comprises a moiety (e.g., a hydroxylamine set) that reacts selectively with a carbonyl- or dicarbonyl-containing molecule in the presence of the accelerator described herein (although such a reaction may be less effective in the absence of the accelerator described herein). ); in a further embodiment, the nucleophilic moiety on the side chain of the unnatural amino acid may be subjected to a nucleophilic attack to generate an oxime-derived protein in the presence of the accelerator described herein (although such a reaction may be less effective in the absence of the accelerator described herein). In a further aspect related to the embodiments described in this paragraph, non-natural amino acid polypeptides resulting from the reaction of a derivative molecule with non-natural amino acid polypeptides in the presence of the accelerator described herein are modified (although such reaction may be less effective in the absence of the accelerator described herein). Further embodiments include any further modifications of previously modified non-natural amino acid polypeptides. The carbonyl assembly is not an aldehyde. In particular, the carbonyl set is an aromatic ketone.

In another aspect, mono-, bi-, and multi-functional linkers for generating derived non-natural amino acid polypeptides are based on an oxime bond, wherein the oxime bond is formed in the presence of the accelerator described herein (although such a reaction may be less effective). in the absence of the accelerator described here). In one embodiment, the molecules are linkers (bi- and multi-functional) that can be used to link carbonyl- or dicarbonyl-containing non-natural amino acid polypeptides to other molecules in the presence of the accelerator described herein, although such a reaction may be less effective. in the absence of the accelerator described here). In another embodiment, the molecules are linkers (bi- and multi-functional) that can be used to link polypeptides of non-natural amino acids containing hydroxylamine to other molecules in the presence of the accelerator described herein (although such a reaction may be less effective in the absence described herein). accelerator). In another embodiment, the carbonyl- or dicarbonyl-containing non-natural amino acid polypeptides comprise a ketone and / or aldehyde side chain. In one embodiment utilizing a hydroxylamine-containing non-natural amino acid polypeptide, the linker molecule comprises a carbonyl or dicarbonyl group on one of its

51998 Β endings; in further embodiments, the carbonyl or dicarbonyl group is selected from an aldehyde set or a ketone set. 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 description, the term second molecule includes, by way of example only, proteins, other polymers (branched and unbranched), small molecules, and assemblies that have also been identified as the desired functionality. In further or additional embodiments, the hydroxylamine-containing linker molecules consist of the same or equivalent clusters at all ends such that upon reaction with a carbonyl- or dicarbonyl-containing non-natural amino acid polypeptide in the presence of the accelerator described herein (although such a reaction may be less effective in the absence of the accelerator described herein), the resulting product being a homomultimerization of a non-natural amino acid polypeptide containing a carbonyl- or dicarbonyl-. In further embodiments, homo-multimerization is homo-dimerization. In further or additional embodiments, carbonyl- or dicarbonyl-containing linker molecules consist of the same or equivalent clusters at all terminations such that upon reaction with a hydroxylamine-containing non-natural amino acid polypeptide in the presence of the accelerator described herein (although such a reaction may be less effective in the absence of the accelerator described herein), the resulting product is homo-multirnerization of a hydroxylamine-containing non-natural amino acid polypeptide. In further embodiments, homo-multimerization is homo-dimerization. In a further embodiment, the non-natural amino acid side chain has orthogonal chemistry to naturally occurring amino acids, allowing the non-natural amino acid to react selectively with hydroxylamine-substituted linker molecules in the presence of the accelerator described herein (although such a reaction may be less effective in the absence of the accelerator described here). In a further embodiment, the non-natural amino acid side chain has orthogonal chemistry to naturally occurring amino acids, allowing the non-natural amino acid to react selectively with carbonyl- or dicarbonyl-substituted linker molecules in the presence of the accelerator described herein (although such a reaction may be less effective in the absence of the accelerator described here). In a further embodiment, the non-natural amino acid side chain comprises an electrophilic moiety that reacts selectively with a hydroxylamine-containing molecule.

51998 Β a linker in the presence of the accelerator described herein (although such a reaction may be less effective in the absence of the accelerator described herein); in a further embodiment, the electrophilic moiety on the side chain of a non-natural amino acid may be subjected to a nucleophilic attack by a hydroxylamine-containing linker molecule to generate an oxidized protein in the presence of the accelerator described herein (although such a reaction may be less effective in the absence of the accelerator described herein). . In a further aspect relating to the embodiments described in this paragraph, related (modified) polypeptides of non-natural amino acids resulting from the reaction of an Inker molecule with non-natural amino acid polypeptides. Further embodiments include any further modification of already linked (modified) non-natural amino acid polypeptides. The carbonyl set is not an aldehyde. In particular, the carbonyl set is an aromatic ketone.

In one aspect, methods for derivatizing proteins via condensation of carbonyl or dicarbonyl and hydroxylamine reactants in the presence of the accelerator described herein (although such a reaction may be less effective in the absence of the accelerator described herein) to obtain an oxime-based product. Included within this aspect are methods for derivatizing proteins based on the condensation of reactants containing carbonyl- or dicarbonyl- and hydroxylamine- to generate an oxidized protein adduct. In additional or further embodiments are methods for derivatizing proteins comprising ketos with hydroxylamine-functionalized polyethylene glycol (PEG) molecules. In yet another additional or further aspect, hydroxylamine-substituted molecules may include proteins, other polymers (branched and unbranched), small molecules, and assemblies that have also been identified as desired functionality. The carbonyl set is not an aldehyde. In particular, the carbonyl set is an aromatic ketone.

Also disclosed are methods for chemically synthesizing substituted hydroxylamine molecules to derive keto-substituted proteins in the presence of the accelerator described herein (although such a reaction may be less effective in the absence of the accelerator described herein). In one aspect, the hydroxylamine-substituted molecule may comprise peptides, other polymers (non-branched and branched), and small molecules. In one aspect, there are methods for producing substituted hydroxylamine molecules suitable for deriving a carbonyl-containing non-natural amino acid polypeptide

51998 Β or dicarbonyl- in the presence of the accelerator described herein (although such a reaction may be less effective in the absence of the accelerator described herein), including, by way of example only, keto-set polypeptides of non-natural amino acids. In a further or additional aspect, non-natural amino acids are incorporated at a specific-position during in vivo protein translation. In a further or additional embodiment, the substituted hydroxylamine molecules allow the derivation at a specific-position of these non-natural amino acids containing a carbonyl- or dicarbonyl-nucleophilic attack of the carbonyl or dicarbonyl assembly to obtain an oxime-derived polypeptide at the position of the specific wherein the oxime-derived polypeptide is formed in the presence of the accelerator described herein (although such a reaction may be less effective in the absence of the accelerator described herein). In a further or additional aspect, the method for preparing a substituted hydroxylamine molecule provides access to a wide range of derived polypeptides at a specific-position. In a further or additional aspect are methods for the synthesis of hydroxylamine-functionalized polyethylene glycol (PEG) molecules.

In another aspect, methods for chemically deriving carbonyl- or dicarbonyl-substituted non-natural amino acid polypeptides, in the presence of the accelerator described herein (although such a reaction may be less effective in the absence of the accelerator described herein), using a bi-functional linker that contains hydroxylamine. In one embodiment, methods are used to attach a hydroxylamine-substituted linker to a carbonyl- or dicarbonyl-substituted protein by a condensation reaction to generate an oxime bond in the presence of the accelerator described herein (although such a reaction may be less effective in the absence of the accelerator 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 polypeptides of non-natural amino acids are derived with a specific position and / or with precise control of the three-dimensional structure, using a bifunctional linker containing hydroxylamine. In one embodiment, such methods are used to attach molecular linkers (mono- and multi-functional) to non-natural amino acid polypeptides containing carbonyl- or dicarbonyl (including by example those containing keto-), wherein at least one from the ends of the linker contains a hydroxylamine set that can bind to non-natural amino acid polypeptides containing carbonyl- or dicarbonyl- via an oxime bond in the presence of

51998 Β the accelerator described here (although such a reaction may be less effective in the absence of the accelerator described here). In a further or additional embodiment, these linkers are used to link a carbonyl- or dicarbonyl-containing non-natural amino acid polypeptide to other molecules, including, for example, proteins, other polymers (branched and non-branched), small molecules and sets also recognized as the desired functionality. The carbonyl set is not an aldehyde. In particular, the carbonyl set is an aromatic ketone.

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

In some embodiments of the invention, the non-natural amino acid polypeptide comprises replacement, addition, or deletion that increases the affinity of the non-natural amino acid polypeptide for the receptor. In some embodiments, the non-natural amino acid polypeptide comprises replacement, addition, or deletion that increases -natural amino acids. In some embodiments of the invention, the non-natural amino acid polypeptide comprises a replacement, addition or deletion that increases the aqueous solubility of the non-natural amino acid polypeptide. In some embodiments of the invention, the non-natural amino acid polypeptide comprises a replacement, addition or deletion that increases the solubility of the non-natural amino acid polypeptide generated in the host cell. In some embodiments of the invention, the non-natural amino acid polypeptide comprises replacement, addition or deletion that modulates protease resistance, serum half-life, immunogenicity, and / or expression relative to the amino acid polypeptide without replacement, addition or deletion.

In some embodiments of the invention, the non-natural amino acid polypeptide is

51998 Β agonist, partial agonist, antagonist, partial antagonist or inverse agonist. In some embodiments of the invention, the agonist, partial agonist, antagonist, partial antagonist, or inverse agonist comprises a non-natural amino acid bound to a water-soluble polymer. In some embodiments of the invention, the aqueous polymer comprises a poly (ethylene glycol) moiety. In some embodiments of the invention, a polypeptide comprising a non-natural amino acid bound to a water-soluble polymer prevents dimerization of the corresponding receptor. In some embodiments of the invention, a polypeptide comprising an unnatural amino acid bound to a water-soluble polymer modulates the binding of the polypeptide to a binding partner. In some embodiments of the invention, a polypeptide comprising a non-natural amino acid bound to a water-soluble polymer modulates one or more properties or activities of the polypeptide.

Methods for preparing a non-natural amino acid polypeptide bound to a water-soluble polymer are also described herein. In some embodiments, the method comprises contacting an isolated non-natural amino acid polypeptide with a water-soluble polymer comprising a portion that reacts with the unnatural amino acid in the presence of the accelerator described herein (although such a reaction may be less effective in the absence described herein). accelerator). In some embodiments of the invention, the inserted non-natural amino acid is reactive with a water-soluble polymer that is otherwise unreactive to any of the 20 common amino acids. In some embodiments of the invention, the aqueous polymer comprises a portion of poly (ethylene glycol). The molecular weights of the polymer can be in a wide range, including between about 100 Da and about 100,000 Da or more. Molecules of polymer weight can be between 100 Yes and 100,000 Yes, including 100,000 Yes, 95,000 Yes, 90,000 Yes, 85,000 Yes, 80,000 Yes, 75,000 Yes, 70,000 Yes, 65,000 Yes, 60,000 Yes, 55,000 Yes, 50,000 Yes, 45,000 Yes, 40,000 Yes, 35,000 Yes, 30,000 Yes, 25,000 Yes, 20,000 Yes, 15,000 Yes, 10,000 Yes, 9,000 Yes, 8,000 Yes, 7,000 Yes, 6,000 Yes, 5,000 Yes, 4,000 Yes, 3,000 Yes, 2,000 Yes, 1,000 Yes, 900 Yes, 800 Yes, 700 Yes, 600 Yes, 500 Yes, 400 Yes, 300 Yes, 200 Yes, and 100 Yes. In some embodiments of the invention, the weight of the polymer molecules is between 100 Da and about 50,000 Da. In some embodiments of the invention, the molecular weight of the polymer is between about 100 Da and about 40,000 Da. In some embodiments of the invention, the molecular weight of the polymer is between about 1,000 Da and about 40,000 Da. In some embodiments of the invention, the molecular weight of the polymer is between about

51998 Β

5,000 Yes and about 40,000 Yes. In some embodiments of the invention, the molecular weight of the polymer is between about 10,000 Da and about 40,000 Da. In some embodiments of the invention, the poly (ethylene glycol) molecule is a branched polymer. The molecular weight of a branched PEG chain can be between about 1,000 Da and about 100,000 Da, including but not limited to 100,000 Yes, 95,000 Yes, 90,000 Yes, 85,000 Yes, 80,000 Yes, 75,000 Yes, 70,000 Yes, 65,000 Yes, 60,000 Yes, 55,000 Yes , 50,000 Yes, 45,000 Yes, 40,000 Yes, 35,000 Yes, 30,000 Yes, 25,000 Yes, 20,000 Yes, 15,000 Yes, 10,000 Yes, 9,000 Yes, 8,000 Yes, 7,000 Yes, 6,000 Yes, 5,000 Yes, 4,000 Yes, 3,000 Yes, 2,000 Yes, and 1,000 Yes. In some embodiments of the invention, the weight of the branched PEG chain molecules is between about 1,000 Da and about 50,000 Da. In some embodiments of the invention, the weight of the branched PEG chain is between about 1,000 Da and about 40,000 Da. In some embodiments of the invention, the weight of the branched PEG chain molecules is between about 5,000 Da and about 40,000 Da. In some embodiments of the invention, the weight of the branched PEG chain molecules is between about 5,000 Da and about 20,000 Da.

Also described herein are compositions comprising a polypeptide comprising at least one non-natural amino acid described herein and a pharmaceutically acceptable carrier. In some embodiments of the invention, the non-natural amino acid is bound to a water-soluble polymer. Also described herein are pharmaceutical compositions comprising a pharmaceutically acceptable carrier and polypeptide, wherein at least one amino acid is substituted with a non-natural amino acid. In some embodiments of the invention, the non-natural amino acid comprises a saccharide moiety. In some embodiments of the invention, the water-soluble polymer is linked to the polypeptide via a saccharide moiety. Also described herein are prodrugs of non-natural amino acids, non-natural amino acid polypeptides, and modified non-natural amino acid polypeptides; further described herein are compositions comprising such prodrugs and a pharmaceutically acceptable carrier. Also described herein are metabolites of non-natural amino acids, non-natural amino acid polypeptides, and modified non-natural amino acid polypeptides; such metabolites may have the desired complementary activity or have a synergistic effect with the activity of non-natural amino acids, non-natural amino acid polypeptides, and modified non-natural amino acid polypeptides. Also described herein is the use of non-natural amino acids, non-natural amino acid polypeptides, and modified non-natural amino acid polypeptides described herein to provide the desired metabolite for

51998 Β organism, including a patient in need of such a metabolite.

Also described herein are libraries of non-natural amino acids described herein or libraries of non-natural amino acid polypeptides described herein, or libraries of modified non-natural amino acid polypeptides described herein, or combinations thereof, wherein members libraries include oxybinding formed in the presence of the accelerator described herein (although such a reaction may be less effective in the absence of the accelerator described herein).

Also described herein are methods for screening libraries for desired activity described herein, or for using sequences to review libraries described herein, or for other libraries of compounds and / or polypeptides and / or polynucleotides for desired activity. The use of such activity data from the screening library 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 for accelerating the conjugation of small molecules, including as an example the conjugation of a hydroxylamine group on one reagent with a carbonyl group on another reagent, wherein no reagent is an unnatural amino acid. In other words, the use of the accelerators described herein is not limited to the further functionalization of non-natural amino acids and non-natural amino acid polypeptides, but may also be used to facilitate the formation of oxime bonds between any two reagents. By way of example, this embodiment includes the use of an accelerator in forming / building dynamic libraries from hydroxylamine-containing reagents and carbonyl-containing reagents. Of course, such dynamic libraries may include non-natural amino acids, but such dynamic libraries are not limited to the inclusion of non-natural amino acids.

Also described herein are methods for extending the therapeutic half-life, serum half-life or circulating time of a polypeptide comprising substituting a non-natural amino acid for any one or more amino acids in the natural polypeptide, and / or adding a non-natural amino acid. into a natural polypeptide, and / or binding of the polypeptide to a water-soluble polymer of the rheko oxime bond formed in the presence of the accelerator described herein (although such a reaction may be less

51998 Β effective in the absence of the accelerator described here).

Also described herein are pharmaceutical compositions comprising a polypeptide comprising a non-natural amino acid containing an oxime bond formed in the presence of the accelerator described herein (although such a reaction may be less effective in the absence of the accelerator described herein) and a pharmaceutically acceptable carrier for use in patient treatment procedures. In some embodiments of the invention, the non-natural amino acid is bound to a water-soluble polymer.

In any of the above aspects or illustrations, the use of an accelerator includes the use of a single accelerator or multiple accelerators. Furthermore, in any of the above aspects or molar ratios, the ratio of accelerator to carbonyl-containing compound includes values ​​between about 0.5: 1 to 5000: 1, including as an example 4000: 1, 3000: 1, 2000: 1, 1000: 1, 500 : 1, 400: 1, 300: 1, 200: 1, 100: 1, 50: 1, 40: 1, 30: 1, 20: 1, 10: 1, 9: 1, 8: 1, 7: 1 , 6: 1, 5: 1,4: 1, 3: 1, 2: 1, 1: 1, 0.9: 1, 0.8: 1, 0.7: 1, 0.6: 1, and 0.5: 1. Furthermore, in any of these aspects or molar representations, the ratio of accelerator to hydroxylamine-containing compound includes values ​​between about 0.5: 1 to 5000: 1, including by way of example, 4000: 1, 3000: 1.2000: 1, 1000: 1, 500: 1, 400: 1, 300: 1, 200: 1, 100: 1, 50: 1, 40: 1, 30: 1, 20: 1, 10: 1, 9: 1, 8: 1, 7: 1, 6: 1, 5: 1, 4: 1, 3: 1, 2: 1, 1: 1, 0.9: 1, 0.8: 1, 0.7: 1, 0.6: 1, and 0.5: 1. Furthermore, in any of the above aspects or illustrations, the accelerator includes compounds that can be substantially removed in vacuo from the resulting oxime-containing compound. Furthermore, in any of the above aspects or embodiments, the accelerator includes compounds comprising an amine moiety, a semi-carbazide moiety, a hydrazine or a hydrazide moiety.

Furthermore, in any of the above aspects or representations, the accelerator is selected from the group consisting of bifunctional aromatic amines, oxoamine derivatives, and compounds having the following structures:

51998 Β

<img file="RS51998B_D0001.tif" />

where R_x, R_y and R_z are selected from the group consisting of: L_x-H, L_x-alkyl, L_x-aryl, L_x-heteroaryl, L_x-alkenyl, L_x-alkynyl, L_x-alkoxy, and L_x-alkylamine, where L_x is a bond, C (= O), C (= NH), and C (= NH) -NH and SO, SO2 wherein the aromatic amine is selected from the group:

Bifunctional aromatic amines:

<img file="RS51998B_D0002.tif" />

<img file="RS51998B_D0003.tif" />

<img file="RS51998B_D0004.tif" />

<img file="RS51998B_D0005.tif" />

and wherein the oxoamine derivative is selected from the group:

Oxoamine derivatives:

<img file="RS51998B_D0006.tif" />

In a further embodiment, the accelerator compounds are selected from the group consisting of h_jn^4,'R. η, νΆϊ KC 0 0 ο¹ where R_x, R_y and R_z are selected from the group consisting of: L_x-H, L_x-alkyl, L_x-aryl, L_x17

51998 Β heteroaryl, L_x-alkenyl, L_x-alkynyl, L_x-alkoxy, and L_x-alkylamine, where L_x is a bond, C (= O), C (= NH), and C (= NH) -NH. Furthermore, in any of the above aspects or illustrations, the accelerator is selected from the compounds shown in Figure 5, Figure 9, or Figure 10, including as an example any of the compounds 6, 8, 10, 7, and 20 of Figure 5. In in any of the above aspects or representations, the accelerator includes an agent that can form a hydrazone after reaction with a carbonyl-containing group. Furthermore, in any of the above aspects, the activity of the accelerator depends on the rate of reaction with the ketone moiety and the stability of the resulting intermediate. Furthermore, in any of the above aspects or illustrations, the pH of the reaction mixture containing the accelerator, the carbonyl-containing compound and the hydroxylamine-containing compound is between about 2.0 and 10; between about 2.0 and 9.0; between about 2.0 and 8.0; between about 3.0 and 7.0; between about 4.0 and 6.0; between 3.0 and 10.0; between about 4.0 and 10.0; between about 3.0 and 9.0; between about 3.0 and 8.0; between about 2.0 and 7.0; between about 3.0 and 6.0; between about 4.0 and 9.0; between about 4.0 and 8.0; between about 4.0 and 7.0; between about 4.0 and 6.5; between about 4.5 and 6.5; about 4.0; about 4.5; about 5.0; about 5.5; about 6.0; about 6.5; and about 7.0. It should be noted, however, that for any pH range described herein, the term between the eye relative to the low and high pH values ​​means that the eye is applied to both the low and high pH values; By way of example only, between about 3.0 and 10.0 is equivalent to between about 3.0 and about 10.0. In addition, unless otherwise stated, for any range specified herein, in which the eye is specified before the lower limit and not before the upper limit (or in the case where the eye is set before the upper limit and not before the lower limit), then this implies that word around occurs before both range limits. Furthermore, in any of the above aspects or representations, the term accelerator includes a compound having at least one of the following: (a) increasing the reaction rate between a carbonyl-containing compound and a hydroxylamine-containing compound while forming an oxime-containing compound, wherein the rate increase is relatively with reaction without accelerator; (b) reducing the activation energy of the reaction between the carbonyl-containing compound and the hydroxylamine-containing compound while forming an oxime-containing compound, wherein the reduction in activation energy is relative to the accelerator-free reaction; (c) increasing the yield of the oxime-containing compound from the reaction of the carbonyl-containing compound with the hydroxylamine-containing compound, wherein the increase in yield is relative to the reaction without accelerator; (d) reducing the temperature at which the carbonyl-containing compound reacts with the hydroxylamine-containing compound to form an oxime-containing compound, wherein the temperature decrease is relative to the reaction without accelerator; (e) reduces time

51998 Β which is required to react a carbonyl-containing compound with a hydroxylamine-containing compound to form an oxime-containing compound, the reduction in time being relative to the reaction without accelerator; (f) reducing the amount of reagent required while forming the oxime-containing compound, wherein the reduction in the amount of reagent is relative to the reaction without accelerator; (g) reducing foreign products resulting from the reaction of a carbonyl-containing compound with a hydroxylamine-containing compound while forming an oxime-containing compound, wherein the reduction of foreign products is relative to the reaction without accelerator; (h) does not irreversibly destroy the tertiary structure of the polypeptide subjected to the oxime formation reaction in the presence of an accelerator (except, of course, where the purpose of the reaction is to destroy such a tertiary structure); (i) may be separated from the oxime-containing compound in vacuo-, and (j) modulate the reaction of the carbonyl-containing compound with the hydroxylamine-containing compound. In further embodiments, the accelerator has at least two of the aforementioned properties, three of the aforementioned properties, four of the aforementioned properties, five of the aforementioned properties, six of the aforementioned properties, seven of the aforementioned properties, eight of the aforementioned properties, nine of the aforementioned properties. properties. In a further embodiment, the accelerator does not have any of the aforementioned properties.

It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only. The scope of the methods and compounds described herein will be limited by the appended claims.

As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly indicates otherwise.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as those commonly understood by one skilled in the art to which the inventions described herein belong. Although any methods, devices similar or equivalent to those described herein may be used in the practice or testing of the inventions described herein, preferred methods, devices, and materials are now described.

The terms alkoxy, alkylamino and alkylthio (or thioalkoxy) are used in their usual sense, and refer to those alkyl groups attached to

51998 Β the rest of the molecule through the corresponding oxygen atom, amino group, or sulfur atom.

The term alkyl, alone or as part of another substituent, means, unless otherwise indicated, a straight-chain or branched chain, or cyclic hydrocarbon radical, or combinations thereof, which may be fully saturated, mono- or polyunsaturated and may include di- and multivalent radicals, which have a number of carbon atoms (i.e., C1-C10 means one to ten carbons). Examples of saturated hydrocarbon radicals include, groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, tbutyl, isobutyl, sec-butyl, cyclohexyl, (cyclohexyl) methyl, cyclopropylmethyl, homologues and isomers of, for example, n -pentyl, n-hexyl, n-heptyl, n-octyl, and the like. An unsaturated alkyl group is one having one or more double bonds 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- and 3-propynyl, 3-butynyl, and more homologues and isomers. The term alkyl, unless otherwise indicated, is also intended to include those alkyl derivatives defined in more detail below, such as heteroalkyl. Alkyl groups that are limited to hydrocarbon groups are called homoalkyl.

The term alkylene alone or as part of a dear substituent means a divalent radical derived from an alkane, as in the example, but not limited to, with the structures CH2CH2- and -CH2CH2CH2CH2-, and further includes those groups described below as heteroalkylene. Typically, the alkyl (or alkylene) group will have from 1 to 24 carbon atoms, with those groups having 10 or fewer carbon atoms being particularly embodied according to the methods and compounds described herein. Lower alkyl, or lower alkylene is a short chain alkyl or alkylene group, which generally has eight or fewer carbon atoms.

The term amino acids refers to natural and non-natural amino acids, as well as amino acid analogs and amino acid mimetics that act in a manner similar to natural 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,

51998 Β proline, serine, threonine, tryptophan, tyrosine, and valine), and pyrrolizine and selenocysteine. The term amino acid analogs refers to compounds having the same basic chemical structure as a natural amino acid, i.e. a carbon bound to hydrogen, a carboxyl group, an amino group, and an R group, such as, homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (such as norleucine) or modified peptide backbones, but retain the same basic chemical composition as the natural amino acid.

Amino acids may be mentioned either by their commonly known three-letter symbols or by single-letter symbols as recommended by the IUPAC-IUB of the Biochemical Commission for Nomenclature. Nucleotides, too, may be mentioned according to their commonly accepted monosyllabic codes.

The amino terminus modification group refers to any molecule that may be attached to the amino terminus of a polypeptide. Similarly, a carboxy termination modification group refers to any molecule that may be attached to the carboxy termination of a polypeptide. Completion modification groups include, but are not limited to, various water-soluble polymers, peptides, or proteins such as serum albumin, or other moieties that increase the half-life of serum peptides.

Any form of antibody other than the full-length form is represented as the antibody fragment. Antibody fragments herein include antibodies that are minor components that exist within full-length antibodies and antibodies that are created. Antibody fragments include but are not limited to Fv, Fc, Fab, and (Fab ')₂, single chain Fv (scFv), diatiels, triantibodies, tetracyclines, bifunctional hybrid antibodies, CDR1, CDR2, CDR3, combinations of CDRa, variable regions, okvim regions, constant regions, heavy chains, light chains, and variable regions, and constructive non-antibodies, bispecific antibodies, and the like (Maynard &amp; Georgiou, 2000, Annu. Rev. Biomed. Eng. 2: 339-76; Hudson, 1998, Curr. Opin. Biotechnol. 9: 395-402). Another functional substructure is the single chain Fv (scFv), which contains immunoglobulin heavy and light chain variable regions covalently linked to a peptide linker (Sz Hu et al., 1996, Cancer Research, 56, 3055-3061). These small (Mr 25,000) proteins generally retain specificity and affinity

51998 Β for antigen in a single polypeptide and provide suitable building blocks for larger, antigen-specific molecules. Unless explicitly stated otherwise, statements and claims that use the term antibody or antibodies specifically include an antibody fragment and antibody fragments.

The term aryl means, unless otherwise indicated, a polyunsaturated, aromatic, hydrocarbon substituent which may be a single ring or multiple rings (including but not limited to 1 to 3 rings) joined together or covalently bonded. The term heteroaryl refers to groups of aryls (or rings) containing from one to four heteroatoms selected from N, O, and S, wherein the nitrogen and sulfur atoms are arbitrarily oxidized, and the nitrogen atom (s) are arbitrarily quatezed. The heteroaryl group may be attached to the rest of the molecule through a heteroatom. Non-limiting examples of aryl and heteroaryl groups include phenyl,

1-naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4imidazolyl, 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-pyrimidyl, 4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl, 1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl, and 6-quinolyl. Substituents for each of the above aryl and heteroaryl ring systems are selected from the group of acceptable substituents described below.

For brevity, the term 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 terms aralkyl or alkaryl mean to include those radicals in which the aryl group is attached to an alkyl group (including but not limited to, benzyl, phenethyl, pyridylmethyl and the like) including those alkyl groups in which the carbon atom (including but not limited to, methylene group) is replaced by, for example, an oxygen atom (including but not limited to, phenoxymethyl, 2-pyridyloxymethyl, 3- (1-naphthyloxy) propyl, and the like).

Bifunctional polymer refers to a polymer containing two discrete functional groups that are able to react specifically with other groups

51998 Β (including but not limited to, amino acid side groups) to form covalent or non-covalent bonds. A bifunctional linker having one functional group reactive with a group on a particular biologically active component, and another group reactive with a group on another biologically active component, can be used to form a conjugate comprising a first biologically active component, a bifunctional linker and a second biologically active component. Many methods and Jinker molecules for binding various compounds to peptides are known. See, e.g., European Patent Application no. 188,256; U.S. Patent no. 4,671,958, 4,659,839, 4,414,148, 4,699,784; 4,680,338; and 4,569,789. A multi-functional polymer refers to a polymer that contains two or more discrete functional groups that are able to react specifically with other groups (including, but not limited to, amino acid side groups) to form covalent or non-covalent bonds. The bi-functional polymer or multi-functional 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 compound-bound molecules and molecules that bind to the compound or compound.

The term biologically active molecule, biologically active part or biologically active agent when used herein means any substance that can affect any physical or biochemical properties of a biological system, pathway, molecule or interaction related to an organism, including viruses, bacteria , bacteriophages, transposons, prions, insects, fungi, plants, animals, and humans. In particular, as used herein, biologically active molecules include, but are not limited to, any substance intended to diagnose, treat, alleviate, treat, or prevent disease in humans or other animals, or otherwise to enhance the physical or mental well-being of humans or animals. . Examples of biologically active molecules include peptides, proteins, enzymes, small drug molecules, heavy drugs, light drugs, carbohydrates, inorganic atoms or molecules, dyes, lipids, nucleosides, radionuclides, oligonucleotides, toxins, cells, viruses, liposomes, microparticles and microparticles . Classes of biologically active agents suitable for use in the methods and compounds described herein include, drugs, prodrugs, radionuclides, imaging agents, polymers, antibiotics, fungicides, antiviral agents, anti-inflammatory agents, anti-tumor agents, cardiovascular agents. , anti-anxiety agents,

51998 Β hormones, growth factors, steroids and microbial toxins.

Cofolding, as used herein, specifically refers to unwinding, reaction, or methods that employ at least two polypeptides that interact with each other and result in the transformation of uncoiled or improperly coiled polypeptides into natural, properly coiled polypeptide.

The comparative framework, as used herein, includes reference to a segment of any number of adjacent sites selected from the group consisting of up to 20 to 600, typically about 50 to about 200, more typically about 100 to about 150 in which the sequence can be compared to by a reference sequence of the same number of adjacent sites after the two sequences are optimally aligned. Methods for classifying sequences for comparison are well known in the art. Optimal sequence alignment for comparison can be performed, including but not limited to, using the local homologous algorithm Smith and Waterman (1970) Adv. Appl. Malh. 2: 482c, using a homologous matching algorithm Needleman and Wunsch (1970) J. Mol. Biol. 48: 443, using a similarity search procedure from Pearson and Lipman (1988) Proc. Nat'l. Acad. Sci. USA 85: 2444, using computer implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the ffisconsin Geneiics Software Package, Genetics Computer Group, 575 Science Dr., Madizon, JVT), or by manual alignment and visual inspection (see, e.g. ., Ausubel et al., Current Protocols in Molecular Biology (1995 supplement).

One example of an algorithm suitable for determining the percentage of 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. Software for conducting BLAST analysis is publicly available through the National Center for Biotechnology Information. The BLAST parameters of the W, T, iX algorithm determine the sensitivity and speed of classification. The BLASTN program (for nucleotide sequences) uses as a given word-length (W) of 11, expectation (E) or 10, M = 5, N = 4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as default a word-length of 3, and an expectation (E) of 10, and a BLOSUM62 scoring matrix (see Henikoff &amp; Henikoff (1992) Proc. Natl. Acad. Sci. USA 89: 10915)

51998 Β alignment (Β) of 50, expectation (E) of 10, M = 5, N = -4, and comparison of both chains. The BLAST algorithm is typically implemented with low complexity and a filter off.

The BLAST algorithm also performs a statistical analysis of the similarity between the 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 smallest sum of probabilities (P (N)), which gives an indication of the probability at which an agreement between two nucleotides or amino acid sequences would accidentally occur. For example, a nucleic acid is considered similar to a reference sequence if the lowest sum of the probabilities compared to the tested nucleic acid with the reference nucleic acid is less than about 0.2, less than about 0.01, and in another embodiment less than about 0.001.

The term conservatively modified variant refers to both amino acid and nucleic acid sequences. With respect to individual nucleic acid sequences, conservatively modified variants refer to those nucleic acids that encode identical or substantially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, into substantially identical sequences. Due to the degeneration of the genetic code, a large number of functionally identical nucleic acids encode any particular protein. For example, the codons GCA, GCC, GCG, and GCU all encode alanine amino acid. Thus, at any position where alanine is specified with a codon, the codon may be altered to any of the correspondingly described codons without altering the encoded polypeptide. Such nucleic acid variations are silent variations, which are a type of conservatively modified variation. Each nucleic acid sequence encoding a polypeptide also describes any possible silent variation of the nucleic acid. One skilled in the art will recognize that each codon in the nucleic acid (except AUG, which is usually the only codon for methionine, and TGG, which is usually the only codon for tryptophan) can be modified to form a functionally identical molecule. Thus, any silent variation of a nucleic acid encoding a polypeptide is implicit in each sequence described.

With respect to the amino acid sequence, one skilled in the art will recognize that individual substitutions, deletions, or additions to a nucleic acid, peptide, polypeptide

51998 Β or a protein sequence that alters, adds or deletes a single amino acid or a small percentage of an amino acid in the encoded sequence is a conservatively modified variant where the modification results in deletion of the amino acid, addition of an amino acid, or replacement of the amino acid with a chemically similar amino acid. Conservative substitution tables that give functionally similar amino acids are well known in the art. Such conservatively modified variants are additional and do not exclude polymorphic variants, homologues between species, and alleles of the methods and compounds described herein.

The following eight groups each contain amino acids that are conservative substitutions for each other:

• 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), Valise (V);

• 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);

• 7) Serine (S), Threonine (T); and • 8) Cysteine ​​(C), Methionine (M) (see, e.g., Creighton, Proteins: Structures and Molecular Properties (WH Freeman &amp; Co .; 2nd edition (December 1993)) The terms cycloalkyl and heterocycloalkyl, alone for alone or in combination with other terms, represent, unless otherwise indicated, the corresponding cyclic variants of alkyl and heteroalkyl.Therefore, cycloalkyl or heterocycloalkyl include saturated, partially unsaturated and fully unsaturated ring bonds. Additionally, for heterocycloalkyl, the heteroatom may occupy a position where the heterocycle is attached to the rest of the molecule. Examples of cycloalkyl include cyclopentyl, cyclohexyl, 1cyclohexenyl, 3-cyclohexenyl and cycloheptyl. Examples of heterocycloalkyl include 1 (1,2,5,6-tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl, 3morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothiene-2- the tetrahydrothiene

3-yl, 1-piperazinyl and 2-piperazinyl. Additionally, the term includes bicyclic and tricyclic

51998 Β ring structures. Similarly, the term heterocycloalkylene alone or as part of another substituent means a divalent radical derived from heterocycloalkyl, and the term cycloalkylene alone or as part of another substituent means a divalent radical derived from cycloalkyl.

A denaturing agent or denaturant, as used herein, is defined as any compound or material that will cause reversible unwinding of a protein. The potency of a denaturing agent or denaturant will be determined by both the properties and concentration of the particular denaturing agent or denaturant. Suitable denaturing agents or denaturing agents may be chaotropes, detergents, water-miscible organic solvents, phospholipids, or a combination of two or more such agents. Suitable chaotropes include urea, guanidine, and sodium thiocyanate. Useful detergents may include, but are not limited to, strong detergents such as sodium dodecyl sulfate, or polyoxyethylene ethers (e.g. Tween or Triton detergents), sarcosyl, mild non-ionic detergents (e.g., digitonin), mild cationic detergents such as N—&gt; 2,3 (Dioleioxy) -propyl-N, N, N-trimethylammonia, mild ionic detergents (prg. sodium collate or sodium deoxycholate) or zwitterionic detergents including sulfobetaine (Zwittergent), 3- (3-chloramidopropyl) dimethylammonium-1-propane sulfate (CHAPS), and 3- (3-chloroamidopropyl) dimethylammonium-2-hydroxy-1-propane sulfonate ). Organic water-miscible solvents such as acetonitrile, lower alkanols (especially C2-C4 alkanol as ethanol or isopropanol), or lower alkanediols (especially C2-C4 alkanediol as ethylene glycol) can be used as denaturants. Phospholipids useful in the methods and compounds described herein may be natural phospholipids such as phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, and phosphatidylinositol or synthetic phospholipid derivatives or variants such as dihexanoylphosphatidylcholine or diheptanoylcholine.

The term desired functionality, as used herein, refers to any or all of the following groups: label; the color; polymer; water-soluble polymer; polyethylene glycol derivative; photo networking tool; cytotoxic compound; medicine; affinity tag; radionuclide; biotin derivative; quantum dot; nanotransmitter; radio transmitter; photoaffinity tag; reactive compound; resin; another protein or polypeptide or polypeptide analog; an antibody or antibody fragment; metal chelator; cofactor; fatty acid;

51998 Β carbohydrate; polynucleotide; DNA; RNA; antisense polynucleotide; saccharide, water-soluble dendrimer, cyclodextrin, biomaterial; nanoparticle; spin mark; fluorophore, a group containing meta); radioactive group; new functional group; a group that acts covalently or non-covalently with other molecules; photoclosed part; stimulating part of actin radiation; ligand; photoisomerizable part; biotin; biotin analog; a group containing a heavy atom; group with the possibility of chemical cleavage; elongated side chain; carbon-bound sugar; redox active agent; amino thio acid; toxic part; isotopically labeled portion; biophysical probe; phosphorescent group; chemiluminescent group; a group of dense electrons; magnetic group; intercalating group; chromophore; energy transfer means; biologically active agent; a tag that can be found; small molecule; inhibitory ribonucleic acid, and any combination of the above.

The term dicarbonyl as used herein refers to a group containing at least two parts selected from the group consisting of -C (O) -, -S (O) -, -S (O)₂-, and -C (S) -, including 1,2-dicarbonyl groups, 1,3-dicarbonyl groups, and 1,4-dicarbonyl groups, 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. Such dicarbonyl groups include diketones, ketoaldehydes, keto acids, ketoesters, and ketothioesters. Additionally, such groups may be part of linear, branched or cyclic molecules. The two groups in the dicarbonyl group may be the same or different, and may include substituents that will produce, for example, an ester, ketone, aldehyde, thioester, or amide, in either of the two moieties.

The term effective amount as used herein refers to that amount of a (modified) non-natural amino acid polypeptide administered that will reduce to some extent one or more symptoms of the disease, condition, or disorder being treated. Compositions comprising the (modified) non-natural amino acid polypeptide described herein may be administered for prophylaxis, amelioration and / or therapeutic treatments.

The terms to improve or amplify mean to increase or prolong either the degree, amount, potency or duration of the desired effect. Therefore, in relation to performance improvement

51998 Β therapeutic agents, the term improvement refers to the ability to increase or prolong, either strength or duration, the effect of other therapeutic agents in the system. Improvement-effective amount, as used herein, refers to an amount sufficient to enhance the effect of another therapeutic agent in the desired system. When used in a patient, the amounts effective for this use depend on the severity and course of the disease, disorder or condition, previous therapy, the patient's health and response to medication, and the judgment of the prescribing physician.

As used herein, the term eukaryote refers to organisms belonging to the phylogenetic domain of eukaryotes such as animals (including but not limited to mammals, insects, reptiles, birds, etc.), ciliates, plants (including but not limited to , monocotyledons, dicotyledons, algae, etc.), fungi, yeasts, flagellates, microsporidia, protists, etc.

The terms functional group, active moiety, activating moiety, leaving moiety, reactive site, chemically reactive moiety, and chemically reactive moiety are used in the art and refer here to distant, defined parts or units of a molecule. The terms are in some ways synonymous in chemistry, and are used here to denote parts of molecules that have some function or activity, and are reactive with other molecules.

The term halogen includes fluorine, chlorine, iodine, and bromine.

The term heteroalkyl, alone or in combination with another term, means, unless otherwise indicated, a stable straight-chain or branched chain, or cyclic hydrocarbon radical, or a combination thereof, and consists of said number of carbon atoms and at least one a heteroatom selected from the group consisting of Ο, N, Si and S, and wherein the nitrogen and sulfur atoms may be arbitrarily oxidized and the nitrogen heteroatom may be optionally quatezed. The heteroatom (i) Ο, N and S and Si can be placed at any internal position of the heteroalkyl group or at the position where the alkyl group is attached to the rest of the molecule. Examples include -CH2-CH2-O-CH3, -CH2-CH2-NH-CH3, -CH₂-CH2-N (CH₃) -CH3, -CH₂-SSN₂SNz, -CH₂-CH₂, -S (O) -CH₃, -CH₂-CH₂-S (O)₂-CH₃, -CH = CH-O-CH₃, -Si (CH₃) 3, CH₂-CH = N-OCH₃, and -CH = CH-N (CH₃) -CH₃. Up to two heteroatoms can be

51998 Β sequentially, such as, for example, -CH2-NH-OCH3 and -SN2-O-8j (SN2) z- Similarly, the term heteroalkylene alone or as part of another substituent means a divalent radical derived from heteroalkylene, as in the example, but is not limited to, -CH2-CH2-S-CH2-CH2i -CH2S-CH2CH2-NH-CH2-. For heteroalkylene groups, the same or different heteroatoms may also occupy each or both ends of the chain (including but not limited to, alkyleneoxy, alkylenedioxy, alkylene amino, alkylendiamino, aminooxyalkylene, and the like). Furthermore, for alkylene and heteroalkylene linking groups, the orientation of the linking group is not implied with the direction in which the formula of the linking group is written. For example, the formula -C (O)₂R'- represents both -C (O)₂R'- and -R'C (O)₂-.

The terms identical or percent identity, in terms of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same. Sequences are substantially identical if they have a percentage of amino acid residue or nucleotide (//., About 60% identity, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% identity in a particular area), when compared and aligned for maximum harmonization across a comparative framework, or a specific area as measured by one of the following sequence comparison algorithms (or other algorithms available to those skilled in the art) or by manual alignment and visual inspection. This definition also refers to the complement of the test sequence. Identity may exist in the region of at least about 50 amino acids or nucleotides, or in the region of 75-100 amino acids and nucleotides, or, where not specified, along the entire sequence of polynucleotides or polypeptides.

For sequence comparison, a sequence typically acts as a reference sequence, with which test sequences are compared. When a sequence comparison algorithm is used, test and reference sequences are inserted into the computer, the coordinates of the subsequence are indicated, if necessary, and the program parameters of the sequence algorithm are indicated. Default program parameters can be used, or alternative parameters can also be used. The sequence algorithm comparison then calculates the percentage of sequence identity for the test sequence relative to the reference sequence, based on the program parameters.

51998 00 The term isolated, when applied to a nucleic acid or protein, means that the nucleic acid or protein is devoid of at least some of the cellular components to which it is naturally associated, or that the nucleic acid or protein is concentrated to a level greater than their concentration for in vivo or in vitro production. It can be in a homogeneous state. Isolated substances may be in either a dry or semi-dry state, or in solution, including but not limited to aqueous solution. It may be a component of a pharmaceutical composition containing additional pharmaceutically acceptable carriers and / or excipients. Purity and homogeneity are typically determined using analytical chemical techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. The protein that is the predominant species present in the composition is substantially purified. In particular, the isolated gene is separated from open reading frames that rotate the gene and encode the protein, except for the gene of interest. The term purified means that the nucleic acid or protein separates essentially one region in the electrophoretic gel. In particular, this may mean that the nucleic acid or protein is at least 85% pure, at least 90% pure, at least 95% pure, at least 99% or more pure.

The term bond or linker is used herein to denote groups or bonds that are commonly formed as a result of a chemical reaction and are typically covalent links or bonds (the process of creating such a bond or linker is used herein as a bond or paired, as well as other synonyms known to a person skilled in the art). Hydrolytically stable bonds mean that the bonds are substantially stable in water and do not react with water at beneficial pH values, including but not limited to, under physiological conditions over an extended period of time, perhaps even indefinitely. Hydrolytically unstable or degradable bonds means that the bonds can be degraded in water or in aqueous solutions, including, for example, blood. Enzymatically unstable or degrading bonds means that the bond can be degraded with one or more enzymes. As understood in the art, PEG and related polymers may include degrading bonds in the polymer backbone or in a linking group between the polymer backbone and one or more terminal functional groups of the polymer molecule. For example, bond esters formed by reacting PEG carboxylic acids or activated PEG carboxylic acids with alcohol groups on a biologically active agent generally hydrolyze under physiological

51998 Β conditions to release the asset. Other hydrolytically degrading bonds include but are not limited to carbonate bonds; imine bonds resulting from the reaction of amines and aldehydes; phosphate estem bonds formed by the reaction of an alcohol with a phosphate group; hydrazone bonds which are the reaction product of hydrazides and aldehydes; acetal bonds which are the reaction product of aldehydes and alcohols; orthoesthemic bonds that are a reaction product of formate and alcohol; peptide bonds formed with an amine group, including but not limited to, a polymer termination such as PEG, and a carboxyl group of the peptide; and oligonucleotide bonds formed with the phosphoramidite group, including but not limited to, the polymer termination, and the 5 'hydroxyl group of the oligonucleotides.

As used herein, the term agent or medium includes any cultured medium, solution, solid, semi-solid, or solid support that may support or contain any host cell, including bacterial host cells, yeast host cells, host cells insect, plant host cells, eukaryotic host cells, mammalian host cells, CHO cells, prokaryotic host cells, E. coli, or Pseudomonas host cells, and cellular contents. Thus, the term may include a medium in which the cultured host cell is, e.g., a medium into which the polypeptide is secreted, including the medium either before or after the proliferation step. The term may also include buffers or reagents comprising host cell lysates, such as in the case where the polypeptide is obtained intracellularly and the host cells are lysed or stimulated to release the polypeptide.

The (modified) non-natural amino acid polypeptide disclosed herein is a derivative of that (modified) non-natural amino acid polypeptide formed when the (modified) non-natural amino acid polypeptide is metabolized. The term active metabolite refers to a biologically active derivative of a (modified) non-natural amino acid polypeptide formed when a (modified) non-natural amino acid polypeptide is metabolized. The term metabolized refers to the sum of the processes (including, but not limited to, hydrolysis reactions and catalytic enzymatic reactions) by which a particular substance is modified with an organism. Further information on metabolism can be obtained from The Pharmacological Basis of Therapeutics, 9th Edition, McGraw-Hill (1996). Metabolites

51998 ) The (modified) non-natural amino acid polypeptide disclosed herein can be identified either by administering to the host (modified) non-natural amino acid polypeptide and analyzing tissue samples from the host, or by incubating the (modified) non-natural amino acid polypeptide with in vitro liver cells and analysis of the resulting compounds.

The term modified, as used herein, refers to the presence of a posttranslatome modification on a polypeptide. The form of the expression (modified) means that the polypeptides in question are arbitrarily modified, i.e., that the polypeptides in question may be modified or unmodified.

As used herein, the term modulated serum half-life means a positive or negative change in the circulating half-life of a modified polypeptide relative to its non-modified form. Serum half-life is measured by taking blood samples at different time points after administration of the polypeptide, and determining the concentration of that molecule in each sample. The correlation of serum concentration with time allows the calculation of serum half-life. The increased serum half-life preferably has at least about a twofold increase, but even a smaller increase may be useful, for example where a satisfactory dosing regimen is provided or where a toxic effect is avoided. In some embodiments of the invention, the increase is at least about threefold, at least about fivefold, or at least about tenfold.

The term modulated therapeutic half-life as used herein means a positive or negative change in the half-life of a therapeutically effective amount of a modified polypeptide, relative to its non-modified form. The therapeutic half-life is measured by measuring the pharmacokinetic and / or pharmacodynamic properties of the polypeptide at various time points after administration. The increased therapeutic half-life preferably provides a particularly useful dosage regimen, a particularly useful total dose, or avoids an undesirable effect. In some embodiments of the invention, increased therapeutic half-life results in increased potency, increased or decreased binding of the modified molecule to its target, increased or decreased degradation of the molecule by enzymes such as proteases, or increased or decreased other parameter or mechanism of action of the non-modified molecule.

51998 Β

10067] As used herein, the term non-eukaryotic refers to non-eukaryotic organisms. For example, a non-eukaryotic organism may belong to the eubacteria (including Escherichia coli, Thermus thermophilus, Bacillus stearoihermophilus, Pseudomonas fluorescens, Pseudomonas aeruginosa, Pseudomonas putida, etc.) of the phylogenetic domain, or Arnascoautus, including Archaeucus, and Halobacterium species NRC-1, Archaeoglobus fulgidus, Pyrococcus furiosus, Pyrococcus horikoshii, Aeigorugit pernix, etc.) phylogenetic domain.

A non-natural amino acid refers to an amino acid that is not one of 20 common amino acids or pyrrolizine or selenocysteine; other terms that can be used interchangeably with the term non-natural amino acid are non-naturally encoded amino acid, non-naturally occurring amino acid, non-naturally occurring amino acid and their various dashed and non-dashed versions. The term non-natural amino acids includes, but is not limited to, naturally occurring amino acids with modification of naturally encoded amino acids (including but not limited to .20 common amino acids or pyrrolizine and selenocysteine) but are not themselves included in the growing polypeptide chain by translational complex. Examples of naturally occurring non-naturally encoded amino acids include N-acetylglucosaminyl-L-serine, N-acetylglucosaminyl-L-threonine, and O-phosphotyrosine.

The term nucleic acid refers to deoxyribonucleotides, deoxyribonukelosides, ribonukelosides or ribonukelotides and their polymers in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids that contain known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and that are metabolized in a similar manner as natural nucleotides. Unless otherwise specifically limited, the term also refers to oligonucleotide analogs that include PNA (peptidonucleic acid), DNA analogs used in antisense technology such as phosphorothioates, phosphoroamidates. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses their conservatively modified variants (including substitutions of degenerate

51998 Β codons) and complement sequences as well as explicitly indicated sequences, specifically, substitutions of the degenerate codon can be achieved by generating sequences in which the third position for one or more selected (or all) codons is substituted with a mixed-base and / or deoxyinosine residue ( Batzer et al., Nucleic Acid Res. 19: 5081 (1991); Ohtsuka et al., J. Biol. Chem. 260: 26052608 (1985); and Rossolini et al., Mol. Cell Probes 8: 91-98 (1994)).

An oxidizing agent, as used herein in relation to protein depletion, is defined as any compound or material capable of removing electrons from an oxidizable compound. Suitable oxidizing agents include oxidized glutathione, cystine, cystamine, oxidized dithiothreitol, oxidized erythreitol, and oxygen. A wide range of oxidizing agents is suitable for use in the methods and compounds described herein.

As used herein, the term polyalkylene glycol refers to polyethylene glycol, polypropylene glycol, polybutylene glycol, and derivatives thereof. The term polyalkylene glycol encompasses both linear and branched polymers and average molecular weights of between 0.1 kDa and 100 kDa. Other embodiments are given, for example, in commercial vendor catalogs, such as the catalog of Shearwater Corporation Polyethylene Glycol and Derivatives for Biomedical Applications (2001).

The terms polypeptide, peptide and protein are used herein as synonyms and refer to a polymer of amino acid residues. That is, the description directed to the polypeptide refers equally to the description of the peptide and the description of the protein, and vice versa. The terms apply to polymers of natural amino acids as well as polymers of amino acids in which one or more amino acid residues is a non-natural amino acid. As used herein, the terms encompass amino acid chains of any length, including full-length proteins, wherein the amino acid residues are linked by covalent peptide bonds.

The term post-translatomo modified refers to any modification of a natural or non-natural amino acid that results in such an amino acid after it is included in the polypeptide chain. The term encompasses, by way of example only, co

51998 Β translational in vivo modifications, co-translational in vitro modifications (as in a cell-free translation system), post-translational in vivo modifications, and posttranslational in vitro modifications.

A prodrug refers to an agent that has been converted to a parent drug in vivo. Prodrugs are often useful because, in some situations, they may be easier to administer than the parent drug. They may, for example, be bioavailable by oral administration while, the parent drug is not. The prodrug may also have improved solubility in pharmaceutical compositions relative to the parent drug. The pro-drug includes a pharmacologically inactive derivative of the active drug, or with reduced-activity. Prodrugs may be intended to modulate the amount of drug or may be a biologically active molecule that reaches a desired site of action through manipulation of drug properties, such as physiochemical, biopharmaceutical, or pharmacokinetic properties. Prodrugs are converted to active drugs within the body through enzymatic or non-enzymatic reactions. Prodrugs may have improved physiochemical properties such as better solubility, improved delivery characteristics, as well as specific targeting of a particular cell, tissue, organ or ligand, and improved therapeutic value. .

In prophylactic applications, compositions comprising a (modified) non-natural amino acid polypeptide may be administered to a patient who is susceptible to or otherwise at risk for a particular disease, disorder or condition. Such an amount is defined as a prophylactically effective amount. In this application, the exact amounts also depend on the patient's health condition, weight, and the like. It is considered within the skill of the art to determine such prophylactically effective amounts by routine experimentation (e.g., clinical trials with increasing doses).

The term protected refers to the presence of a protecting group or moiety that prevents the reaction of a chemically reactive functional group under certain reaction conditions. The protecting group will vary depending on the type of chemically reactive group to be protected. For example, if the chemically reactive group is an amine or hydrazide, the protecting group may be selected from the group of tert-butyloxycarbonyl (t-Boc) and 9-fluorenylmethoxycarbonyl (Fmoc). If the chemically reactive group is a thiol, the protecting group may be orthopyridyldisulfide. If it is chemical

51998 A reactive carboxylic acid group, such as butanoic or propanoic acid, or a hydroxyl group, the protecting group may be a benzyl or alkyl group such as methyl, ethyl, or tert-butyl. Other protecting groups known in the art may also be used in or with the methods and compounds described herein, including photolabile groups such as Nvoc and MeNvoc.

By way of example, blocking / protecting groups may be selected from:

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alil n_g

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Bn

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Cbz

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aloc

H₃C ^

Me

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Vos (N₃S) zS

N₃S_No. , SN₃(NzSJzS-³'^.

O (H₃C)₃Yes

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t-butyl TBDMS (SN₃) zS ^⁰G ^

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_NzH acetyl

Teoc

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Other protecting groups are described in Greene and Wuls, Prolective Groups in Organic Synthesis, 3rd Ed., John Wiley &amp; Sons, New York, NY, 1999.

Recombinant host cell or host cell refers to a cell that includes an exogenous polynucleotide, regardless of the insertion method used, for example, direct entry, transduction, f-mating, or other methods known in the art to create a recombinant host cell. . An exogenous polynucleotide may be maintained as a non-integrated vector, for example, a plasmid, or alternatively, may be

51998 Β integrated into the host genome.

A reducing agent, as used herein in relation to protein depletion, is defined as any compound or material that maintains sulfhydryl groups in a reduced state and reduces intra- or intermolecular disulfide bonds. Suitable reducing agents include, but are not limited to, dithiothreitol (DTT), 2 mercaptoethanol, dithioerythritol, cysteine, cysteamine (2-aminoethanthiol), and reduced glutathione. A wide range of reducing agents is suitable for use in the methods and compounds described herein.

The consideration as used herein describes a process, reaction, or process that transforms a disulfide bond containing polypeptides from an improperly coiled or uncoiled state into a natural or properly coiled conformation with respect to disulfide bonds.

The term "selectively (or specifically) hybridizes to refers to the binding, duplication, or hybridization of a molecule to a particular nucleotide sequence only under stringent hybridization conditions when that sequence is present in a complex mixture (including, but not limited to, the entire cell or library." DNA or RNA).

The term stringent hybridization conditions refers to the hybridization of DNA, RNA, or PNA sequences, other nucleic acid mimetics, or combinations thereof under low ionic strength and high temperature conditions known in the art. Typically, under stringent conditions, the probe will hybridize to its target subsequence in a complex nucleic acid mixture (including, but not limited to, the entire cell or DNA or RNA library) but will not hybridize to other sequences in the complex mixture. Strict conditions are sequence dependent and will be different in different circumstances. Longer sequences specifically hybridize at higher temperatures. A comprehensive guide to nucleic acid hybridization is found in Tijssen, Laboratory Techniques in Biochemistry and Molecular BiologyHybridization with Nucleic Probes, ”An Overview of Principles of Hybridization and the Strategy of Nucleic Acid Assays (1993). In general, strict conditions are selected to be about 5-10 ° C lower than the melting point (T_m) for the specific sequence of the code

51998 Β ionic strength pH. T_m is the temperature (at determined ionic strength, pH and nucleic concentration) at which 50% of the probe is complemented with a target to hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at T_m, 50% of the sample is occupied in equilibrium). Strict conditions may be those where the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30 ° C for short tests (including but not limited to, 10 to 50 nucleotides) and at least about 60 ° C for long probes (including but not limited to, more than 50 nucleotides). Strict conditions can also be achieved with the addition of destabilizing agents such as formamide. For selective or specific hybridization, the positive signal may be at least twice as strong as the background, arbitrarily 10 times as strong as the background of the hybridization. An example of stringent hybridization conditions may be as follows: 50% formamide, 5X SSC, and 1% SDS, incubation at 42 ° C, or 5X SSC, 1% SDS, incubation at 65 ° C, with washing in 0.2H SSC, and 0.1% SDS at 65 ° C. Such rinses may be performed for 5.15, 30, 60, 120 or more minutes.

The term subject as used herein refers to an animal, in some embodiments of the invention on a mammal, and in other embodiments on a human, which is the object of treatment, observation or experiment.

The term substantially purified refers to a polypeptide that may be substantially or substantially free of components that normally accompany or interact with a protein that can be found in the natural environment, i. a natural cell, or a host cell in the case of a recombinantly produced polypeptide. A polypeptide that may be substantially free of cellular material includes protein compositions having 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% (dried weight) of the contaminating protein. When the polypeptide or variant thereof is recombinantly derived from host cells, the protein may be present in 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 dried weight of the cells. When the polypeptide or variant thereof is recombinantly derived from host cells, the protein may be present in the cultured medium at about 5 g / L, about 4 g / L, about 3 g / L, about 2 g / L, about 1 g / L, about

51998 Β

750mg / L, about 500mg / L, about 250mg / L, about 100mg / L, about 50mg / L, about 100mg / L, or about 100mg / L or less than the dried weight of the cells. Thus, a substantially purified polypeptide as obtained by the methods described herein may have a purity level of 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%, preferably, a purity level of at least about 75%, 80%, 85%, and more preferably, a purity level of at least about 90%, a purity level of at least about 95%, a level a purity of at least about 99% or more as determined by appropriate procedures such as SDS / PAGE analysis, RP-HPLC, SEC, and capillary electrophoresis.

The term substituents includes non-interfering substituents. Non-interfering substituents are those groups which give stable compounds. Suitable non-interfering substituents or radicals include, but are not limited to, halo, C1-C10 alkyl, C2-C10 alkenyl, C₂-C 10 alkynyl, C 1 -C 10 alkoxy, C 5 -C 11₂ aralkyl, C3-C12 cycloalkyl, C4-C1₂cycloalkenyl, phenyl, substituted phenyl, toluene, xienyl, biphenyl, C₂-C 12 alkoxyalkyl, C 5 Cl 2 alkoxyaryl, C 5 -C 11₂ aryloxyalkyl, C7-C1₂ oxyaryl, C1-C6 alkylsulfonyl, C1-C10 alkylsulfonyl, - (CH₂)_m-O- (C 1 -C 10 alkyl) wherein m is from 1 to 8, aryl, substituted aryl, substituted alkoxy, fluoroalkyl, heterocyclic radical, substituted heterocyclic radical, nitroalkyl, -NO₂, -CN, -NRC (O) - (C1-C10 alkyl), -C (O) - (C1-C10 alkyl), C₂-C 10 alkylthioalkyl, -C (O) O- (C 1 -C 6)₀ alkyl), -OH, -SO₂, = S, -COOH, -NR₂, carbonyl, -C (O) (C1-C10 alkyl) -CF3, -C (O) -CF3, -C (O) NR2, - (C1-C10 aryl) -S-₆-C 10 aryl), -C (O) - (C₆Cio aryl), - (CH₂) m-O- (CH₂)_m-O- (C 1 -C 10 alkyl) wherein each m is from 1 to 8, -C (O) NR₂, -C (S) NR₂, -SO₂NR₂, -NRC (O) NR₂, -NRC (S) NR₂, their salts, and the like. Each R group in the preceding list is independently selected from the group consisting of H, alkyl or substituted alkyl, aryl or substituted aryl, or alkaryl. Where substituent groups are specified with their conventional chemical formulas, written from left to right, they equally encompass chemically identical substituents that would result from writing the structure from right to left, for example, -CH₂O-is equivalent to -OCH₂-, Substituents for alkyl and heteroalkyl radicals (including those groups often referred to as alkylene, alkenyl, heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl) may be one or more of various groups selected from, but which are not limited to: -OR, = O, = NR, = N-OR, -NR₂,

51998 Β

SR, -halogen, -SiR₃, -OC (O) R, -C (O) R, -CO₂R, -CONR₂, -OC (O) NR₂, -NRC (O) R, NR-C (O) NR₂, -NR (O)₂R 1 -NR-C (NR₂) = NR, -S (O) R, -S (O)₂R, -S (O)₂NR₂, -nrso₂r, -CN and -NO₂ with a number ranging from zero to (2m '+ 1), where m' is the total number of carbon atoms in such a radical. Each R group in the preceding list is independently selected from the group consisting of hydrogen, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, including aryl substituted with 1-3 halogens, substituted or unsubstituted alkyl, alkoxy or thioalkoxy groups, or aralkyl groups. When two R groups are attached to the same nitrogen atom, they can be combined with the nitrogen atom to form a 5-, 6-, or 7-membered ring. For example, -NR₂is intended to include, but 1-pyrrolidinyl and 4-morpholinyl. From the foregoing discussion of substituents, one skilled in the art will appreciate that the term alkyl is intended to include groups that include carbon atoms attached to groups other than hydrogen groups, such as haloalkyl (including -CF₃ and -CH₂CF₃) and acyl (including -C (O) CH₃, -C (O) CF.₃ and -C (O) CH₃=).

Similarly to the alkyl radical substituents described, the substituents for the aryl and heteroaryl groups are different and are selected from -OR = O = NR, N-OR, -NR₂, -SR, -halogen, SiR₃, -OC (O) R, -C (O) R, -CO₂R, -CONR₂, -OC (O) NR₂, -NRC (O) R, -NR-C (O) NR₂, NR (O)₂R 1 -NR-C (NR₂) = NR, -S (O) R, -S (O)₂R, -S (O)₂NR₂, -NRSO₂R 1 is -CN, -NO₂, -R, -N₃, -CH (Ph)₂, fluoro (C1-C4) alkoxy, and fluoro (C1-C4) alkyl, with a number ranging from zero to the total number of open valences in the aromatic ring system; and wherein each R group in the preceding list is independently selected from hydrogen, alkyl, heteroalkyl, aryl and heteroaryl.

In therapeutic applications, compositions comprising a (modified) non-natural amino acid polypeptide are administered to a patient already suffering from a disease, condition or disorder, in an amount sufficient to treat or at least partially prevent the symptoms of the disease, condition or disorder. Such an amount is defined as a therapeutically effective amount, and will depend on the severity and course of the disease, condition or disorder, previous therapy, the patient's medical condition and response to medications, and the judgment of the prescribing physician. It is considered within the skill of the art to determine such therapeutically effective amounts by routine experimentation (e.g., clinical trials with increasing doses).

51998 00 The term treatment is used when referring to either prophylaxis and / or therapeutic treatments.

The term water-soluble polymer as used herein refers to any polymer that is soluble in aqueous solvents. Binding of the water-soluble polymer to the polypeptide may result in changes including, but not limited to, increased or modulated serum half-life, or increased or modulated therapeutic half-life relative to the unmodified form, modulated immunogenicity, modulated physical binding characteristics such as aggregation and formation multimers, altered receptor binding, altered binding to one or more binding partners, and altered receptor dimerization or multimerization. A water-soluble polymer may or may not have its own biological activity. Suitable polymers include, but are not limited to, polyethylene glycol, polyethylene glycol propionaldehyde, mono C1-C10 alkoxy, or aryloxy derivatives thereof (described in U.S. Pat. No. 5,252,714, which is incorporated herein by reference), monomethoxy-polyethylene glycol, polyvinyl pyrrolidone, polyvinyl alcohol, polyamino acids, maleic divinyl ether anhydride, A- (2-Hydroxypropyl) -methacrylamide, dextran, dextyletrane sulfide derivatives including dextran / ethylene oxide copolymer, polyoxyethylated polyol, heparin, heparin fragments, polysaccharides, oligosaccharides, glycans, cellulose and cellulose derivatives, including but not limited to methylcellulose and carboxymethyl cellulose, starch and starch derivatives, polypeptides, polyalkylene glycol and their derivatives, copolymers of polyalkylene glycol and their derivatives, polyvinyl ethyl ethers, and alpha-beta-poly [(2-hydroxyethyl) -DL-aspartame , and the like, or mixtures thereof. Examples of such water-soluble polymers include polyethylene glycol and albumin serum. In some embodiments of the invention, the aqueous polymer comprises a poly (ethylene glycol) moiety. The molecular weights of the polymer can be in a wide range, including between 100 Da and 100,000 Da or more. Molecular weights of polymers can be in between

100 Yes and 100,000 Yes, including 100,000 Yes, 95,000 Yes, 90,000 Yes, 85,000Yes,

80,000 Yes, 75,000 Yes, 70,000 Yes, 65,000 Yes, 60,000 Yes, 55,000 Yes, 50,000Yes,

45,000 Yes, 40,000 Yes, 35,000 Yes, 30,000 Yes, 25,000 Yes, 20,000 Yes, 15,000Yes,

10,000 Yes, 9,000 Yes, 8,000 Yes, 7,000 Yes, 6,000 Yes, 5,000 Yes, 4,000 Yes, 3,000 Yes, 2,000 Yes, 1,000 Yes, 900 Yes, 800 Yes, 700 Yes, 600 Yes, 500 Yes, 400 Yes, 300 Yes , 200

51998 Β

Yes, and 100 Yes. In some embodiments of the invention, the weight of the polymer molecules is between 100 Da and about 50,000 Da. In some embodiments of the invention, the molecular weight of the polymer is between about 100 Da and about 40,000 Da. In some embodiments of the invention, the molecular weight of the polymer is between about 1,000 Da and about 40,000 Da. In some embodiments of the invention, the molecular weight of the polymer is between about 5,000 Da and about 40,000 Da. In some embodiments of the invention, the molecular weight of the polymer is between about 10,000 Da and about 40,000 Da. In some embodiments of the invention, the poly (ethylene glycol) molecule is a branched polymer. The molecular weight of a branched PEG chain can be between about 1,000 Da and 100,000 Yes, including 100,000 Yes, 95,000 Yes, 90,000 Yes, 85,000 Yes, 80,000 Yes, 75,000 Yes, 70,000 Yes, 65,000 Yes, 60,000 Yes, 55,000 Yes, 50,000 Yes, 45,000 Yes, 40,000 Yes, 35,000 Yes, 30,000 Yes, 25,000 Yes, 20,000 Yes, 15,000 Yes, 10,000 Yes, 9,000 Yes, 8,000 Yes, 7,000 Yes, 6,000 Yes, 5,000 Yes, 4,000 Yes, 3,000 Yes, 2,000 Yes, and 1,000 Yes . In some embodiments of the invention, the weight of the branched PEG chain molecules is between about 1,000 Da and about 50,000 Da. In some embodiments of the invention, the weight of the branched PEG chain molecules is between about 1,000 Da and about 40,000 Da. In some embodiments of the invention, the weight of the branched PEG chain molecules is between about 5,000 Da and about 40,000 Da. In some embodiments of the invention, the weight of the branched PEG chain molecules is between about 5,000 Da and about 20,000 Da.

Unless otherwise indicated, conventional methods of mass spectroscopy, NMR, HP LC, protein chemistry, biochemistry, recombinant DNA techniques and pharmacology, known in the art, have been used.

Compounds (including, but not limited to, non-natural amino acids, (modified) non-natural amino acid polypeptides, and reagents for preparing any of the foregoing compounds) listed herein include isotopically-labeled compounds that are identical to those listed. in the various formulas and structures listed here, but due to the fact that one or more atoms are replaced by an atom having an atomic mass or mass number which is different from the atomic mass or mass number normally found in nature. Examples of isotopes that may be included in the present compounds include the corresponding isotopes of hydrogen, carbon, nitrogen, oxygen, fluorine and chlorine such as²H, ³H, ¹³C, ¹⁴C, ¹⁵N, ¹⁸ABOUT, ^l7ABOUT,³⁵S, ¹⁸F, ³⁶C1. Certain isotopically-labeled compounds described herein, for example those in which they are included

51998 Β radioactive isotopes as ³H i ^I4C, are useful in drug and / or substrate distribution tests in tissue. Furthermore, substitution with isotopes such as deuterium, i.e.²H, may provide certain therapeutic benefits that result in greater metabolic stability, for example increased in vivo half-life or decreased dosing requirements.

Some of said compounds (including, non-natural amino acids, (modified) non-natural amino acid polypeptides and reagents for the preparation of any of the foregoing) have asymmetric carbon atoms and may therefore exist as enantiomers or diastereomers, Diastereomers mixtures can be separated into their individual diastereomers on the basis of their physicochemical differences using known methods, for example, by chromatography and / or fractional crystallization. The enantiomers may be separated by converting the enantiomeric mixture into a diastereomeric mixture by aiding the reaction with a suitable optically active compound (e.g., an alcohol) which separates 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 part of the compositions described herein.

In additional or further embodiments, the compounds described herein (including, but not limited to, non-natural amino acids, (altered) non-natural amino acid polypeptides, and reagents for preparing any of the foregoing compounds) are metabolized upon administration to the body. which is necessary for the production of a metabolite which is then used to obtain the desired effect, including the desired therapeutic effect. In further or additional embodiments, the metabolites of unnatural amino acids and (modified) polypeptides of non-natural amino acids are active.

The methods and formulations described herein include the use of V-oxides, crystalline forms (also known as polymorphs), or pharmaceutically acceptable salts of non-natural amino acids and (modified) non-natural amino acid polypeptides. In some situations, non-natural amino acids and (modified) non-natural amino acid polypeptides may exist as tautomers. All tautomers are included within the non-natural amino acids and (modified) non-natural amino acid polypeptides described herein. Additionally, the non-natural amino acids described herein and (modified)

51998 Β Non-natural amino acid polypeptides may exist in undissolved as well as in dissolved forms with pharmaceutically acceptable solvents such as water, ethanol, and the like. The dissolved forms of non-natural amino acids and (modified) non-natural amino acid polypeptides disclosed herein are also believed to be disclosed herein.

Those skilled in the art will recognize that some of said compounds (including non-natural amino acids, (modified) non-natural amino acid polypeptides, and reagents for preparing any of the foregoing) may exist in several tautomic forms. All such tautomeric forms are considered as part of the compositions described herein. Also, for example, all enol-keto forms of any of the compounds (including, but not limited to, non-natural amino acids, (modified) non-natural amino acid polypeptides, and reagents for preparing any of the aforementioned compounds) are considered herein to be part of of the compositions described herein.

Some of said compounds (including non-natural amino acids, (modified) non-natural amino acid polypeptides and reagents for preparing any of the foregoing) are acidic and may form a salt with a pharmaceutically acceptable cation. Some of these compounds (including non-natural amino acids, (modified) non-natural amino acid polypeptides, and reagents for preparing any of the foregoing compounds) may be basic and, therefore, may form a salt with a pharmaceutically acceptable anion. All such salts, including di-salts, are within the scope of the compositions described herein and may be prepared by conventional methods. For example, salts can be obtained by contacting acidic and basic formations, either in an aqueous, non-aqueous or partially aqueous medium. The salts are recovered using at least one of the following techniques: filtration, precipitation with non-solvents followed by filtration, evaporation of the solvent, or, in the case of aqueous solutions, lyophilization.

Salts, for example, include: (1) acid addition salts, obtained with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphomic acid, and the like; or obtained with organic acids such as acetic acid, propionic acid, hexanoic acid,

51998 Β cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumamic acid, tartaric acid, citric acid, benzoic acid, 3- (4-hydroxybenzoyl) benzoic 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, 4,4'-methylbenbis- (3-hydroxy-2-ene-1-carboxylic acid), 3-phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid, lauryl sulfumic acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid, and the like; (2) salts formed when an acidic proton is present in the parent compound are either replaced by a metal ion, e.g., an alkali metal ion, an alkaline earth ion, or an aluminum ion; or align with the 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.

It is to be understood that reference to a salt includes solvent addition forms or crystalline forms thereof, especially solvates or polymorphs. Solvates contain either stoichiometric or non-stoichiometric amounts of solvent, and are often obtained during the crystallization process. Hydrates are obtained when the solvent is water, and alcoholates are obtained when the solvent is alcohol. Polymorphs include different crystal packing arrangements for the same compound composition elements. Polymorphs typically have different X-ray diffraction patterns, infrared spectrum, melting point, density, strength, crystal shape, optical and electrical properties, stability, and solubility. Various factors such as recrystallization solvent, recrystallization rate, and storage temperature can cause a single crystalline form to dominate.

BRIEF DESCRIPTION OF THE DRAWINGS A better understanding of the features and advantages of the subject processes and compositions can be achieved by reference to the following detailed description illustrating embodiments, using the principles of our methods, compositions, devices and apparatus,

51998 Uz and with drawings that:

FIGURE 1 shows a schematic representation of the relationship of certain aspects to the methods, compositions, strategies, and techniques described herein.

FIGURE 2 shows a non-limiting example of a one-step SDS-PAGE dimerization reaction analysis for scFv 108 using a 2 K homobifunctional hydroxylamine PEG linker with different molar ratios: 1) scFvjinker = 1.6: 1, with acetic hydrazide; 2) scFv: Iinker = 2: 1, with acetic hydrazide; 3) scFvdinker = 2.4: 1, with acetic hydrazide; 4) scFv: linker = 2: 1, without acetic hydrazide; 5) scFv.linker = 2: 1, with acetic hydrazide without PEG linker.

FIGURE 3 shows a non-limiting example of SDS-PAGE analysis of scFv-pAcF and 30 K mono hydroxylamine PEG conjugation 1) a standard for 100% initial scFvpAcF; 2) standard for 20% of initial scFv-pAcF; 3) standard for 10% of initial scFvpAcF; 4) scFv: PEG = 1: 3 with 20 mM acetic hydrazide; 5) scFv: PEG = 1: 3 without acetic hydrazide; 6) scFv: PEG = 1: 5 with 20 mM acetic hydrazide; 7) scFv: PEG = 1: 5 without acetic hydrazide.

FIGURE 4 shows a non-limiting example of SDS-PAGE analysis of scFv-pAcF and 30 K mono hydroxylamine PEG conjugation with different concentrations of acetic hydrazide. 1) scFv-pAcF: PEG = 1: 2.5 mM acetic hydrazide; 2) scFv-pAcF: PEG = 1: 2, 20 mM acetic hydrazide; 3) scFv-pAcF: PEG 1: 2, 80 mM acetic hydrazide; 4) scFv-pAcF: PEG = 1: 5, without acetic hydrazide; 5) 10% scFv-pAcF standard; 6) standard of 20% scFv-pAcF; 7) standard of 100% scFv-pAcF.

FIGURE 5 shows non-limiting examples of accelerators that can be used in the methods, reactions, and syntheses described herein.

FIGURE 6 shows a non-limiting example of SDS-PAGE analysis compared to oxime preparation in the presence of various accelerators; the track number corresponds to the number of accelerators in FIGURE 5 and the last track is the control reaction without accelerator.

51998 01 FIGURE 7 shows a non-limiting example of SDS-PAGE conjugation analysis for hGH-pAcF with 30 K monohydroxylamine PEG with accelerators 7 and 20: 1) hGHpAcF: PEG = 1: 2 with accelerator 7; 2) hGH-pAcF PEG = 1: 2 with accelerator 20; 3) hGH-pAcF: PEG = 1: 2 without accelerator; 4) hGH-pAcF: PEG = 1: 5 without accelerator.

FIGURE 8 shows a non-limiting example of LCMS analysis for hGH incubated with different concentrations of acetic hydrazide accelerator: A) total LCMS trace; B) mass spectrum of hGH without accelerator; C) mass spectrum for hGH with 200 mM acetic hydrazide accelerator.

FIGURE 9 shows non-limiting examples of accelerators that can be used in the methods, reactions, and syntheses described herein.

FIGURE 10a shows a non-limiting reaction of a ketone model with a hydroxylamine model in the presence of an accelerator to obtain an oxime model; FIGURE 10b shows non-limiting examples of accelerators that can be used in the methods, reactions and syntheses described herein.

FIGURE 11 shows a non-limiting set of oxime yields for a reaction model performed without and with the various accelerators described herein.

DETAILED DESCRIPTION OF THE INVENTION

I. Introduction [0113]

Recently, completely new technologies have been reported in protein science, which promise to overcome many of the limitations associated with position-specific modifications of proteins. Specifically, new components have been added to the protein biosynthetic devices of prokaryotes Escherichia coli (E. coli) (e.g., L · IVang, et al., (2001), Science 292: 498-500), and eukaryotes Sacchromyces cerevisiae (S. cerevisiae). ) (e.g., J. Chin et al., Science 301: 964-7 (2003)), which enabled the in vivo incorporation of unnatural amino acids into proteins. Numerous new amino acids with new chemical, physical or biological properties, including photoaffinity and photoisomeric amino acid labels, photonaturated amino acids (see, e.g., Chin,

51998 Β

JW, et al. (2002) Proc. Natl. Acad. Sci. USA 99: 11020-11024; and Chin, JW, et al. (2002) J. Am. Chem. Soc. 124: 9026-27); keto amino acids, and glycosylated amino acids have been successfully incorporated both with high binding into proteins in E. coli and in yeast in response to the yantami codon, TAG, using this methodology. See, e.g., JW Chin et al., (2002), Journal of the American Chemical Society 124: 9026-902; JW Chin, &amp; PG Schultz, (2002), ChemBioChem 3 (11): 1135-1137; JW Chin, et al. (2002), PNAS United States of America 99: 11020-11024; and, L. IVang, &amp; PG Schultz, (2002), Chem. Comm., 1: 1-11. These studies have shown that it is possible to selectively and routinely introduce chemical functional groups not found in proteins, which are chemically inert to all functional groups found in 20 common, genetically encoded amino acids (i.e., natural amino acids) and which can be used to they respond effectively and selectively form stable covalent links.

Chemical functional groups not found in natural amino acids include carbonyl groups, such as ketones and aldehydes, and hydroxylamine groups. A portion of the hydroxylamine is reacted with a carbonyl group such as a ketone and an aldehyde to give a relatively stable oxime; this coupling (hydroxylamine with a carbonyl group) therefore provides means for further functionalization of the non-natural amino acid polypeptide. One example of such pairing is shown below:

protein

<img file="RS51998B_D0015.tif" />

H₂N '⁰~ R

<img file="RS51998B_D0016.tif" />

For example, when either a portion of the hydroxylamine or a carbonyl group is included in a non-natural amino acid polypeptide and reacts with a reagent containing that other member of the pair, the non-natural amino acid polypeptide may be functionalized with the oxime group-forming reagent. Although suitable for protein functionalization reactions, the standard oxime formation reaction can be made even more efficient which would allow, for example, the use of lower amounts

51998 Β reactants and reduced the time to complete the reaction. Therefore, the development of accelerators is highly desirable.

II. Overview Figure 1 is one embodiment for the compositions, methods, and techniques described herein. The carbonyl-containing compound was selected for reaction with the hydroxylamine-containing compound to give the oxime-containing compound. The carbonyl-containing compound includes non-natural amino acids, polypeptides, oligonucleotides, polymers (including polyethylene glycol only as an example), reagents, linker groups, groups containing further functionality, and combinations thereof; the present invention provides a number of examples of groups containing further functionality. The hydroxylamine-containing compound includes unnatural amino acids, polypeptides, oligonucleotides, polymers (including as an example polyethylene glycol), reagents, linker groups, groups containing further functionality, and combinations thereof; the present invention provides a number of examples of groups containing further functionality. The oxime-containing compound includes non-natural amino acids, polypeptides, oligonucleotides, polymers (including polyethylene glycol only as an example), reagents, linker groups, groups containing further functionality, and combinations thereof; the present invention provides a number of examples of groups containing further functionality. An accelerator is added to the reaction mixture of the hydroxylamine-containing compound and the carbonyl-containing compound, the accelerator having at least one of the following properties: (a) increasing the reaction rate between the carbonyl-containing compound and the hydroxylamine-containing compound to give the oxime-containing compound; where the increase in velocity is relative to the reaction without accelerator; (b) reducing the activation energy of the reaction between the carbonyl-containing compound and the hydroxylamine-containing compound to give the oxime-containing compound, wherein the reduction in activation energy is relative to the accelerator-free reaction; (c) increasing the yield of the oxime-containing compound from the reaction of the carbonyl-containing compound with the hydroxylamine-containing compound, wherein the increase in yield is relative to the reaction without accelerator; (d) reducing the temperature at which the carbonyl-containing compound is reacted with the hydroxylamine-containing compound to give the oxime-containing compound, wherein the temperature reduction is relative to the reaction without accelerator; (e) reducing the time required for the carbonyl-containing compound to react with the hydroxylamine-containing compound to give the oxime-containing compound, wherein the reduction in time is relative to the accelerator-free reaction; (f) decreases

51998Β the amount of reagent required to obtain the oxime group on the non-natural amino acid polypeptide, wherein the reduction in the amount of reagent is relative to the reaction without accelerator; (g) reducing foreign products resulting from the reaction of a carbonyl-containing compound with a hydroxylamine-containing compound to obtain an oxime-containing compound, wherein the reduction of foreign products is relative to the reaction without accelerator; (h) does not irreversibly destroy the tertiary structure of the polypeptide that is subjected to the oxime formation reaction in the presence of an accelerator (except, of course, where the purpose of the reaction is to destroy such a tertiary structure); (i) may be separated in vacuo from the oxime-containing compound; and (j) modulates the reaction of a carbonyl-containing compound with a hydroxylamine-containing compound. In a further embodiment, the accelerator does not have any of the aforementioned properties. Arbitrarily, a number of accelerators described herein have been tested and the accelerator has been selected based on its possession of at least one of the aforementioned properties. Optionally, the reaction characteristics (e.g., yield of the oxime-containing compound) may be further optimized with at least one of the following: (a) varying the amount of accelerator, (b) varying the amount of carbonyl-containing compound, (c) varying the amount of hydroxylamine-containing compound. , (d) changing the reaction temperature, (e) changing the pH of the reaction, and (f) changing the solvent in the reaction mixture. Optionally, additional accelerators are tested under optimized reaction conditions, or the selection and optimization steps are reversed, or the selection and optimization steps are repeated in an iterative manner. The carbonyl-containing compound and the hydroxylamine-containing compound are reacted in the presence of an accelerator to give the oxime-containing compound. Optionally, the progress of the reaction is monitored by detection means, including chromatography as an example. The oxime-containing compound obtained by reacting the carbonyl-containing compound and the hydroxylamine-containing compound in the presence of an accelerator can be isolated arbitrarily, purified and labeled. The accelerator can be removed from the oxime-containing compound by a variety of methods, including by way of example only filtration, in vacuo techniques, chromatography, membrane bioseparation, electrophoresis, precipitation of the oxime-containing compound, distillation, or combinations thereof. Thus, in one embodiment described herein, the accelerators can be removed in vacuo from the oxime-containing material; however, in other embodiments described herein, the accelerators may be removed using any of the foregoing methods (or any combination thereof). Isolation and purification means the removal from the material in the reaction mixture of at least one compound containing non-oximes.

At one level, tools (methods, compounds, techniques) for creating and using a polypeptide comprising at least one non-natural amino acid or a modified non-natural amino acid with an oxime group obtained in the presence of an accelerator are described herein. although such a reaction may be less effective in the absence of the accelerator described herein). Such non-natural amino acids may contain further functionality, including the desired functionality.

Also described herein are non-natural amino acids that have or may be modified to contain an oxime moiety formed in the presence of an accelerator (although such a reaction may be less effective in the absence of the accelerator described herein). Included in this aspect are methods for preparing, purifying, labeling and using such non-natural amino acids. In another aspect, methods, strategies and techniques for incorporating at least one such non-natural amino acid into a polypeptide are described herein. Also included in this aspect are methods for preparing, purifying, labeling and using such polypeptides comprising at least one such non-natural amino acid. Also included in this aspect are compositions and methods for preparing, purifying, labeling and using polynucleotides (including DNA and RNA) that can be used to prepare, at least in part, a polypeptide containing at least one non-natural amino acid that can react, in the presence of accelerator (although such a reaction may be less effective in the absence of the accelerator described herein) to obtain an oxime-containing non-natural amino acid polypeptide, including such a polypeptide that has been modified. Also included in this aspect are compositions and methods for preparing, purifying, labeling and using cells that can express such polynucleotides that can be used to prepare, at least in part, a polypeptide comprising at least one non-natural amino acid.

Also within the scope of the accelerators described herein are methods, compositions, strategies, and techniques for reacting a reagent with a non-natural amino acid (containing a carbonyl or dicarbonyl group, a hydroxylamine group, or protected forms thereof) that is part of a polypeptide to provide any of the aforementioned post-translational modifications. In general, the resulting post-translatomo modified

The 51998Β non-natural amino acid polypeptide will contain at least one oxime group; the resulting modified oxime-containing non-natural amino acid polypeptide may be subjected to subsequent modification reactions. Also included in this aspect are methods for selecting, obtaining, optimizing, purifying, labeling, and using such accelerators that can be used with any such posttranslational modifications of such non-natural amino acids.

A non-natural amino acid containing a polypeptide may contain 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 non-natural amino acids that containing an oxime group (or protected or masked forms thereof), where at least one oxime group is obtained in the presence of the accelerators described herein, and further, such an oxime-containing non-natural amino acid polypeptide may optionally contain at least a non-natural amino acid polypeptide containing one carbonyl or dicarbonyl group, a hydroxylamine group, or protected forms thereof. Unnatural amino acids may be the same or different, for example, 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 different sites in the protein containing 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more different non-natural amino acids. In certain embodiments, at least one, but less than all, single amino acid found in the natural version of the protein is substituted with a non-natural amino acid.

The methods and compositions of a non-natural amino acid described herein comprise conjugates of substances having a wide range of functional groups (provided that at least one conjugate is chemically linked to a non-natural amino acid via an oxime group obtained in the presence of accelerators described herein), substituents or a group, with other substances including but not limited to the desired functionality.

In another aspect of the compositions, methods, techniques and strategies, methods for studying or using any of the foregoing (modified) non-natural amino acid polypeptides are described herein. Examples within this aspect include therapeutic, diagnostic, test, industrial, cosmetic, plant biological, environmental, energy, and / or military uses.

51998 Β uses that may benefit from a polypeptide containing a (modified) non-natural polypeptide or amino acid protein.

III. Post-translational modifications of non-natural components of an amino acid polypeptide in the presence of at least one accelerator Methods, compositions, techniques and strategies have been developed to include non-natural amino acids at a specific position during in vivo protein translation. By incorporating a non-natural amino acid with side chain chemistry that is orthogonal to those naturally occurring amino acids, this technology enables the derivation of recombinant proteins at a specific position. As a result, the main advantage of the methods, compositions, techniques and strategies described herein is that the derived proteins can now be obtained as defined homogeneous products. However, the methods, compositions, reaction mixtures, techniques, and strategies described herein that include an accelerator are not limited to non-natural amino acid polypeptides obtained by in vivo protein translation techniques, but include non-natural amino acid polypeptides obtained by any technique , including by way of example only protein ligation expression, chemical synthesis, ribozyme-based techniques (see, eg, here the section entitled Expression in Other Systems). For convenience, the term post-translational modification, when directed to the use of an accelerator to obtain an oxime bond on a non-natural amino acid polypeptide, includes non-natural amino acid polypeptides obtained by any method, including any in vivo and in vitro method, as well as those described herein and known to those skilled in the art.

The ability to incorporate non-natural amino acids into recombinant proteins significantly expands the chemistry that can be used for derivation. More specifically, deriving a protein to obtain an oxime bond on a portion of a non-natural amino acid polypeptide offers several advantages. First, natural amino acids generally do not form oxime bonds, and therefore reagents intended to form oxime bonds will react at a specific position with the non-natural component of the amino acid polypeptide (assuming, of course, that the non-natural amino acid and the corresponding reagent are designed to form oxime). connection), hence the ability to

51998 Β Derived proteins at the selected position give a unique homogeneous product as opposed to mixtures of derived proteins obtained using earlier technology. Second, oxime conjugates are stable under biological conditions, indicating that oxime-altered proteins are suitable candidates for therapeutic applications. Third, the stability of the resulting oxime bond can be adjusted based on the identity (ie, functional group and / or structure) of the non-natural amino acid on which the oxime bond is formed. Thus, in some embodiments of the invention, the oxime bond to the non-natural amino acid polypeptide has a decay half-life of less than one hour, in other embodiments less than 1 day, in other embodiments less than 2 days, in other embodiments less than 1 week, and in subsequent embodiments. more than 1 week. In still further embodiments, the resulting oxime is stable for at least two weeks under mildly acidic conditions, in other embodiments, the resulting oxime is stable for at least 5 days under mildly acidic conditions. In other embodiments, the non-natural amino acid polypeptide is stable for 1 day at a pH between about 2 and 8; in other embodiments, with a pH between about 2 to 6; in another embodiment, with a pH between about 2 to 4. In other embodiments, using the strategies, methods, compositions, and techniques described herein, one skilled in the art will be able to synthesize an oxime bond to a non-natural amino acid polypeptide with a decay half-life adjusted to the needs of one skilled in the art (eg, for therapeutic prolonged-release use, either for diagnostic use, or for industrial or military use).

Formation of a non-natural amino acid comprising an unnatural amino acid oxime or polypeptide from reaction (a) a carbonyl-containing non-natural amino acid or a carbonyl-containing non-natural amino acid polypeptide and a hydroxylamine-containing reagent, or ( b) a hydroxylamine-containing non-natural amino acid or a hydroxylamine-containing non-natural amino acid polypeptide and a carbonyl-containing reagent can be improved by adding an accelerator to the reaction mixture. An accelerator is a compound having at least one of the following properties: (a) increasing the reaction rate between a carbonyl-containing compound and a hydroxylamine-containing compound to give an oxime-containing compound, where the rate increase is relative to the reaction without an accelerator; (b) reducing the activation energy of the reaction between the carbonyl-containing compound and the hydroxylamine-containing compound to give the oxime-containing compound, where the reduction

51998 Β activation energies relative to the reaction without accelerators; (c) increasing the yield of the oxime-containing compound from the reaction of the carbonyl-containing compound with the hydroxylamine-containing compound, wherein the increase in yield is relative to the reaction without accelerator; (d) reducing the temperature at which the carbonyl-containing compound is reacted with the hydroxylamine-containing compound to give the oxime-containing compound, where the temperature reduction is relative to the reaction without accelerator; (e) reducing the time required for the carbonyl-containing compound to react with the hydroxylamine-containing compound to give the oxime-containing compound, the reduction in time being relative to the accelerator-free reaction; (f) reducing the amount of reagent required to form the oxime group on the non-natural amino acid polypeptide, where the reduction in the amount of reagent is relative to the reaction without accelerator; (g) reducing the foreign products resulting from the reaction of the carbonyl-containing compound with the hydroxylamine-containing compound to give the oxime-containing compound, wherein the reduction of the foreign products is relative to the reaction without accelerator; (h) does not irreversibly destroy the tertiary structure of the polypeptide that is subjected to the oxime formation reaction in the presence of an accelerator (except, of course, where the purpose of the reaction is to destroy such a tertiary structure); (i) may be separated in vacuo from the oxime-containing compound; and (j) modulates the reaction of a carbonyl-containing compound with a hydroxylamine-containing compound. In further embodiments, the accelerator has at least two of the aforementioned properties, three of the aforementioned properties, four of the aforementioned properties, five of the aforementioned properties, six of the aforementioned properties, seven of the aforementioned properties, eight of the above properties . In a further embodiment, the accelerator does not have any of the aforementioned properties.

The use of an accelerator includes the use of a single accelerator or multiple accelerators. Additionally, the molar ratio of accelerator to carbonyl-containing compound includes values ​​between about 0.5: 1 to 5000: 1, including by way of example only 4000: 1, 3000: 1, 2000: 1, 1000: 1, 500: 1, 400: 1, 300: 1, 200: 1, 100: 1, 50: 1, 40: 1, 30: 1, 20: 1, 10: 1, 9: 1, 8: 1, 7: 1, 6: 1, 5: 1, 4: 1, 3: 1, 2: 1, 1: 1, 0.9: 1, 0.8: 1, 0.7: 1, 0.6: 1, and 0.5: 1. Furthermore, the molar ratio of accelerator to hydroxylamine-containing compound includes values ​​between about 0.5: 1 to 5000: 1, including by way of example only 4000: 1, 3000: 1, 2000: 1, 1000: 1, 500: 1, 400: 1, 300: 1, 200: 1, 100: 1, 50: 1, 40: 1, 30: 1, 20: 1, 10: 1, 9: 1, 8: 1, 7: 1, 6: 1, 5: 1, 4: 1, 3: 1, 2: 1, 1: 1,0.9: 1,0.8: 1, 0.7: 1,0.6: 1, and 0.5: 1. Furthermore, the accelerator

51998 Β includes compounds that can be substantially removed in vacuo from the resulting oxime-containing compound. Furthermore, the accelerator includes compounds containing a diamine moiety, a semi-carbazide moiety, a hydrazine, or a hydrazide moiety.

Furthermore, in any of the above aspects or representations, the accelerator is selected from the group consisting of bifunctional aromatic amines, oxoamine derivatives, and compounds

<td>having the following structures:</td><td>i^v</td>

<td></td><td>^RX'' N '^N'R. n</td>

<img file="RS51998B_D0017.tif" />

<img file="RS51998B_D0018.tif" />

<img file="RS51998B_D0019.tif" />

where R_x, R_y and R_z are selected from the group consisting of: L_x-H, L_x-alkyl, L_x-aryl, L_x-heteroaryl, L_x-alkenyl, L_x-alkynyl, L_x-alkoxy, and L_x-alkylamine, where L_x is a bond, C (= O), C (= NH), C (= NH) -NH and SO, SO₂, where the aromatic amine is selected from the group:

Bifunctional aromatic amines:

<img file="RS51998B_D0020.tif" />

<img file="RS51998B_D0021.tif" />

51998 Wherein and where the oxoamine derivative is selected from the group:

Oxoamine derivatives:

Me^NABOUT '^Me Me^NABOUT '^{E t} Me ^ HG⁸

Furthermore, the accelerator includes compounds selected from the group consisting of:

«A,„ .D, where R_x, R_y and R_z are selected from the group consisting of: L_x-H, L_x-alkyl, L_x-aryl, L_xheteroaryl, L_x-alkenyl, L_x-alkynyl, L_x-alkoxy, and L_x-alkylamine, where L_x is a bond, C (= O), C (= NH), and C (= NH) -NH. Furthermore, in any of the above aspects or illustrations, the accelerator is selected from the compounds listed in Figure 5, Figure 9, or Figure 10, including as an example any of the compounds 6, 8, 10, 7 and 20 of Figure 5 , In either in any of the above aspects or representations, the accelerator includes an agent that can form a hydrazone after reaction with a carbonyl-containing group. Furthermore, in any of the above aspects, the activity of the accelerator depends on the rate of reaction with the ketone moiety and the stability of the resulting intermediate. Furthermore, in any of the above aspects or illustrations, the pH of the reaction mixture containing the accelerator, the carbonyl-containing compound and the hydroxylamine-containing compound is between about 2.0 and 10; between about 2.0 and 9.0; between about 2.0 and 8.0; between about 3.0 and 7.0; between about 4.0 and 6.0; between about 3.0 and 10.0; between about 4.0 and 10.0; between about 3.0 and 9.0; between about 3.0 and 8.0; between about 2.0 and 7.0; between about 3.0 and 6.0; between about 4.0 and 9.0; between about 4.0 and 8.0; between about 4.0 and 7.0; between about 4.0 and 6.5, between about 4.5 and 6.5; about 4.0; about 4.5; about 5.0; about 5.5; about 6.0; about 6.5; and about 7.0.

The non-natural amino acid polypeptides described above are useful for, including but not limited to, novel therapeutics, diagnostics, catalytic enzymes, industrial enzymes, binding proteins (including antibodies and antibody fragments), and including but not limited to, study of structure and function. See, eg, Dougherty, (2000) Unnatural Amino Acids as Probes of Protein Structure and

51998 Β

Function, Current Opinion in Chemical Biology, 4: 645-652. Other uses for the non-natural amino acid polypeptides described above include, by way of example only, test uses, cosmetic, plant biological uses, environmental uses, energy uses, and / or military uses. However, the non-natural amino acid polypeptides described above may be subjected to further modifications to contain new or modified functionalities, including manipulating the therapeutic effect of the polypeptide, improving the safety profile of the polypeptide, adjusting the pharmacokinetics, pharmacology and / or pharmacodynamics of the polypeptide. bioavailability, increase in serum half-life, increase in therapeutic half-life, modulation of immunogenicity, modulation of biological activity, or prolongation of circulation time), provide additional functionality to the polypeptide, the inclusion of a label or recognizable signal in the polypeptide, facilitating the isolation of the properties of the polypeptide, and any combination of the aforementioned modifications.

The methods, compositions, strategies, and techniques described herein are not limited to a particular type, class, or family of polypeptides or proteins. In fact, virtually any polypeptide may include at least one non-natural amino acid described herein. According to example, the polypeptide may be homologous to a therapeutic protein selected from the group consisting of: alpha-1 antitrypsin, angiostatin, antihemolytic factor, antibody, antibody fragments, apolipoprotein, apoprotein, atrial natriuretic factor, atrial natriuretic polypeptide, atrial peptide, SHS chemokine, T39765, NAP2, ENA-78, gro -b, gro-c, IP -10, GCP-2, NAP-4, SDF-1, PF4, MIG, calcitonin, c-kit ligand, cytokine, CC chemokine, monocyte chemoattractive protein-1, monocyte chemoattractive protein-2, monocyte chemoattractive protein-3, monocyte inflammatory protein-1 alpha, monocyte inflammatory proteins and beta, RANTES, 1309, R83915, R91733, HCCl, T58847, D31065, T64262, CD40, CD40 ligand, c-kit ligand, collagen, colony stimulating factor (CSF), complement factor 5a, complement inhibitor, complement receptor 1, cytokine, epithelial neutrophil activating peptide-78, MIP16, MCP-1, epidermal growth factor (EGF), epithelial neutrophil activating peptide, erythropoietin (EPO), exfoliating toxin, Factor IX, Factor VII, Factor VIII, Factor X, fibroblast growth factor (FGF), fibrinogen, fibronectin, 4-helical package protein, GCSF, glp-1, GM-CSF, glucocerebrosidase, gonadotropin, growth factor, growth factor receptor, grf, hedgehog protein, hemoglobin, hepatocyte growth factor (hGF ), hirudin,

51998 Β

Human growth hormone (hGH), human serum albumin, ICAM-1, ICAM-1 receptor, LFA-1, LFA-1 receptor, insulin, insulin-like growth factor (IGF), IGF-I, IGF-II, interferon IFN ), IFN-alpha, IFN-beta, IFN-gamma, any molecule interferon-like or member of the IFN family, interleukin (IL), IL-1, IL-2, IL-3, IL-4, IL-5 , IL-6, IL-7, IL-8, IL9, IL-10, IL-11, IL-12, keratinocyte growth factor (KGF), lactoferrin, leukemia inhibition factor, luciferase, neurturin, neutrophil inhibition factor (NIF) ,, oncostatin M, osteogenic protein, oncogenic product, paracitonin, parathyroid hormone, PD-ECSF, PDGF, peptide hormone, pleiotropin, protein A, protein G, pth, pyrogenic exotoxin A, pyrogenic exotoxin B, pyrogenic exotoxin C, ruu, relaxin, renin , SCF, small biosynthetic protein, soluble complement receptor I, soluble I-CAM 1, soluble interleukin receptor, soluble TNF receptor, somatomedin, somatostatin, somatotropin, streptokinase, superantigens, staphylococcal enterotoxin, FLT, SEA, SEB, SECl, 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, tumor necrosis factor, 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, p53, tat, fos, myc, jun, myb, rel, estrogen receptor, progesterone receptor, testosterone receptor, aldosterone receptor, LDL receptor, and corticosterone. The non-natural amino acid polypeptide may also be homologous to any polypeptide member of the supergen growth hormone family.

Such modifications include the inclusion of further functionality on an unnatural component of an amino acid polypeptide, including the desired functionality.

Thus, as an example, a non-natural amino acid polypeptide comprising any of the following amino acids may be further modified in the presence of the accelerators described herein using the methods and compounds described herein:

(a)

51998 Β

<img file="RS51998B_D0022.tif" />

where:

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 heterocycloalkylene, substituted heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene , aralkylene, or substituted aralkylene;

B is optional, and when present the linker is selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O- (alkylene or substituted alkylene) -, -S-, -S- (alkylene or substituted alkylene) -, -S (O) k- where k is 1,2, or 3, -S (O) k (alkylene or substituted alkylene) -, -CO ) -, -C (O) - (alkylene or substituted alkylene) -, -C (S) -, -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) kN (R') - , -N (R ') C (O) N (R') -, -N (R ') C (S) N (R') -, -N (R ') S (O)_kN (R ') -, -N (R') - N =, -C (R ') = N-, C (R') = NN (R ') -, -C (R') = NN =, -C (R ')₂-N = N-, and -C (R ')₂-N (R ') - N (R') -, wherein each R 'is independently H, alkyl, or substituted alkyl;

Yes

<img file="RS51998B_D0023.tif" />

51998 Β

<img file="RS51998B_D0024.tif" />

R is N, 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, the two Rs optionally form heterocycloalkyl;

R 1 is optional, and when present, is H, an amino protecting group, a resin; and R2 is optional, and when present, is OH, an ester protecting group, a resin; each of R 3 and R 4 is independently H, halogen, lower alkyl, or substituted lower alkyl, or R 3 and R 4 or two R₃ the groups optionally form cycloalkyl or heterocycloalkyl;

or ABJR groups together form a bicyclic or tricyclic cycloalkyl or heterocycloalkyl containing at least one carbonyl group, including a dicarbonyl group, a protected carbonyl group, including a protected dicarbonyl group, or a masked carbonyl group, including a masked dicarbonyl group;

or the -JR group together forms a monocyclic or bicyclic cycloalkyl or heterocycloalkyl containing at least one carbonyl group, including a dicarbonyl group, a protected carbonyl group, including a protected dicarbonyl group, or a masked carbonyl group, including a masked dicarbonyl group;

(b)

<img file="RS51998B_D0025.tif" />

51998 Β where:

R is alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;

R 1 is optional, and when present is H, an amino protecting group, a resin; and R₂ is optional, and when present, is OH, an ester protecting group, a resin; and each R 1 is independently selected from the group consisting of H, halogen, alkyl, substituted alkyl, -N (R ')₂, - C (O) kR 'where k is 1,2, or 3, -C (O) N (R')₂, -OR ', and S (O) kR', wherein each R'is independently H, alkyl, or substituted alkyl;

(c)

<img file="RS51998B_D0026.tif" />

where:

A is optional, and when the presence of lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocylenealkylene, substituted lower substituted heteroarylene, alkarylene, substituted alkarylene , aralkylene, or substituted aralkylene;

B is optional, and when present the linker is selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O- (alkylene or substituted alkylene) -, -S-, -S- (alkylene or substituted alkylene) -, -S (O) k- where k is 1,2, or 3, -S (O) k (alkylene or substituted alkylene) -, -C ) -, -C (O) - (alkylene or substituted alkylene) -, -C (S) -, -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) kN (R') - , -N (R ') C (O) N (R') -, -N (R ') C (S) N (R') -, -N (R ') S (O)_kN (R ') -, -N (R') - N =, -C (R ') = N-, C (R') = NN (R ') -, -C (R') = NN =, -C (R ')₂-N = N-, and -C (R ')₂-N (R ') - N (R') -, where

51998 Β each R'is independently H, alkyl, or substituted alkyl;

K is -NR₆R₇ or -N = CR₆R₇;

R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;

R 1 is optional, and when present is H, an amino protecting group, a resin; and R2 is optional, and when present, is OH, an ester protecting group, a resin; each of R 3 and R 4 is independently H, halogen, lower alkyl, or substituted lower alkyl, or R 3 and R 4 or two R₃ the groups optionally form cycloalkyl or heterocycloalkyl;

each of Rj and R₇ is independently selected from the group consisting of H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, polyalkylene oxide, substituted polyalkylene oxide, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkaryl, substituted alkaryl, aralkyl, and substituted aralkyl, -C (O) R, -C (O) 2R, -C (O) N (R)₂, wherein each R is independently hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, alkaryl, substituted alkaryl, aralkyl, or substituted aralkyl; or R6 or R₇ is LX, where X is selected from the group consisting of the desired functionality; and L is optional, and when present the linker is selected from the group consisting of alkylene, substituted alkylene, alkenylene, substituted alkenylene, -O-, -O- (alkylene or substituted alkylene) -, S-, -S-, alkylene or substituted alkylene) -, -S (O) k- where k is 1,2, or 3, -S (O) r (alkylene or substituted alkylene) -, -C (O) -, -C (O) - (alkylene or substituted alkylene) -, -C (S) -, -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 ( ABOUT)_kN (R ') -, -N (R') - N =, -C (R ') = N-, -C (R') = NN (R ') -, -C (R') = NN = , -C (R ')₂-N = N-, and -C (R ')₂-N (R ') - N (R') -, wherein each R 'is independently H, alkyl, or substituted alkyl;

(d)

51998 XXX (XXX);

<img file="RS51998B_D0027.tif" />

<img file="RS51998B_D0028.tif" />

where;

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 heterocycloalkylene, substituted heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene , aralkylene, or substituted aralkylene;

R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;

R 1 is optional, and when present, is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide;

R 2 is optional, and when present, is OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide;

X 1 is 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 (e)

<img file="RS51998B_D0029.tif" />

Rj

<img file="RS51998B_D0030.tif" />

<img file="RS51998B_D0031.tif" />

<img file="RS51998B_D0032.tif" />

<img file="RS51998B_D0033.tif" />

(ХХХХ),

51998 Β where:

A is optional, and when lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene is present; alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene;

M is -C (R₃)-,

<td>(b&gt;</td><td></td><td>(b)</td>

<td></td><td>R₃</td><td>-YYY</td>

<td> 1</td><td> 7 &lt;</td><td> . 1 $</td>

<td></td><td>-c— ' \ '</td><td>&gt; (b) C = C— | (b)</td>

<td>(a) 7 R «</td><td>R «</td><td>(a) $ R, .</td>

<img file="RS51998B_D0034.tif" />

<td></td><td></td><td>(b)</td>

<td>(b)</td><td></td><td>σνν</td>

<td>&gt; / W</td><td>R₃</td><td>1 s</td>

<td> 1</td><td> / 5</td><td>C = C - S (b)</td>

<td></td><td>-c - 1 (b) X</td><td>/ Ι&gt; X</td>

<td></td><td> (^a) &gt;</td><td>(a) &gt;</td>

<td>(b) 0-</td><td>—C-5 1 5</td><td>(b) ; (b) S-</td><td> /^R\ —C - 1 (b)</td>

<td></td><td> 1</td><td></td><td>1 i / W</td>

<td></td><td>(a)</td><td>, or</td><td>(a)</td>

where (a) denotes attachment to group A and (b) denotes attachment to the corresponding carbonyl groups, R₃ and R₄ are independently selected from H, halogen, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl, or R₃ and R₄ or two R₃ groups or two R₄ the groups optionally form cycloalkyl or heterocycloalkyl;

R is H, halogen, alkyl, substituted alkyl cycloalkyl, or substituted cycloalkyl; T₃ is a bond, C (R) (R), O, or S, and R is H, halogen, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;

R 1 is optional, and when present, is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; i

R2 is optional, and when present, is OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide.

51998 In one aspect of the methods and compounds described herein, compositions comprising at least one protein with at least one, including but not limited to, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten or more non-natural amino acids that are posttranslatomo-modified. Post-translatomo-modified non-natural amino acids may be the same or different, including but not limited to, that 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 different sites in the protein containing 1,2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13 , 14, 15, 16, 17, 18, 19, 20, or more different post-translatomo-modified non-natural amino acids. In another aspect, the composition comprises a protein with at least one, but less of all, single amino acid present in the protein substituted with posttranslatomo-modified non-natural amino acid. For a particular protein with more than one post-translatomo-modified non-natural amino acid, posttranslatomo-modified non-natural amino acids may be identical or different (including but not limited to, a protein that includes two or more different types of post -translatomo- modified pe-natural amino acids, or may include two of the same post-translatomo-modified non-natural amino acids). For a particular protein with more than two post-translatomo-modified non-natural amino acids, the posttranslatomo-modified non-natural amino acids may be the same, different, or a combination of multiple post-translatomo-modified non-natural amino acids of the same species with at least one different post-translatomo-modified unnatural amino acid.

A. Methods for Post-Translational Modification of Non-Natural Amino Acid Polypeptides in the Presence of at least One Accelerator: Reactions of Carbonyl-Containing Non-Natural Amino Acids with Hydroxylamite-Containing Reagents The side chains of natural amino acids lack highly electrophilic positions . Thus, the insertion of a non-natural amino acid with an electrophile-containing side chain, including, by way of example only, an amino acid containing a carbonyl or dicarbonyl group such as a ketone, allows derivation at a specific position of that side chain by nucleophilic attack on the carbonyl or dicarbonyl

51998 Β group. In the case where the attacking nucleophile is hydroxylamine, an oxidized protein will be created. Methods for derivation and / or further modification can be performed with the polypeptide purified before the derivation step or after the derivation step. Furthermore, kog derivation can occur under mildly acidic or slightly basic conditions, including as an example, a pH between about 2-8, or a pH between about 4-8.

The formation of a non-natural amino acid containing an oxime or non-natural amino acid polypeptide from the reaction of a carbonyl-containing non-natural amino acid or a carbonyl-containing non-natural amino acid polypeptide and a hydroxylamine-containing reagent can be improved by adding an accelerator to the reaction mixture. An accelerator is a compound having at least one of the following properties: (a) increasing the reaction rate between a carbonyl-containing compound and a hydroxylamine-containing compound while forming an oxime-containing compound, wherein the rate increase is relative to the reaction without an accelerator; (b) reducing the activation energy of the reaction between the carbonyl-containing compound and the hydroxylamine-containing compound while forming an oxime-containing compound, where the reduction in activation energy is relative to the accelerator-free reaction; (c) increasing the yield of the oxime-containing compound from the reaction of the carbonyl-containing compound with the hydroxylamine-containing compound, wherein the increase in yield is relative to the reaction without accelerator; (d) reducing the temperature at which the carbonyl-containing compound reacts with the hydroxylamine-containing compound to form an oxime-containing compound, where the temperature decrease is relative to the reaction without accelerator; (e) reducing the time required for the carbonyl-containing compound to react with the hydroxylamine-containing compound while forming an oxime-containing compound, where the reduction in time is relative to the accelerator-free reaction; (f) reducing the amount of reagent required to form the oxime group on the non-natural amino acid polypeptide, where the reduction in the amount of reagent is relative to the reaction without accelerator; (g) reducing foreign products resulting from the reaction of a carbonyl-containing compound with a hydroxylamine-containing compound while forming an oxime-containing compound, where the reduction of foreign products is relative to the reaction without accelerator; (h) does not irreversibly destroy the tertiary structure of the polypeptide that is subjected to the oxime formation reaction in the presence of an accelerator (except, of course, where the purpose of the reaction is to destroy such a tertiary structure); (i) may be separated in vacuo from the oxime-containing compound; and (j) modulates the reaction of a carbonyl-containing compound with a hydroxylamine-containing compound. In further embodiments,

51998 Β The accelerator has at least two of the above properties, three of the above properties, four pre-specified properties, five of the above properties, six of the above properties, seven of the above properties, eight of the above properties, nine of the above properties, and all of the above properties. In a further embodiment, the accelerator does not have any of the aforementioned properties.

The use of accelerators includes the use of single accelerators or multiple accelerators. Additionally, the molar ratio of accelerator to carbonyl-containing compound includes values ​​between about 0.5: 1 to 5000: 1, including by way of example only 4000: 1, 3000: 1, 2000: 1, 1000: 1, 500: 1, 400: 1, 300: 1, 200: 1, 100: 1, 50: 1, 40: 1, 30: 1, 20: 1, 10: 1, 9: 1, 8: 1, 7: 1, 6: 1, 5: 1, 4: 1, 3: 1, 2: 1, 1: 1, 0.9: 1, 0.8: 1, 0.7: 1, 0.6: 1, and 0.5: 1. Furthermore, the molar ratio of accelerator to hydroxylamine-containing compound includes values ​​between about 0.5: 1 to 5000: 1, including by way of example only 4000: 1, 3000: 1, 2000: 1, 1000: 1, 500: 1, 400: 1, 300: 1, 200: 1, 100: 1, 50: 1, 40: 1, 30: 1, 20: 1, 10: 1, 9: 1, 8: 1, 7: 1, 6: 1, 5: 1, 4: 1, 3: 1, 2: 1, 1: 1, 0.9: 1, 0.8: 1, 0.7: 1, 0.6: 1, and 0.5: 1. Furthermore, the accelerator includes compounds that can be substantially removed in vacuo from the resulting oxime-containing compound. Furthermore, the accelerator includes compounds containing a diamine moiety, a semi-carbazide moiety, a hydrazine moiety, or a hydrazide moiety.

Furthermore, in any of the following aspects or representations, the accelerator is selected from the group consisting of bifunctional aromatic amines, oxoamine derivatives, and compounds having the following structures:

Ry

<img file="RS51998B_D0035.tif" />

<img file="RS51998B_D0036.tif" />

<img file="RS51998B_D0037.tif" />

oo

51998 Β

<img file="RS51998B_D0038.tif" />

<img file="RS51998B_D0039.tif" />

where R_x, R_y and R_z are selected from the group consisting of: L_x-H, L_x-alkyl, L_x-aryl, L_xheteroaryl, L_x-alkenyl, L_x-alkynyl, L_x-alkoxy, and L_x-alkylamine, where L_x is a bond, C (= O), C (= NH), C (= NH) -NH and SO, SO2, where the aromatic amine is selected from the group:

Bifunctional aromatic amines:

<img file="RS51998B_D0040.tif" />

<img file="RS51998B_D0041.tif" />

and where in a further embodiment, the oxoamine derivative is selected from the group:

Oxoamine derivatives:

<img file="RS51998B_D0042.tif" />

<img file="RS51998B_D0043.tif" />

<img file="RS51998B_D0044.tif" />

<img file="RS51998B_D0045.tif" />

<img file="RS51998B_D0046.tif" />

Furthermore, the accelerator includes compounds selected from the group consisting of:

<img file="RS51998B_D0047.tif" />

<img file="RS51998B_D0048.tif" />

n S ^R.^r

M'R, _{η? Γ}^Ν_Ί1ι where R_x, R_y and R_z are selected from the group consisting of: L_x-H, L_x-alkyl, L_x-aryl, L_xheteroaryl, L_x-alkenyl, L_x-alkynyl, L_x-alkoxy, and L_x-alkylamine, where L_x is a bond, C (= O), C (= NH), and C (= NH) -NH. Furthermore, in any of the above aspects or illustrations, the accelerator is selected from the compounds shown in Figure 5, Figure 9 or Figure 10, including as an example any of the compounds 6, 8, 10, 7 and 20 of Figure 5. In any of the foregoing aspects or representations, the accelerator includes an agent that can form a hydrazone after

51998 Β reactions with a carbonyl-containing group. Furthermore, in any of the above aspects, the activity of the accelerator depends on the rate of reaction with the ketone moiety and the stability of the resulting intermediate. Furthermore, in any of the above aspects or illustrations, the pH of the reaction mixture containing the accelerator, the carbonyl-containing compound and the hydroxylamine-containing compound is between about 2.0 and 10; between about 2.0 and 9.0; between about 2.0 and 8.0; between about 3.0 and 7.0; between about 4.0 and 6.0; between about 3.0 and 10.0; between about 4.0 and 10.0; between about 3.0 and 9.0; between about 3.0 and 8.0; between about 2.0 and 7.0; between about 3.0 and 6.0; between about 4.0 and 9.0; between about 4.0 and 8.0; between about 4.0 and 7.0; between about 4.0 and 6.5; between about 4.5 and 6.5; about 4.0; about 4.5; about 5.0; about 5.5; about 6.0; about 6.5; and about 7.0.

The method of protein derivative based on the reaction of a protein containing a carbonyl or dicarbonyl with a hydroxylamine-substituted molecule has various advantages. First, the hydroxylamines are subjected to condensation with compounds containing carbonyl- or di-carbonyl at a pH between about 2 and 8 (in further embodiments at a pH between about 4 and 8) to generate oxime adducts. Under these conditions, the side chains of natural amino acids are unreactive. Second, such selective chemistry allows derivatization of recombinant proteins at a specific position: derived proteins can now be obtained as defined homogeneous products. Third, the mild conditions required for the reaction of the hydroxylamine described herein with the carbonyl or dicarbonyl-containing polypeptides described herein do not generally irreversibly destroy the tertiary structure of the polypeptide (except, of course, where the purpose of the reaction is to destroy such a tertiary structure). Finally, although the hydroxylamine amino group appears to be metabolized by E. coli, the condensation of hydroxylamine with carbonyl- or dicarbonyl-containing molecules generates oxime adducts that are stable under biological conditions.

By way of example, the following non-natural amino acids are carbonyl- or dicarbonyl-containing amino acid types that are reactive with the hydroxylamine-containing reagents described herein to give a non-natural amino acid or oxime-containing polypeptide in the presence of that described herein. accelerator (although such a reaction may be less effective in the absence of the accelerator described here):

51998 Β

<img file="RS51998B_D0049.tif" />

(I) where:

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 heterocycloalkylene, substituted heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene , aralkylene, or substituted aralkylene;

B is optional, and when present the linker is selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O- (alkylene or substituted alkylene) -, -S-, -S- (alkylene or substituted alkylene) -, -S (O) k- where k is 1,2, or 3, -S (O) k (alkylene or substituted alkylene) -, -CO ) -, -C (O) - (alkylene or substituted alkylene) -, -C (S) -, - 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 ') - (alkylene or substituted alkylene) -, N (R') CO- (alkylene or substituted alkylene) -, -N (R ') C (O) O-, -S (O) kN (R') - , -N (R ') C (O) N (R') -, -N (R ') C (S ) N (R') -, -N (R ') S (O)_kN (R ') -, -N (R') - N =, -C (R ') = N-, C (R') = NN (R ') -, -C (R') = NN =, -C (R ')₂-N = N-, and -C (R ')₂-N (R ') - N (R') -, wherein each R 'is independently H, alkyl, or substituted alkyl;

Yes

<img file="RS51998B_D0050.tif" />

<img file="RS51998B_D0051.tif" />

R

<img file="RS51998B_D0052.tif" />

51998Β or \ ζ + Ν

<img file="RS51998B_D0053.tif" />

R is Η, 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, the two Rs optionally form heterocycloalkyl;

R 1 is optional, and when present is H, an amino protecting group, a resin; and R2 is optional, and when present, is OH, an ester protecting group, a resin; each of R3 and R₄ is independently H, halogen, lower alkyl, or substituted lower alkyl, or R₃ and R₄ or two R₃ the groups optionally form cycloalkyl or heterocycloalkyl;

or ABJR groups together form a bicyclic or tricyclic cycloalkyl or heterocycloalkyl containing at least one carbonyl group, including a dicarbonyl group, a protected carbonyl group, including a protected dicarbonyl group, or a masked carbonyl group, including a masked dicarbonyl group;

or the -JR group together forms a monocyclic or bicyclic cycloalkyl or heterocycloalkyl containing at least one carbonyl group, including a dicarbonyl group, a protected carbonyl group, including a protected dicarbonyl group, or a masked carbonyl group, including a masked dicarbonyl group.

10136] According to an example for the above purposes, the compounds of Formula (I) include compounds having the structure:

51998 Β

<img file="RS51998B_D0054.tif" />

where:

R is alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;

R 1 is optional, and when present, is H, an amino protecting group, a resin; i

R2 is optional, and when present, is OH, an ester protecting group, a resin; and each Ra is independently selected from the group consisting of H, halogen, alkyl, substituted alkyl, -N (R ') 2-C (O) kR' where k is 1, 2, or 3, -C (O) NR 1) 2, -OR ', and -S (O) r R', wherein each R 1 is independently H, alkyl, or substituted alkyl.

By way of example, for the aforementioned purposes, the compounds of Formula (I) include compounds having the structure:

<img file="RS51998B_D0055.tif" />

where;

A is optional, and when lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocylenkylene, substituted heteroalkylene, alkyloxyalkylene, a substituted, substituted heteroarylene, alkarylene , substituted alkarylene, aralkylene, or substituted aralkylene;

51998 Β

R is alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;

R 1 is optional, and when present, is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide;

R 2 is optional, and when present, is OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide;

X 1 is 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 a further example, for the above purposes, the compounds according to

Formulas (I) include compounds having the structure according to Formula ():

<img file="RS51998B_D0056.tif" />

R₃

<img file="RS51998B_D0057.tif" />

ο (HHHH), where:

A is optional, and when lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocylenkylene, substituted heteroalkylene, alkyloxyalkylene, a substituted, substituted heteroarylene, alkarylene , substituted alkarylene, aralkylene, or substituted aralkylene;

Mje-C (R₃)-&gt;

(»1 _R

II _s___s_|_ad(a) LR «R.

Φ)

<img file="RS51998B_D0058.tif" />

φ)

<img file="RS51998B_D0059.tif" />

φ) llg

<img file="RS51998B_D0060.tif" />

51998 Β

<td>(b) |</td><td>Rj</td><td>(b) I</td><td></td><td>/ X</td><td>(b)</td><td> /\</td>

<td> 1</td><td>/ s</td><td></td><td rowspan="2">(b) O-</td><td>—C - ί</td><td>: m s-</td><td>—C—? (b)</td>

<td></td><td>c— | (b) X</td><td></td><td>1 's / VV'</td><td></td><td> 1 ^ς«LLR</td>

<td></td><td>(a)</td><td>(a)</td><td></td><td>(a)</td><td>, or</td><td>(a)</td>

where (a) denotes attachment to group A and (b) denotes attachment to the corresponding carbonyl groups, R₃ and R₄ are independently selected from H, halogen, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl, or R₃ and R₄ or two R₃ groups or two R 1 groups optionally form cycloalkyl or heterocycloalkyl;

R is alkyl substituted alkyl, cycloalkyl, or substituted cycloalkyl;

T₃ is a bond, C (R) (R), O, or S, and R is H, halogen, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;

R 1 is optional, and when present, is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; i

R₂ is optional, and when present, is OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide.

The types of polypeptides containing such non-natural amino acids containing carbonyl or dicarbonyl are practically unlimited as long as the non-natural amino acid containing carbonyl or dicarbonyl is present on the polypeptide so that the hydroxylamine reagent can react with the carbonyl or a dicarbonyl group and does not create the resulting modified non-natural amino acid that destroys the tertiary structure of the polypeptide (with the exception, of course, where such destruction is the purpose of the reaction).

By way of example, the following hydroxylamine-containing reagents are of the hydroxylamine-containing reagent type that are reacted with the carbonyl- or dicarbonyl-containing non-natural amino acids described herein to give a non-natural oxime-containing amino acid in the presence described herein. accelerator (although such a reaction may be less effective in the absence of the accelerator described here):

51998 Β

W (XIX) where:

each X is independently H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, alkylalkoxy, substituted alkylalkoxy, polyalkylene oxide, substituted polyalkylene oxide, aryl, substituted aryl, heteroaryl, substituted a substituted alkyl , substituted aralkyl, - (alkylene or substituted alkylene) -ON (R) 2, - (alkylene or substituted alkylene) -C (O) SR, - (alkylene or substituted alkylene) SS- (aryl or substituted aryl), -C (O) R, -C (O) 2R, or -C (O) N (R) 2, each R being independently hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, alkaryl, substituted alkaryl, aralkyl, or substituted aralkyl;

or each X is independently selected from the group consisting of the desired functionality; each L is independently selected from the group consisting of alkylene, substituted alkylene, alkenylene, substituted alkenylene, -O-, -O- (alkylene or substituted alkylene) -, -S-, -S- (alkylene or substituted alkylene) -, - S (O) k- where k is 1,2, or 3, S (O) k (alkylene or substituted alkylene) -, -C (O) -, -C (O) - (alkylene or substituted alkylene) - , -C (S) -, -C (S) - (alkylene or substituted alkylene) -, -N (R ') -, -NR' - (alkylene or substituted alkylene) -, -C (O) N ') -, -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 -, - N (R') CO) O- alkylene or substituted alkylene, -S (O)_kN (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)_kN (R ') -, -N (R') - N =, -C (R ') = N-, -C (R') = NN (R ') -, -C (R') = NN = , -C (R ')₂-N = N-, and -C (R ')₂-N (R ') - N (R') -;

Li is arbitrary, and when present is -C (R ')_p-NR'-C (O) O- (alkylene or substituted alkylene) - wherein p is 0.1 or 2;

each R 1 is independently H, alkyl, or substituted alkyl;

W is N (R₈)₂, where each R 5 is independently H or an amino protecting group; and is 1 to 3; provided that the L-Li-W together give at least one hydroxylamine

51998Β a group that can react with a carbonyl group (including dicarbonyl) on a non-natural amino acid or a (modified) non-natural amino acid polypeptide.

In one illustrative embodiment, the hydroxylamine-derived reagent is added to a buffer solution (pH 2-8) of a carbonyl-containing non-natural amino acid polypeptide. The resulting non-natural amino acid polypeptide containing the oxime was purified by HPLC, FPLC or exclusion chromatography.

In a further or alternative illustrative embodiment, the molar ratio of the compound of Formula (I) to the compound of Formula (XIX) is about 1: 2; 1: 1; 1.5: 1; 1.5: 2; 2: 1; 1: 1.5; 2: 1.5; or 1.5 to2.

In one embodiment, multiple bond chemisms can be reacted at a specific position with a carbonyl- or dicarbonyl-substituted non-natural amino acid polypeptide to form an oxime bond in the presence of the accelerator described herein (although such a reaction may be less effective in the absence of accelerator). In one embodiment, the bonding methods described herein use linkers comprising hydroxylamine functionality at at least one bond termination (mono, bi- or multi-functional). Condensation of hydroxylamine-derived bonds with a ketosubstituted protein generates a stable oxime bond. Bi- and / or multi-functional linkers (eg, hydroxylamine with one or more, other binding chemicals) allow a compound at a specific position with different molecules (eg, with other proteins, polymers or small molecules) to a non-natural amino polypeptide acids, while mono-functional linkers (hydroxylamine-substituted at all ends) facilitate dimerization or oligomerization at a specific position of a non-natural amino acid polypeptide. By combining these binding strategies with the in vivo translation technology described herein, it becomes possible to specify three-dimensional structures of chemically-developed proteins.

B. Methods for post-translational modification of non-natural amino acid polypeptides in the presence of at least one accelerator: reactions of non-natural amino acids containing hydroxylamine with reagents containing carbonyl

51998 Post The post-translational modification techniques and compositions described above can also be used with non-natural amino acids containing hydroxylamine that react with reagents containing carbonyl or dicarbonyl reagents to obtain modified polypeptides of non-natural amino acids containing oxime in the presence of the accelerator described herein (although such a reaction may be less effective in the absence of the accelerator described herein).

The formation of a non-natural amino acid containing an oxime or non-natural amino acid polypeptide from a hydroxylamine-containing non-natural amino acid reaction or a hydroxylamine-containing non-natural amino acid polypeptide and a carbonyl-containing reagent can be improved by adding an accelerator to the reaction mixture. An accelerator is a compound having at least one of the following properties: (a) increasing the reaction rate between a carbonyl-containing compound and a hydroxylamine-containing compound while forming an oxime-containing compound, wherein the rate increase is relative to the reaction without an accelerator; (b) reducing the activation energy of the reaction between the carbonyl-containing compound and the hydroxylamine-containing compound while forming an oxime-containing compound, where the reduction in activation energy is relative to the accelerator-free reaction; (c) increasing the yield of the oxime-containing compound from the reaction of the carbonyl-containing compound with the hydroxylamine-containing compound, wherein the increase in yield is relative to the reaction without accelerator; (d) reducing the temperature at which the carbonyl-containing compound reacts with the hydroxylamine-containing compound to form an oxime-containing compound, where the temperature decrease is relative to the reaction without accelerator; (e) reducing the time required for the carbonyl-containing compound to react with the hydroxylamine-containing compound while forming an oxime-containing compound, where the reduction in time is relative to the accelerator-free reaction; (f) reducing the amount of reagent required to form the oxime group on the non-natural amino acid polypeptide, where the reduction in the amount of reagent is relative to the reaction without accelerator; (g) reducing foreign products resulting from the reaction of a carbonyl-containing compound with a hydroxylamine-containing compound while forming an oxime-containing compound, where the reduction of foreign products is relative to the reaction without accelerator; (h) does not irreversibly destroy the tertiary structure of the polypeptide that is subjected to the oxime formation reaction in the presence of an accelerator (except, of course, where the purpose of the reaction is to destroy such a tertiary structure); (i)

51998Β may be separated in vacuo from the oxime-containing compound; and (j) modulates the reaction of a carbonyl-containing compound with a hydroxylamine-containing compound. In further embodiments, the accelerator has at least two of the aforementioned properties, three of the aforementioned properties, four of the aforementioned properties, five of the aforementioned properties, six of the aforementioned properties, seven of the aforementioned properties, eight of the aforementioned properties , nine of the aforementioned properties. properties. In a further embodiment, the accelerator does not have any of the aforementioned properties.

The use of accelerators includes the use of single accelerators or multiple accelerators. Additionally, the molar ratio of accelerator to carbonyl-containing compound includes values ​​between about 0.5: 1 to 5000: 1, including by way of example only 4000: 1, 3000: 1, 2000: 1, 1000: 1, 500: 1, 400: 1, 300: 1, 200: 1, 100: 1, 50: 1, 40: 1, 30: 1, 20: 1, 10: 1, 9: 1, 8: 1, 7: 1, 6: 1, 5: 1, 4: 1, 3: 1, 2: 1, 1: 1, 0.9: 1, 0.8: 1, 0.7: 1, 0.6: 1, and 0.5: 1. Furthermore, the molar ratio of accelerator to hydroxylamine-containing compound includes values ​​between about 0.5: 1 to 5000: 1, including by way of example only 4000: 1.3000: 1, 2000: 1, 1000: 1, 500: 1, 400: 1, 300: 1, 200: 1, 100: 1, 50: 1, 40: 1, 30: 1, 20: 1, 10: 1, 9: 1, 8: 1, 7: 1, 6: 1, 5: 1.4: 1, 3: 1, 2: 1, 1: 1, 0.9: 1, 0.8: 1, 0.7: 1, 0.6: 1, and 0.5: 1. Furthermore, the accelerator includes compounds that can be substantially removed in vacuo from the resulting oxime-containing compound. Furthermore, the accelerator includes compounds containing a diamine moiety, a semi-carbazide moiety, a hydrazine, or a hydrazide moiety.

Furthermore, in any of the above aspects or representations, the accelerator is selected from the group consisting of bifunctional aromatic amines, oxoamine derivatives, and compounds having the following structures:

H ^g „A.-Γ rV

51998 Β

<img file="RS51998B_D0061.tif" />

<img file="RS51998B_D0062.tif" />

<img file="RS51998B_D0063.tif" />

where R_x, R_y and R₂ are selected from the group consisting of: L_x-H, L_x-alkyl, L_x-aryl, L_xheteroaryl, L_x-alkenyl, L_x-alkynyl, L_x-alkoxy, and L_x-alkylamine, where L_x is a bond, C (= O), C (= NH), C (= NH) -NH and SO, SO₂ where the aromatic amine is selected from the group:

Bifunctional aromatic amines:

<img file="RS51998B_D0064.tif" />

<img file="RS51998B_D0065.tif" />

<img file="RS51998B_D0066.tif" />

and where the oxoamine derivative is selected from the group:

Oxoamine derivatives:

<img file="RS51998B_D0067.tif" />

Furthermore, the accelerator includes compounds selected from the group consisting of:

XX 0 „J r where R_x, R_y and R_z are selected from the group consisting of: L_x-H, L_x-alkyl, L_x-aryl, L_xheteroaryl, L_x-alkenyl, L_x-alkynyl, L_x-alkoxy, and L_x-alkylamine, where L_x is a bond, C (= O), C (= NH), and C (= NH) -NH. Furthermore, in any of the above aspects or illustrations, the accelerator is selected from the compounds shown in Figure 5, Figure 9, or Figure 10, including as an example any of the compounds 6, 8, 10, 7 and 20 of Figure 5 .In any which of the following

51998 Β aspects or representations, the accelerator includes an agent that can form a hydrazone after reaction with a carbonyl-containing group. Furthermore, in any of the above aspects, the activity of the accelerator depends on the rate of reaction with the ketone moiety and the stability of the resulting intermediate. Furthermore, in any of the above aspects or illustrations, the pH of the reaction mixture containing the accelerator, the carbonyl-containing compound and the hydroxylamine-containing compound is between about 2.0 and 10; between about 2.0 and 9.0; between about 2.0 and 8.0; between about 3.0 and 7.0; between about 4.0 and 6.0; between about 3.0 and 10.0; between about 4.0 and 10.0; between about 3.0 and 9.0; between about 3.0 and 8.0; between about 2.0 and 7.0; between about 3.0 and 6.0; between about 4.0 and 9.0; between about 4.0 and 8.0; between about 4.0 and 7.0; between about 4.0 and 6.5; between about 4.5 and 6.5; about 4.0; about 4.5; about 5.0; about 5.5; about 6.0; about 6.5; and about 7.0.

The method of protein derivation based on the reaction of a hydroxylamine-containing protein with a carbonyl- or dicarbonyl-substituted molecule has various advantages. First, the hydroxylamines are subjected to condensation with compounds containing carbonyl- or di-carbonyl at a pH between about 2 to 8 (in further embodiments at a pH between about 4 to 8) to generate oxime adducts. Under these conditions, the side chains of natural amino acids are unreactive. Second, such selective chemistry allows derivatization of recombinant proteins at a specific position: derived proteins can now be obtained as defined homogeneous products. Third, the mild conditions required for the action of the carbonyl- or dicarbonyl-containing reagents described herein with the hydroxylamine-containing polypeptides described herein generally do not irreversibly destroy the tertiary structure of the polypeptide (unless, of course, the purpose of the reaction is to destroy such tertiary structure). Finally, although the hydroxylamine amino group appears to be metabolized by E. coli, condensation of carbonylyl-dicarbonyl-containing reagents with hydroxylamine-containing amino acids generates oxime adducts that are stable under biological conditions.

By way of example, the following non-natural amino acids are of the hydroxylamine-containing amino acid type that are reactive with the carbonyl- or dicarbonyl-containing reagents described herein to give a non-natural amino acid or oxime-containing polypeptide in the presence herein of the accelerator described (although such a reaction may be less effective in the absence of the accelerator described here):

51998 Β

<img file="RS51998B_D0068.tif" />

where:

A is optional, and when lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocylenkylene, substituted heteroalkylene, alkyloxyalkylene, a substituted, substituted heteroarylene, alkarylene , substituted alkarylene, aralkylene, or substituted aralkylene;

B is optional, and when present the linker is selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O- (alkylene or substituted alkylene) -, -S-, -S- (alkylene or substituted alkylene) -, -S (O) k- where k is 1,2, or 3, -S (O) k (alkylene or substituted alkylene) -, -C ) -, -C (O) - (alkylene or substituted alkylene) -, -C (S) -, -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) kN (R') - , -N (R ') C (O) N (R') -, -N (R ') C (S) N (R') ~, -N (R ') S (O)_kN (R ') -, -N (R') - N =, -C (R ') = N-, C (R') = NN (R ') -, -C (R') = NN =, -C (R ')₂-N = N-, and -C (R ')₂-N (R ') - N (R&gt;, wherein each R'is independently H, alkyl, or substituted alkyl;

KjeNH₂;

R 1 is optional, and when present, is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide; i

R₂ is optional, and when present, is OH, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide;

each of R₃ and R 4 is independently H, halogen, lower alkyl, or substituted lower alkyl, or R₃ and R4 or two R₃ the groups optionally form cycloalkyl or

51998 Β heterocycloalkyl.

The types of polypeptides containing such non-natural amino acids containing hydroxylamine are practically unlimited as long as the non-natural amino acid containing hydroxylamine is present on the polypeptide so that the carbonyl or dicarbonyl reagent can react with the hydroxylamine group and does not create the resulting modified no. -a natural amino acid that destroys the tertiary structure of the polypeptide (with the exception, of course, where such destruction is the purpose of the reaction).

By way of example, the following carbonyl or dicarbonyl-containing reagents are carbonyl or dicarbonyl-containing reagent species that react with the hydroxylamine-containing unnatural amino acids described herein to give a non-natural amino acid or oxime-containing polypeptide in the presence of the accelerator described herein. (although such a reaction may be less effective in the absence of the accelerator described here):

h - l] - c - w ^Jp (XIX) where:

each X is independently H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, alkylalkoxy, substituted alkylalkoxy, polyalkylene oxide, substituted polyalkylene oxide, aryl, substituted aryl, heteroaryl, substituted a substituted alkyl , substituted aralkyl, - (alkylene or substituted alkylene) -ON (R)₂, - (alkylene or substituted alkylene) -C (O) SR, - (alkylene or substituted alkylene) SS- (aryl or substituted aryl), -C (O) R, -C (O) 2R, or -C) N (R)₂, wherein each R is independently hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, alkaryl, substituted alkaryl, aralkyl, or substituted aralkyl;

or each X is independently selected from the group consisting of the desired functionality; each L is independently selected from the group consisting of alkylene, substituted alkylene, alkenylene, substituted alkenylene, -O-, -O- (alkylene or substituted alkylene) -, -S-, -S- (alkylene or substituted alkylene) -, - S (O) k- where k is 1,2, or 3, 84

51998 Β

S (O) k (alkylene or substituted alkylene) -, -C (O) -, -C (O) - (alkylene or substituted alkylene) -, -C (S) -, -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 -, - N (R') C (O) O- (alkylene or substituted alkylene) -, -S (O)_kN (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)_kN (R ') -, -N (R') - N =, -C (R ') = N-, -C (R') = NN (R ') -, -C (R') = NN = , -C (R ') 2-N = N-, and -C (R') 2-N (R ') - N (R') -;

Li is arbitrary, and when present, is -C (R ')_p-NR'-C (O) O- (alkylene or substituted alkylene) - wherein p is 0.1, or 2;

each R 1 is independently H, alkyl, or substituted alkyl;

W is -JR, where

<img file="RS51998B_D0069.tif" />

<img file="RS51998B_D0070.tif" />

<img file="RS51998B_D0071.tif" />

<img file="RS51998B_D0072.tif" />

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, the two Rs optionally form heterocycloalkyl; and n is 1 to 3;

provided that the L-Li-W together provide at least one carbonyl group (including a dicarbonyl group) that can react with a hydroxylamine group on a non-natural amino acid or a (modified) non-natural amino acid polypeptide.

51998 In one illustrative embodiment, the hydroxylamine-derived reagent is added to a buffer solution (pH 2-8) of a non-natural amino acid polypeptide comprising a hydroxylamine and an accelerator. The resulting non-natural amino acid polypeptide containing the oxime was purified by HPLC, FPLC or exclusion chromatography.

In a further or alternative illustrative embodiment, the molar ratio of the compound of Formula (XIV) to the compound of Formula (XIX) is about 1: 2; 1: 1; 1.5: 1; 1.5: 2; 2: 1; 1: 1.5; 2: 1.5; or 1.5 to 2.

In one embodiment, multiple bond chemisms can react at a specific position with a hydroxylamine-substituted non-natural amino acid polypeptide. In one embodiment, the bonding methods described herein use linkers comprising carbonyl or dicarbonyl functionality at at least one bond termination (mono, bi- or multi-functional). Condensation of carbonyl- or dicarbonyl-derived bonds with a hydroxylamine-substituted protein generates a stable oxime bond. Bi- and / or multi-functional linkers (eg, carbonyl or dicarbonyl with one or more, other binding chemicals) allow a compound at a specific position with different molecules (eg, with other proteins, polymers or small molecules) to a non- polypeptide. natural amino acids, while mono-functional linkers (carbonyl- or dicarbonyl-substituted at all ends) facilitate dimerization or oligomerization at a specific position of a non-natural amino acid polypeptide. By combining these binding strategies with the in vivo translation technology described herein, it becomes possible to specify three-dimensional structures of chemically digested proteins.

C. Example of Adding Functionality in the Presence of at least One Accelerator: Macromolecular Polymers Paired with Non-Natural Amino Acid Polypeptides Various modifications of the non-natural amino acid polypeptides described herein can be performed using the compositions, methods, techniques, and strategies described herein. These modifications include the insertion of further functionality on a non-natural amino acid polypeptide component via an oxime bond obtained in the presence of

51998 Β the accelerator described herein (although such a reaction may be less effective in the absence of the accelerator described herein), including but not limited to, the desired functionality. As an illustrative example of the compositions, methods, techniques, and strategies described herein, the following description will focus on adding macromolecular polymers to a non-natural amino acid polypeptide with the understanding that the compositions, methods, techniques, and strategies described are also applicable (with appropriate modifications if necessary, and which one skilled in the art can do with the data provided herein) to add other functionalities, including those listed above.

Formation of macromolecular polymers paired via an oxime bond with a non-natural amino acid polypeptide from (a) reacting a hydroxylamine-containing non-natural amino acid polypeptide and a carbonyl-containing reagent, or (b) reacting a non-natural amino acid -containing polypeptide carbonyl and a hydroxylamine-containing reagent can be improved by adding an accelerator to the reaction mixture. Such an accelerator is a compound having at least one of the following properties: (a) increasing the reaction rate between the carbonyl-containing compound and the hydroxylamine-containing compound while forming an oxime-containing compound, where the rate increase is relative to the accelerator-free reaction; (b) reducing the activation energy of the reaction between the carbonyl-containing compound and the hydroxylamine-containing compound while forming an oxime-containing compound, where the reduction in activation energy is relative to the accelerator-free reaction; (c) increasing the yield of the oxime-containing compound from the reaction of the carbonyl-containing compound with the hydroxylamine-containing compound, wherein the increase in yield is relative to the reaction without accelerator; (d) reducing the temperature at which the carbonyl-containing compound reacts with the hydroxylamine-containing compound to form an oxime-containing compound, where the temperature decrease is relative to the reaction without accelerator; (e) reducing the time required for the carbonyl-containing compound to react with the hydroxylamine-containing compound while forming an oxime-containing compound, where the reduction in time is relative to the accelerator-free reaction; (f) reducing the amount of reagent required to form the oxime group on the non-natural amino acid polypeptide, where the reduction in the amount of reagent is relative to the reaction without accelerator; (g) reducing foreign products resulting from the reaction of a carbonyl-containing compound with a hydroxylamine-containing compound while forming an oxime-containing compound, where the reduction of foreign products is relative to the reaction without accelerator; (h) does not irreversibly destroy the tertiary

51998 Β the structure of a polypeptide subjected to an oxime formation reaction in the presence of an accelerator (except, of course, where the purpose of the reaction is to destroy such a tertiary structure); (i) may be separated in vacuo from the oxime-containing compound; and (j) modulates the reaction of a carbonyl-containing compound with a hydroxylamine-containing compound. In further embodiments, the accelerator has at least two of the aforementioned properties, three of the aforementioned properties, four of the aforementioned properties, five of the aforementioned properties, six of the aforementioned properties, seven of the aforementioned properties, eight of the above listed properties. In a further embodiment, the accelerator does not have any of the aforementioned properties.

The use of an accelerator includes the use of a single accelerator or multiple accelerators. Additionally, the molar ratio of accelerator to carbonyl-containing compound includes values ​​between about 0.5: 1 to 5000: 1, including by way of example only 4000: 1, 3000: 1, 2000: 1, 1000: 1, 500: 1, 400: 1, 300: 1, 200: 1, 100: 1, 50: 1, 40: 1, 30: 1, 20: 1, 10: 1, 9: 1, 8: 1, 7: 1, 6: 1, 5: 1, 4: 1, 3: 1, 2: 1, 1: 1, 0.9: 1, 0.8: 1, 0.7: 1, 0.6: 1, and 0.5: 1. Furthermore, the molar ratio of accelerator to hydroxylamine-containing compound includes values ​​between about 0.5: 1 to 5000: 1, including by way of example only 4000: 1, 3000: 1, 2000: 1, 1000: 1, 500: 1, 400: 1, 300: 1, 200: 1, 100: 1, 50: 1, 40: 1, 30: 1, 20: 1, 10: 1, 9: 1, 8: 1, 7: 1, 6: 1, 5: 1, 4: 1, 3: 1; 2: 1, 1: 1,0.9: 1, 0.8: 1, 0.7: 1, 0.6: 1, and 0.5: 1. Furthermore, the accelerator includes compounds that can be substantially removed in vacuo from the resulting oxime-containing compound. Furthermore, the accelerator includes compounds containing a diamine moiety, a semi-carbazide moiety, a hydrazine, or a hydrazide moiety.

Furthermore, in any of the above aspects or representations, the accelerator is selected from the group consisting of bifunctional aromatic amines, oxoamine derivatives, and compounds having the following structures:

I ^RxvN^N'R_t n

51998 Β

<img file="RS51998B_D0073.tif" />

Η

<img file="RS51998B_D0074.tif" />

<img file="RS51998B_D0075.tif" />

ο

<img file="RS51998B_D0076.tif" />

where R_x, R_y and R₂ are selected from the group consisting of: L_x-H, L_x-alkyl, L_x-aryl, L_x heteroaryl, L_x-alkenyl, L_x-alkynyl, L_x-alkoxy, and L_x-alkylamine, where L_x is a bond, C (= O), C (= NH), C (= NII) -NH and SO, SO₂, where the aromatic amine is selected from the group:

Bifunctional aromatic amines:

<img file="RS51998B_D0077.tif" />

<img file="RS51998B_D0078.tif" />

<img file="RS51998B_D0079.tif" />

and where the oxoamine derivative is selected from the group:

Oxoamine derivatives:

<img file="RS51998B_D0080.tif" />

<img file="RS51998B_D0081.tif" />

<img file="RS51998B_D0082.tif" />

<img file="RS51998B_D0083.tif" />

<img file="RS51998B_D0084.tif" />

<img file="RS51998B_D0085.tif" />

Furthermore, the accelerator includes compounds selected from the group consisting of:

»„ Ju ',,, jy _H, A. „0;, where R_x, R_y and R_z are selected from the group consisting of: L_x-H, L_x-alkyl, L_x-aryl, L_xheteroaryl, L_x-alkenyl, L_x-alkynyl, L_x-alkoxy, and L_x alkylamine, where L_x is a bond, C (= O), C

51998 Β (= ΝΗ), and C (= NH) -NH. Furthermore, in any of the above aspects or illustrations, the accelerator is selected from the compounds shown in Figure 5, Figure 9, or Figure 10, including as an example any of the compounds 6, 8, 10, 7 and 20 of Figure 5 In any of the above aspects or representations, the accelerator includes an agent that can form a hydrazone after reaction with a carbonyl-containing group. Furthermore, in any of the above aspects, the activity of the accelerator depends on the rate of reaction with the ketone moiety and the stability of the resulting intermediate. Furthermore, in any of the above aspects or illustrations, the pH of the reaction mixture containing the accelerator, the carbonyl-containing compound and the hydroxylamine-containing compound is between about 2.0 and 10; between about 2.0 and 9.0; between about 2.0 and 8.0; between about 3.0 and 7.0; between about 4.0 and 6.0; between about 3.0 and 10.0; between about 4.0 and 10.0; between about 3.0 and 9.0; between about 3.0 and 8.0; between about 2.0 and 7.0; between about 3.0 and 6.0; between about 4.0 and 9.0; between about 4.0 and 8.0; between about 4.0 and 7.0; between about 4.0 and 6.5; between about 4.5 and 6.5; about 4.0; about 4.5; about 5.0; about 5.5; about 6.0; about 6.5; and about 7.0.

A wide variety of macromolecule polymers and other molecules described herein can be paired with non-natural amino acid polypeptides to modulate the biological properties of a non-natural amino acid polypeptide (or a corresponding natural amino acid polypeptide), and / or confer new biological properties on the non-natural amino acid polypeptide. -natural amino acids (or the corresponding natural amino acid polypeptide). These macromolecule polymers can be coupled to a non-natural amino acid polypeptide via an oxime bond on a non-natural amino acid.

The water-soluble polymer can be paired with the non-natural amino acid polypeptides described herein. The water-soluble polymer can be paired with a non-natural amino acid with an oxime bond. In some cases, the non-natural amino acid polypeptides described herein comprise one or more non-natural amino acids bound to a water-soluble polymer and one or more naturally occurring amino acids bound to a water-soluble polymer. Covalent binding of hydrophilic polymers to a biologically active molecule is an approach to increase water solubility (as in the physiological environment), bioavailability, increase serum half-life, increase therapeutic half-life, modulate immunogenicity, modulate biological activity, or extend circulation time of a biologically active molecule, including proteins ,,

51998 Β peptides, and especially hydrophobic molecules. Additional important features for such hydrophilic polymers include biocompatibility, lack of toxicity, and lack of immunogenicity. Preferably, for the therapeutic use of the final product composition, the polymer will be pharmaceutically acceptable.

Examples of suitable hydrophilic polymers include: polyalkyl ethers and their alkoxy-coated analogs (eg, polyoxyethylene glycol, polyoxyethylene / propylene glycol, and their methoxy or ethoxy-coated analogs, especially polyoxyethylene glycol, the latter also known as polyethylene glycol PEG); polyvinylpyrrolidone; polyvinylalkyl ether; polyoxazolines, polyalkyl oxazolines and polyhydroxyalkyl oxazolines; polyacrylamides, polyalkyl acrylamides, and polyhydroxyalkyl acrylamides (eg, polyhydroxypropylmethacrylamides and their derivatives); polyhydroxyalkyl acrylate; polysialic acids and their analogues; hydrophilic peptide sequences; polysaccharides and their derivatives, including dextran and dextran derivatives, eg, carboxymethyldextran, dextran sulfate, aminodextran; cellulose and its derivatives, eg, carboxymethyl cellulose, hydroxyalkyl cellulose; chitin and its derivatives, eg, chitosan, succinyl chitosan, carboxymethylkitin, carboxymethylkitosan; hyaluronic acid and its derivatives; starch; alginates; chondroitin sulfate; albumin; pullulan and carboxymethyl pullulan; polyamino acids and their derivatives, eg, polyglutamic acids, polylysines, polyaspartic acids, polyaspartamides; maleic anhydrous copolymers such as: styrene maleic anhydride copolymer, divinylethyl ether maleic anhydride copolymer; polyvinyl alcohol; their copolymers; terpolymers thereof; mixtures thereof; and derivatives for the aforesaid. The water-soluble polymer may be of any structural form including but not limited to linear, branched or branched. In some embodiments of the invention, water-soluble backbone polymers with from 2 to about 300 terminations are particularly useful. Multifunctional polymer derivatives include, but are not limited to, lineame polymers having two endings, each ending being attached to functional groups that may be the same or different. In some embodiments of the invention, the aqueous polymer comprises a poly (ethylene glycol) moiety. The molecular weight of the polymer can be in a wide range, including but not limited to, between about 100 Da and about 100,000 Da or more. Molecules of polymer weight can be between about 100 Da and about 100,000 Da, including, 100,000 Yes, 95,000 Yes, 90,000 Yes, 85,000 Yes, 80,000 Yes, 75,000 Yes, 70,000 Yes, 65,000 Yes, 60,000 Yes, 55,000 Yes, 50,000 Yes,

51998 Β

45,000 Yes, 40,000 Yes, 35,000 Yes, 30,000 Yes, 25,000 Yes, 20,000 Yes, 15,000 Yes, 10,000 Yes, 9,000 Yes, 8,000 Yes, 7,000 Yes, 6,000 Yes, 5,000 Yes, 4,000 Yes, 3,000 Yes, 2,000 Yes, 1,000 Yes , 900 Yes, 800 Yes, 700 Yes, 600 Yes, 500 Yes, 400 Yes, 300 Yes, 200 Yes, and 100 Yes. In some embodiments of the invention, the molecular weight of the polymer is between about 100 Da and about 50,000 Da. In some embodiments of the invention, the molecular weight of the polymer is between about 100 Da and about 40,000 Da. In some embodiments of the invention, the molecular weight of the polymer is between about 1,000 Da and about 40,000 Da. In some embodiments of the invention, the molecular weight of the polymer is between about 5,000 Da and about 40,000 Da. In some embodiments of the invention, the molecular weight of the polymer is between about 10,000 Da and about 40,000 Da. In some embodiments of the invention, the poly (ethylene glycol) molecule is a branched polymer. The molecular weight of a branched PEG chain can be between about 1,000 Da and about 100,000 Da, including but not limited to, 100,000 Yes, 95,000 Yes, 90,000 Yes, 85,000 Yes, 80,000 Yes, 75,000 Yes, 70,000 Yes, 65,000 Yes, 60,000 Yes, 55,000 Yes, 50,000 Yes, 45,000 Yes, 40,000 Yes, 35,000 Yes, 30,000 Yes, 25,000 Yes, 20,000 Yes, 15,000 Yes, 10,000 Yes, 9,000 Yes, 8,000 Yes, 7,000 Yes, 6,000 Yes, 5,000 Yes, 4,000 Yes, 3,000 Yes, 2,000 Yes, and 1,000 Yes. In some embodiments of the invention, the weight of the branched PEG chain molecules is between about 1,000 Da and about 50,000 Da. In some embodiments of the invention, the weight of the branched PEG chain molecules is between about 1,000 Da and about 40,000 Da. In some embodiments of the invention, the weight of the branched PEG chain molecules is between about 5,000 Da and about 40,000 Da. In some embodiments of the invention, the weight of the branched PEG chain molecules is between about 5,000 Da and about 20,000 Da. Those skilled in the art will recognize that the foregoing list for backbones that are substantially soluble in water is by no means exhaustive and is only illustrative, and that all polymeric materials having the qualities described above may be considered suitable for use in the processes described herein . and preparations.

As described above, one example of a hydrophilic polymer is poly (ethylene glycol), abbreviated PEG, which is widely used in pharmaceuticals, in artificial implants, and for other applications where biocompatibility, lack of toxicity, and lack of immunogenicity are important. . The polymer: polypeptide embodiments described herein will use PEG as an example of a hydrophilic polymer with the understanding that other hydrophilic polymers may be similarly used in such embodiments.

51998 EG PEG is a well-known, water-soluble polymer that is commercially available or can be obtained by opening-ring polymerization of ethylene glycol according to methods well known in the art (Sandler and Karo, Polymer Synlhesis, Academic Press, New York, Vol 3, pp. 138-161). PEG is typically pure, colorless, odorless, water-soluble, heat-resistant, inert to many chemicals, does not hydrolyze or decay, and is generally non-toxic. Poly (ethylene glycol) is considered to be biocompatible, which means that PEG is able to coexist with living tissues or organisms without causing harm. More specifically, PEG is essentially non-immunogenic, implying that PEG does not tend to produce an immune response in the body.

When attached to a molecule that has some desirable function in the body, such as biologically active agents, PEG tends to mask that agent and can reduce or eliminate any immune response so that the body can tolerate the presence of that agent. PEG conjugates tend not to produce a significant immune response or cause clotting or other adverse effects.

The term PEG is widely used to encompass all polyethylene glycol molecules, regardless of size or modification at the PEG termination, and can be represented as bound to a non-natural amino acid polypeptide of the formula:

XO- (CH₂CH₂O) n-CH₂CH2-Y where n is 2 to 10,000 and X is H or a modification of the termination, including but not limited to, C 1-4 alkyl, a protecting group, or a terminal functional group. The term PEG includes, but is not limited to, poly (ethylene glycol) in any of its forms, including bifunctional PEG, multi-armed PEG, derived PEG, branched PEG, branched PEG (with each chain having a molecular weight of about 1 kDa up to about 100 kDa, from about 1 kDa to about 50 kDa, or from about 1 kDa to about 20 kDa), suspended PEG (ie, PEG or related polymers having one or more functional groups suspended on a polymer backbone), or PEG with degrading bonds. In one embodiment, the PEG where n is from about 20 to about 2000 is suitable for use in the methods and compounds described herein. In some embodiments of the invention, aqueous

51998 Β The polymer contains a portion of poly (ethylene glycol). The molecular weight of the polymer can be in a wide range, including but not limited to, between about 100 Da and about 100,000 Da or more. Molecules of polymer weight can be between 100 Yes and 100,000 Yes, including 100,000 Yes, 95,000 Yes, 90,000 Yes, 85,000 Yes, 80,000 Yes, 75,000 Yes, 70,000 Yes, 65,000 Yes, 60,000 Yes, 55,000 Yes, 50,000 Yes, 45,000 Yes, 40,000 Yes, 35,000 Yes, 30,000 Yes, 25,000 Yes, 20,000 Yes, 15,000 Yes, 10,000 Yes, 9,000 Yes, 8,000 Yes, 7,000 Yes, 6,000 Yes, 5,000 Yes, 4,000 Yes, 3,000 Yes, 2,000 Yes, 1,000 Yes, 900 Yes , 800 Yes, 700 Yes, 600 Yes, 500 Yes, 400 Yes, 300 Yes, 200 Yes, and 100 Yes. In some embodiments of the invention, the weight of the polymer molecules is between 100 Da and about 50,000 Da. In some embodiments of the invention, the molecular weight of the polymer is between about 100 Da and about 40,000 Da. In some embodiments of the invention, the molecular weight of the polymer is between about 1,000 Da and about 40,000 Da. In some embodiments of the invention, the molecular weight of the polymer is between about 5,000 Da and about 40,000 Da. In some embodiments of the invention, the molecular weight of the polymer is between about 10,000 Da and about 40,000 Da. In some embodiments of the invention, the poly (ethylene glycol) molecule is a branched polymer. The molecular weight of a branched PEG chain can be between about 1,000 Da and 100,000 Yes, including 100,000 Yes, 95,000 Yes, 90,000 Yes, 85,000 Yes, 80,000 Yes, 75,000 Yes, 70,000 Yes, 65,000 Yes, 60,000 Yes, 55,000 Yes, 50,000 Yes, 45,000 Yes, 40,000 Yes, 35,000 Yes, 30,000 Yes, 25,000 Yes, 20,000 Yes, 15,000 Yes, 10,000 Yes, 9,000 Yes, 8,000 Yes, 7,000 Yes, 6,000 Yes, 5,000 Yes, 4,000 Yes, 3,000 Yes, 2,000 Yes, and 1,000 Yes. In some embodiments of the invention, the weight of the branched PEG chain molecules is between about 1,000 Da and about 50,000 Da. In some embodiments of the invention, the weight of the branched PEG chain molecules is between about 1,000 Da and about 40,000 Da. In some embodiments of the invention, the weight of the branched PEG chain molecules is between about 5,000 Da and about 40,000 Da. In some embodiments of the invention, the weight of the branched PEG chain molecules is between about 5,000 Da and about 20,000 Da. A wide range of PEG molecules are described in, including but not limited to, the Shearwater Polymers, Inc. catalog. and catalog Nectar Therapeutics.

Specific examples of terminal functional groups in the literature include Nsuccinimidyl carbonates (see, eg, U.S. Pat. No. 5,281,698, 5,468,478), amines (see, eg, Buckmann et al. Macromol. Chem. 182: 1379 (1981), Zalipsky et al. al. Eur. Polim. J. 19: 1177 (1983)), hydrazide (see, eg, Andresz et al. Macromol. Chem.

51998 Β

179: 301 (1978)), succinimidyl propionate and succinimidyl butanoate (see, eg, Olson et al. Poly (ethylene glycol) Chemistry &amp; Biological Applications, pp. 170-181, Harris &amp; Zalipsky Eds., ACS, Washington, DC, 1997; see also U.S. Pat. No. 5,672,662), succinimidyl succinate (see, eg, Abuchowski et al. Cancer Biochem. Biophys. 7: 175 (1984) and Joppich et al. Macromol. Chem. 180: 1381 (1979), succinimidyl ester (see, eg, US Pat. No. No. 4,670,417), benzotriazole carbonates (see, eg, U.S. Pat. No. 5,650,234), glycidyl ether (see, eg, Pitha et al., Eur. J Biochem. 94:11 (1979), Elling et al., Biotech. Appl. Biochem. 13: 354 (1991), oxycarbonylimidazole (see, eg, Beauchamp, et al., Anal. Biochem. 131: 25 (1983), Tondelli et al. J. Controlled Release 1: 251 (1985)), p. -nitrophenyl carbonate (see, eg, Veronese, et al., Appl. Biochem. Biotech., 11: 141 (1985); and Sartore et al., Appl. Biochem. Biolech., 27:45 (1991)), an aldehyde (see, eg, Harris et al. J. Polim. Sci. Chem. Ed. 22: 341 (1984), U.S. Pat. No. 5,824,784, U.S. Pat. No. 5,824,784). .5,824,784). . No. 5,252,714), maleimide (see, eg, Goodson et al. Bio / Technology 8: 343 (1990), Romani et al. Chemistry of Peptides and Proteins 2:29 (1984)), and Vogan, Synlhetic Comm. 22: 2417 (1992)), orthopyridyl disulfide (see, eg, Woghiren, et al. Bioconj. Chem. 4: 314 (1993)), acrylol (see, eg, Sawhney et al., Macromolecules, 26: 581 (1993)), vinylsulfone (see, eg, U.S. Pat. No. 5,900,461).

In some cases, PEG terminates at one end with hydroxy or methoxy, ie, X is H or SN 2 (methoxy PEG). Alternatively, the PEG may terminate with a reactive group, thus forming a bifunctional polymer. Typical reactive groups may include those reactive groups commonly used to react with functional groups found in 20 common amino acids (including maleimide groups, activated carbonates (including but not limited to, pnitrophenyl ester), activated esters (including N-hydroxysuccinimide) ,, p-nitrophenyl ester) and aldehydes) as well as groups that are inert to 20 common amino acids but that can specifically react with complementary functional groups found in non-natural amino acids to form an oxime group in the presence of the accelerator described herein although such a reaction may be less effective in the absence of the accelerator described herein); examples of the latter include, carbonyl or dicarbonyl and hydroxylamine groups.

51998 It has been observed that the dear PEG termination, shown in the above formula with Y, will bind either directly or indirectly to the polypeptide via a non-natural amino acid. When Y is a hydroxylamine group, then the hydroxylamine-containing PEG reagent can be reacted with a non-natural amino acid containing a carbonyl- or dicarbonyl- in the polypeptide to form a PEG group linked to the polypeptide via an oxime bond in the presence of the accelerator described herein (although such a reaction may be less efficient in the absence of the accelerator described here). When Y is a carbonyl or dicarbonyl group, then a carbonyl- or dicarbonyl-containing PEG reagent may be reacted with a hydroxylamine-containing non-natural amino acid in the polypeptide to form a polypeptide-linked PEG group via an oxime bond in the presence of the accelerator described herein (although such the reaction may be less efficient in the absence of the accelerator described herein).

Heterobifunctional derivatives are also particularly useful when it is desirable to link different molecules to each polymer termination. For example, omega-N-aminoN-azido PEG will allow attachment of a molecule having an activated electrophilic group, such as an aldehyde, ketone, activated ester, activated carbonate, and so on, to one PEG termination and a molecule having an acetylene group to another PEG termination.

In some embodiments of the invention, a strong nucleophile (including hydroxylamine) can be reacted with a carbonyl group, including ketone groups found in a non-natural amino acid to form an oxime in the presence of the accelerator described herein (although such a reaction may be less effective in the absence of described accelerator); the subsequent oxime group may in some cases be further reduced by treatment with an appropriate reducing agent. Alternatively, a strong nucleophile may be included in the polypeptide via a non-natural amino acid and may be used to preferably react with a carbonyl group, including ketone groups present in the water-soluble polymer to form an oxime in the presence of the accelerator. described herein (although such a reaction may be less effective in the absence of the accelerator described here). In general, at least one termination of the PEG molecule is available for reaction with a non-natural amino acid.

51998 01 The backbone polymer can be linear or branched. Branched backbone polymers are generally known in the art. Typically, the branched polymer has a portion of a central branching core and a plurality of linear polymer chains attached to the central branching core. PEG is used in branched forms which can be obtained by adding ethylene oxide to various polyols, such as glycerol, glycerol oligomers, pentaerythritol and sorbitol. The central part for branching can also be obtained from several amino acids, such as lysine. Branched poly (ethylene glycol) can be represented in general form as R (-PEG-OH)_m in which R is derived from a part of the nucleus, glycerol, glycerol oligomers, or pentaerythritol, represents the number of branches. Multiple branched PEG molecules, such as those described in US Pat. no. 5,932,462 5,643,575; 5,229,490; 4,289,872; US Pat. application 2003/0143596; WO 96/21469; and WO 93/21259, can also be used as backbone polymers.

The branched PEG may also be in the form of a branched PEG represented by PEG (-YCHZ2)._n, where Y is a linking group, n is 100-1,000 (ie, the average molecule weight is between about 5 kDa to about 40 kDa), and Z is an activated end group attached to the CH chain of atoms of defined length. Another branched form, suspended PEG, has reactive groups, such as carboxyl, along the PEG backbone instead of at the end of the PEG chains.

In order to increase the desired properties of PEG, the total molecular weight and hydration state of PEG polymers or polymers bound to the biologically active molecule must be high enough to give favorable characteristics typically associated with PEG polymer binding, such as increased water solubility and circulating half-life, until they adversely affect the bioactivity of the parent molecule.

The methods and compositions described herein can be used to prepare substantially homogeneous preparations of polymer protein conjugates. Essentially homogeneous as used herein means that the polymer-protein conjugate molecules are observed to be greater than half of the total protein. The polymeriprotein conjugate has biological activity, and the substantially homogeneous PEGylated polypeptide compositions described herein are those that are sufficiently homogeneous to have the advantage of a homogeneous composition, eg, ease of clinical application in predictability of pharmacokinetics.

51998 As used herein, and when considering a hydrophilic polymer: polypeptide / protein conjugates, the term therapeutically effective amount refers to an amount that is beneficial to the patient. The amount will vary from one individual to long and will depend on a number of factors, including the overall physical condition of the patient and the underlying cause of the disease or condition. A therapeutically effective amount of the subject compositions can be readily determined by those skilled in the art using publicly available materials and methods. By way of example only, a therapeutically effective amount may be an amount that increases the hematocrit in anemic patients; it may be an amount that reduces tumor size in cancer patients; it can be an amount that increases insulin levels in diabetics, it can be an amount that reduces pain in patients suffering from some form of chronic pain.

The number of water-soluble polymers bound to the (modified) non-natural amino acid polypeptide described herein (PEGylation or glycosylation range) can be adjusted to alter (including but not limited to, increase or decrease) pharmacological, pharmacokinetic, or pharmacodynamic characteristic as well as in vivo half-life. In some embodiments of the invention, the half-life of the polypeptide is increased by at least about 10, 20, 30, 40, 50, 60, 70, 80, 90 percent, double, fivefold, 10-fold, 50-fold , or at least about 100-fold in relative to the unmodified polypeptide.

In one embodiment, a carbonyl- or dicarbonyl-containing non-natural amino acid polypeptide is modified in the presence of an accelerator, with a PEG derivative containing a hydroxylamine terminal attached directly to the PEG backbone, to form an oxime bond (although such a reaction may be less effective in the absence of the accelerator described herein). In some embodiments of the invention, the hydroxylamine-terminated PEG derivative will have the following structure:

RO- (CH₂CH₂O) nO- (CH₂)_m-O-NH₂ where R is simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10 in is 100-1,000 (ie, the average molecular weight is between about 5 kDa to about 40 kDa). Molecules

51998 Β The weight of the polymer can be a wide range, including but not limited to, between about 100 Da and about 100,000 Da or more. The molecular weight of the polymer may be between about 100 Yes and about 100,000 Yes, including 100,000 Yes, 95,000 Yes, 90,000 Yes, 85,000 Yes, 80,000 Yes, 75,000 Yes, 70,000 Yes, 65,000 Yes, 60,000 Yes, 55,000 Yes, 50,000 Yes , 45,000 Yes, 40,000 Yes, 35,000 Yes, 30,000 Yes, 25,000 Yes, 20,000 Yes, 15,000 Yes, 10,000 Yes, 9,000 Yes, 8,000 Yes, 7,000 Yes, 6,000 Yes, 5,000 Yes, 4,000 Yes, 3,000 Yes, 2,000 Yes, 1,000 Yes, 900 Yes, 800 Yes, 700 Yes, 600 Yes, 500 Yes, 400 Yes, 300 Yes, 200 Yes, and 100 Yes. In some embodiments of the invention, the molecular weight of the polymer is between about 100 Da and about 50,000 Da. In some embodiments of the invention, the molecular weight of the polymer is between about 100 Da and about 40,000 Da. In some embodiments of the invention, the molecular weight of the polymer is between about 1,000 Da and about 40,000 Da. In some embodiments of the invention, the molecular weight of the polymer is between about 5,000 Da and about 40,000 Da. In some embodiments of the invention, the molecular weight of the polymer is between about 10,000 Daioko 40,000 Da.

In another embodiment, the carbonyl- or dicarbonyl-containing non-natural amino acid polypeptide is modified with a PEG derivative comprising a hydroxylamine terminal portion attached to the PEG backbone by an amide bond to form an oxime bond (although such a reaction may be less effective in the absence of the accelerator described herein) with further pairing to the PEG backbone by an amide bond. In some embodiments of the invention, PEG derivatives with a hydroxylamine-terminus have the following structure:

RO- (CH₂CH₂O) nO- (CH₂)₂-NH-C (O) (CH₂)_m-O-NH₂ where R is simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10 and n is 100-1,000 (ie, the average molecular weight is between about 5 kDa to about 40 kDA).

In another embodiment, the carbonyl- or dicarbonyl-containing non-natural amino acid polypeptide is modified with a branched PEG derivative comprising a hydroxylamine terminal portion to form an oxime bond (although such a reaction may be less effective in the absence of that described herein ). accelerator), with each branched PEG chain having an average molecular weight in the range

51998 Β from about 10 kDa to about 40 kDa, and in other embodiments, from about 5 kDa to about 20 kDa. In some embodiments of the invention, PEG derivatives containing a hydroxylamine group will have the following structure:

[RO- (CH₂CH2O)_n-O- (CH2) 2-C (O) -NH-CH2-CH2] 2CH-X- (CH2) mO-NH₂ where R is simple alkyl (methyl, ethyl, propyl, etc.), X is optionally NH, O, S, C (O) or is not present, m is 2-10 in is 100-1,000. Molecular weights of a branched PEG chain can be between about 1,000 Da and about 100,000 Yes, including 100,000 Yes, 95,000 Yes, 90,000 Yes, 85,000 Yes, 80,000 Yes, 75,000 Yes, 70,000 Yes, 65,000 Yes, 60,000 Yes, 55,000 Yes, 50,000 Yes, 45,000 Yes, 40,000 Yes, 35,000 Yes, 30,000 Yes, 25,000 Yes, 20,000 Yes, 15,000 Yes, 10,000 Yes, 9,000 Yes, 8,000 Yes, 7,000 Yes, 6,000 Yes, 5,000 Yes, 4,000 Yes, 3,000 Yes, 2,000 Yes, and 1,000 Yes. In some embodiments of the invention, the weight of the branched PEG chain molecules is between about 1,000 Da and about 50,000 Da. In some embodiments of the invention, the weight of the branched PEG chain molecules is between about 1,000 Da and about 40,000 Da. In some embodiments of the invention, the weight of the branched PEG chain molecules is between about 5,000 Da and about 40,000 Da. In some embodiments of the invention, the weight of the branched PEG chain molecules is between about 5,000 Da and about 20,000 Da.

Several reviews and monographs on functionalization and conjugation for PEG are available. See, for example, Harris, Macromol. Chem. Phys. C25: 325373 (1985); Scouten, Methods in Enzymology 135: 30-65 (1987); Wong et al., Epgute Microb. Technol. 14: 866-874 (1992); Delgado et al., Critical Reviews in Therapeutic Drug Carrier Systems 9: 249-304 (1992); Zalipsky, Bioconjugale Chem. 6: 150-165 (1995). Methods for activating polymers can also be found in WO 94/17039, US Pat. no. No. No. 5,324,844, WO 94/18247, WO 94/04193, U.S. Pat. no. No. No. 5,219,564, U.S. Pat. no. No. No. 5,122,614, WO 90/13540, U.S. Pat. no. No. No. 5,281,698, further to WO 93/15189, as well as for conjugation between activated polymers and enzymes including but not limited to coagulation factor VIII (WO 94/15625), hemoglobin (WO 94/09027), oxygen-carrying molecules (US Pat. No. 4,412,989), ribonucleases and superoxide dismutase (Veronese et al., App. Biochem. Biotech. 11: 141-52 (1985)).

100

51998 If necessary, the PEGylated non-natural amino acid polypeptide described herein obtained by hydrophobic chromatography may be further purified by one or more methods known to those skilled in the art including, but not limited to, affinity chromatography; anion- or cation-exchange chromatography (using, including but not limited to, DEAE SEPHAROSE); silicon chromatography; Reverse phase HPLC; gel filtration (using, including but not limited to, SEPHADEX G-75); hydrophobic interaction chromatography; exclusion chromatography, metal chelate chromatography; ultrafiltration / diafiltration; ethanol precipitation; ammonia sulphate precipitation; chromatofocusing; shift chromatography; by electrophoresis (including but not limited to preparative isoelectric focusing), differential solubility (including but not limited to ammonia sulfate precipitation) or extraction. Visible molecular weight can be estimated by GPC comparison with globular protein standards (Preneta AZ, PROTEIN PURIFICATION METHODS, A PRACTICAL APPROACH. (Harris &amp; Angal, Eds.) IRL Press 1989, 293-306). The purity of non-natural amino acid polypeptide conjugates: PEG can be assessed with proteolytic degradation (including, but not limited to, trypsin cleavage) followed by mass spectrometry analysis. Pepinsky RB, et. al., J. Pharmacol. &amp; Ehr. Ther. 297 (3): 1059-66 (2001).

D. Use of Binding Groups and Applications, Including Polypeptide Dimers and Multimers In addition to adding the desired functionality directly to a non-natural amino acid polypeptide, a portion of the non-natural amino acid polypeptide may first be modified with a multifunctional (eg , bi-, tri-, a tetra-) linker molecule which was then further modified. This m eans that at least one kgaj of the multifunctional linker molecule reacts with at least one non-natural amino acid in the polypeptide and at least one other end of the multifunctional linker that is available for further functionalization. If all ends of the multifunctional linker are identical, then (depending on the stoichiometric conditions) homomultimers of the non-natural amino acid polypeptide can be formed. If the ends of a multifunctional linker have different chemical reactivities, then at least one end of the multifunctional linker

101

The 51998® group may react to bind to a non-natural amino acid polypeptide and the other end may subsequently react with a variety of functionalities, including, by way of example only, the desired functionality.

The multifunctional linker group has a general structure:

X - l! —C - W&gt; P (XIX) where:

each X is independently NH2, -C (= O) R.9, -SR 'or -JR, where R9 is H or OR', where

Yes

<img file="RS51998B_D0086.tif" />

or

<img file="RS51998B_D0087.tif" />

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, the two Rs optionally form heterocycloalkyl;

each R 1 is independently H, alkyl, or substituted alkyl;

each L is independently selected from the group consisting of alkylene, substituted alkylene, alkenylene, substituted alkenylene, -O-, -O- (alkylene or substituted alkylene) -, -S-, -S- (alkylene or substituted alkylene) -, - S (O) k- where k is 1, 2, or 3, S (O) k (alkylene or substituted alkylene) -, -C (O) -, -C (O) - (alkylene or substituted alkylene) - , -C (S) -, -C (S) - (alkylene or substituted alkylene) -, -N (R ') -, -NR' - (alkylene)

102

51998 Β 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) -, -NR' ) CO- (alkylene or substituted alkylene) -, N (R ') C (O) O-,

-N (R) C (O) O- (alkylene or substituted alkylene) -, -S (O) kN (R ') -, -N (R') C (O) N (R ') -, -N (R ') C (O) N (R') - (alkylene or substituted alkylene) -, -N (R ') C (S) N (R') -&gt; -N (R ') S (O) kN (R ') -, -N (R') - N =, -C (R ') = N-, -C (R') = NN (R ') -&gt; -C (R') = NN =, -C (R ') 2-N = N-, and -C (R') 2-N (R ') - N (R') -;

Li is arbitrary, and when present, is -C (R ')_p-NR'-C (O) O- (alkylene or substituted alkylene) - wherein p is 0, 1, or 2;

W is NH₂, -C (= O) R₉, -SR 'or -JR; and is 1 to 3 provided that X and L-Li-W together independently give at least one of the following (a) a hydroxylamine group capable of reacting with a carbonyl (including dicarbonyl) group on a non-natural amino acid or (modified ) non-natural amino acid polypeptide; (b) a carbonyl group (including a dicarbonyl group) is capable of reacting with a hydroxylamine group on a non-natural amino acid or on a (modified) non-natural amino acid polypeptide; or (c) a carbonyl group (including a dicarbonyl group) is capable of undergoing a substitution reaction with an oxime group on a non-natural amino acid or a (modified) non-natural amino acid polypeptide.

In a further or alternative illustrative embodiment, the molar ratio of the compound of Formula (I) or Formula (XIV) to the multifunctional linker of Formula (XIX) is about 1: 2; 1: 1; 1.5: 1; 1.5: 2; 2: 1; 1: 1.5; 2: 1.5; or 1.5 to 2.

A bifunctional homolinker in which the linker has two identical kgai, ie, hydroxylamine groups, can be used to form paired polypeptides by forming oxime bonds, where the formation of such paired polypeptides is carried out in the presence of at least one accelerator (although the oxime bond can also be obtained with a reduced reaction rate in the absence of an accelerator). The use of the accelerator described herein in the formation of polypeptide dimers and multimers is expected

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51998 Β provides significant benefits, because the stoichiometric ratio of the linker with the first polypeptide, or the ratio of the linker- (first polypeptide) complex to the second polypeptide will be closer to stoichiometric in the presence of an accelerator than without an accelerator (or , further, as the molar ratio of accelerator increases, this will of the above reactants be closer to stoichiometric). The stoichiometric ratio (or molar ratio) is an important factor in polypeptide modification due to the cost of reagents (including polypeptides and conjugation molecules) and purification difficulties. Thus, the use of the accelerators indicated herein can be used to reduce the cost and waste resulting from modifying a non-natural amino acid polypeptide, including forming polypeptide dimers or multimers, or linking any desired group or functionality to the polypeptide.

Such a linker can be used to form a homodimer of a non-natural amino acid polypeptide containing carbonyl- or dicarbonyl- to form two oxime bonds, each or both of which is obtained in the presence of at least one accelerator (although the oxime bond may also be obtained with reduced reaction rate in the absence of an accelerator). Alternatively, if one end of such a linker is protected, then such a partially protected linker can be used to bind the unprotected hydroxylamine termination to a carbonyl- or dicarbonyl-containing non-natural amino acid polypeptide via an oxime bond, leaving the other protected kgaj available for further binding reactions. followed by removal of protection. Alternatively, careful manipulation of the stoichiometry of the reagent can give a similar result, (heterodimer), although this is the result in which the desired heterodimer is likely to be contaminated with some homodimer.

Such a linker can also be used to form a homodimer of a hydroxylamine-containing non-natural amino acid polypeptide to form two oxime bonds, either or both of which are formed in the presence of at least one accelerator (although an oxime bond may also be obtained with reduced reaction rate in the absence of accelerators). Alternatively, if one end of such a linker is protected, then such a partially protected linker can be used to bind the unprotected carbonyl terminus to a hydroxylamine-containing non-natural amino acid polypeptide via an oxime bond, leaving the other protected kgaj available for further binding reactions.

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51998 Β followed by removal of protection. Alternatively, careful manipulation of the stoichiometry of the reagent can give a similar result, (heterodimer), although this is the result in which the desired heterodimer is likely to be contaminated with some homodimer.

Multifunctional heteroliners in which each linker has more than one type of terminal reaction group, ie, hydroxylamine, oxime and thioester groups, can be used to form paired polypeptides by forming at least one oxime bond, where the oxime bond formation is performed with the presence of at least one accelerator. Such a linker can be used to form heterodimers of a non-natural amino acid polypeptide using the accelerator-promoted oxime-promoted chemistry discussed herein.

The methods and compounds described herein also disclose polypeptide combinations, such as homodimers, heterodimers, homomultimers, or heteromultimers (ie, trimers, tetramers, etc.). By way of example only, the following description focuses on GH supergenic family members, however, the methods, techniques and compositions described in this section can be applied to virtually any other polypeptide that can provide a benefit in the form of dimers and multimers, including by way of example only: alpha-1 antitrypsin, angiostatin, antihemolytic factor, antibody, antibody fragments, apolipoprotein, apoprotein, atrial natriuretic factor, atrial natriuretic polypeptide, atrial peptide, SHS chemokine, T39765, NAP-2, ENA-78,, gro-b, gro- c, IP-10, GCP-2, NAP-4, SDF1, PF4, MIG, calcitonin, c-kit ligand, cytokine, CC chemokine, monocyte chemoattractive protein-1, monocyte chemoattractive protein-2, monocyte chemoattractive protein-3, monocyte inflammatory protein-1 alpha, monocyte inflammatory proteins and beta, RANTES, 1309, R83915, R91733, FICCl, T58847, D31065, T64262, CD40, 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), exfoliating toxin, Factor IX, Factor VII, Factor VIII, Factor X ,, fibroblast growth factor (FGF), fibrinogen, fibronectin, 4-helical package protein, G-CSF, glp-1, GM-CSF, glucocerebrosidase, gonadotropin, growth factor, growth factor receptor, grf, hedgehog protein, hemoglobin, hepatocyte growth factor (hGF), hirudin, human growth hormone (hGH), human serum

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51998 Β albumin, ICAM-1, ICAM-1 receptor, LFA-1, LFA-1 receptor, insulin, insulin-like growth factor (IGF), IGF-I, IGF-II, interferon (IFN), IFN-alpha, IFN -beta, IFN-gamma, any molecule interferon-like or member of the IFN family, 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 inhibition factor, luciferase, neurturin, neutrophil inhibition factor (NIF), oncostatin M, osteogenic protein ,, oncogenic product, paracytonin, parathyroid hormone, PD-ECSF, PDGF, peptide hormone, pleiotropin, protein A, protein G, pth, pyrogenic exotoxin A, pyrogenic exotoxin B, pyrogenic exotoxin C, ruu, relaxin, renin, SCF, small biosynthetic protein , soluble complement receptor I, soluble I-CAM 1, soluble interleukin receptor, soluble TNF receptor, somatomedin, somatostatin, somatotropin, streptokinase, superantigens, staphylococcal enterotoxin, FLT, SEA, SEB, SECl, SEC2 steroid hormone receptor, superoxide dismutase, toxic shock syndrome toxin, thymosin alpha 1, plasminogen tissue activator, tumor growth factor (TGF), tumor necrosis factor, tumor necrosis factor, 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, p53, tat, fos, myc, jun, myb, rel, estrogen receptor, progesterone receptor, testosterone receptor, aldosterone receptor, LDL receptor, and corticosterone. The non-natural amino acid polypeptide may also be homologous to any polypeptide member of the supergen growth hormone family.

Therefore, encompassed within the methods, techniques, and compositions described herein are members of a GH supergene family of polypeptides comprising one or more non-natural amino acids linked to another member of the GH supergene family or a variant thereof, or any other polypeptide that is not a member of the family. -GH supergene or a variant thereof, either direct to the backbone of the polypeptide or rgeko linker. Due to their increased molecular weight compared to monomers, members of the GH supergene family of dimer or multimer conjugates may exhibit new or desired properties, including but not limited to different pharmacological, pharmacokinetic, or pharmacodynamic properties, modulated therapeutic half-lives, or modulated plasma half-lives. on a monomemic member of the GH supergene family. In some embodiments of the invention, the dimers of the GH supergene family member described herein will modulate the receptor dimerization of the GH supergene family member. In other embodiments, the dimers described herein

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51998 Β or multimers of a member of the GH supergene family will act as a receptor antagonist, agonist or modulator of a member of the GH supergene family.

In some embodiments of the invention, the methods and compounds described herein provide multimers containing one or more GH supergenic family members formed by reaction with activated water-soluble polymers having the structure:

R- (CH2CH₂O) nO- (CH₂m is where n is from about 5 to 3,000, m is 2-10, X may be hydroxylamine or containing a carbonyl or dicarbonyl diode, and R is a backing group, a functional group, or a leaving group which may be the same or different as X. R may be, for example, a functional group selected from the group consisting of hydroxyl, protected hydroxyl, alkoxyl, N-hydroxysuccinimidyl ester, 1-benzotriazolyl ester, N-hydroxysuccinimidyl carbonate, 1-benzotriazolyl carbonate, acetal, aldehyde, aldehyde hydrates, alkene acrylate, methacrylate, acrylamide, active sulfone, gamine, aminooxy, protected amine, hydrazide, protected hydrazide, protected thiol, carboxylic acid, protected carboxylic acid, isocyanate, isothiocyanate, maleimide, vinylsulfon, dithiopyridine, vinylpyridine, iodoacetamide, epoxide, glyoxals, diones, mesylates, tosylates, and tresylate, alkene, and ketones.

Using the chemistry described in detail herein, one skilled in the art can design a linker in which at least one functional group can form an oxime group, in the presence of the accelerator disclosed herein, with a non-natural amino acid polypeptide; other functional groups on the linker may use others known in chemistry, including nucleophilic / electrophilic chemistry known in organic chemistry.

The formation of polypeptide dimers or multimers linked together via at least one oxime group forms (a) a reaction of a non-natural amino acid polypeptide containing hydroxylamine and a carbonyl-containing reagent, or (b) a reaction of a non-natural amino acid polypeptide containing carbonyl and reagent containing hydroxylamine, can be improved by adding accelerators to the reaction mixture. Such an accelerator is a compound having at least one of the following properties: (a)

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51998Β increases the reaction rate between the carbonyl-containing compound and the hydroxylamine-containing compound while forming an oxime-containing compound, where the increase in rate is relative to the reaction without accelerator; (b) reducing the activation energy of the reaction between the carbonyl-containing compound and the hydroxylamine-containing compound while forming an oxime-containing compound, where the reduction in activation energy is relative to the accelerator-free reaction; (c) increasing the yield of the oxime-containing compound from the reaction of the carbonyl-containing compound with the hydroxylamine-containing compound, wherein the increase in yield is relative to the reaction without accelerator; (d) reducing the temperature at which the carbonyl-containing compound reacts with the hydroxylamine-containing compound to form an oxime-containing compound, where the temperature decrease is relative to the reaction without accelerator; (e) reducing the time required for the carbonyl-containing compound to react with the hydroxylamine-containing compound while forming an oxime-containing compound, thereby reducing the time relative to the accelerator-free reaction; (f) reducing the amount of reagent required to form the oxime group on the non-natural amino acid polypeptide, where the reduction in the amount of reagent is relative to the reaction without accelerator; (g) reducing foreign products resulting from the reaction of a carbonyl-containing compound with a hydroxylamine-containing compound while forming an oxime-containing compound, where the reduction of foreign products is relative to the reaction without accelerator; (h) does not irreversibly destroy the tertiary structure of the polypeptide that is subjected to the oxime formation reaction in the presence of an accelerator (except, of course, where the purpose of the reaction is to destroy such a tertiary structure); (i) may be separated in vacuo from the oxime-containing compound; and (j) modulates the reaction of a carbonyl-containing compound with a hydroxylamine-containing compound. In further embodiments, the accelerator has at least two of the aforementioned properties, three of the aforementioned properties, four of the aforementioned properties, five of the aforementioned properties, six of the aforementioned properties, seven of the aforementioned properties, eight of the above properties . In a further embodiment, the accelerator does not have any of the aforementioned properties.

The use of accelerators includes the use of single accelerators or multiple accelerators. Additionally, the molar ratio of accelerator to carbonyl-containing compound includes values ​​between about 0.5: 1 to 5000: 1, including by way of example only 4000: 1, 3000: 1, 2000: 1, 1000: 1, 500: 1, 400: 1, 300: 1, 200: 1, 100: 1, 50: 1, 40: 1, 30: 1, 20: 1, 10: 1, 9: 1, 8: 1, 7: 1, 6: 1, 5: 1, 4: 1, 3: 1, 2: 1, 1: 1, 0.9: 1, 0.8: 1, 0.7: 1, 0.6: 1, and 0.5: 1.

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51998 Β

Furthermore, the molar ratio of accelerator to hydroxylamine-containing compound includes values ​​between about 0.5: 1 to 5000: 1, including by way of example only 4000: 1, 3000: 1,

2000: 1, 1000: 1, 500: 1, 400: 1, 300: 1, 200: 1, 100: 1, 50: 1, 40: 1, 30: 1, 20: 1, 10: 1, 9: 1, 8: 1, 7: 1, 6: 1, 5: 1, 4: 1, 3: 1,2: 1, 1: 1, 0.9: 1,0.8: 1, 0.7: 1, 0.6: 1, and 0.5: 1. Furthermore, the accelerator includes compounds that can be substantially removed in vacuo from the resulting oxime-containing compound. Furthermore, the accelerator includes compounds containing a diamine moiety, a semi-carbazide moiety, a hydrazine, or a hydrazide moiety.

Furthermore, in any of the following aspects or representations, the accelerator is selected from the group consisting of bifunctional aromatic amines, oxoamine derivatives, and compounds having the following structures:

n_; n

n₂g

<img file="RS51998B_D0088.tif" />

<img file="RS51998B_D0089.tif" />

<img file="RS51998B_D0090.tif" />

ο

<img file="RS51998B_D0091.tif" />

where R_x, R_y and R_z are selected from the group consisting of: L_x-H, L_x-alkyl, L_x-aryl, L_xheteroaryl, L_x-alkenyl, L_x-alkynyl, L_x-alkoxy, and L_x-alkylamine, where L_x is a bond, C (= O), C (= NH), C (= NH) -NH and SO, SO2, where the aromatic amine is selected from the group:

Bifunctional aromatic amines:

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51998 Β

<img file="RS51998B_D0092.tif" />

and where the oxoamine derivative is selected from the group:

Oxoamine derivatives:

<img file="RS51998B_D0093.tif" />

Furthermore, the accelerator includes compounds selected from the group consisting of:

<img file="RS51998B_D0094.tif" />

<img file="RS51998B_D0095.tif" />

where R_x, R_y and R_z are selected from the group consisting of: L_x-H, L_x-alkyl, L_x-aryl, L_xheteroaryl, L_x-alkenyl, L_x-alkynyl, n-alkoxy, and n-alkylamine, where L_x is a bond, C (= O), C (= NH), and C (= NH) -NH. Furthermore, in any of the above aspects or illustrations, the accelerator is selected from the compounds shown in Figure 5, Figure 9, or Figure 10 including as an example any of the compounds 6, 8, 10, 7, and 20 of Figure 5 In any of the above aspects or representations, the accelerator includes an agent that can form a hydrazone after reaction with a carbonyl-containing group. Furthermore, in any of the above aspects, the activity of the accelerator depends on the rate of reaction with the ketone moiety and the stability of the resulting intermediate. Furthermore, in any of the above aspects or illustrations, the pH of the reaction mixture containing the accelerator, the carbonyl-containing compound and the hydroxylamine-containing compound is between about 2.0 and 10; between about 2.0 and 9.0; between about 2.0 and 8.0; between about 3.0 and 7.0; between about 4.0 and 6.0; between about 3.0 and 10.0; between about 4.0 and 10.0; between about 3.0 and 9.0; between about 3.0 and 8.0; between about 2.0 and 7.0; between about 3.0 and 6.0; between about 4.0 and 9.0; between about 4.0 and 8.0; between about 4.0 and 7.0; between about 4.0 and 6.5; between about 4.5 and 6.5; about 4.0; about 4.5; about 5.0; about 5.5; about 6.0; about 6.5; and about 7.0.

Expression in alternative systems Several strategies have been applied to introduce non-natural amino acids into

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51998 Β proteins in non-recombinant host cells, mutagenized host cells, or in non-cell systems. These systems are also suitable for use in preparing non-natural amino acid polypeptides described herein. Amino acid derivation with reactive side chains such as Lys, Cys and Tug results in the conversion of lysine to N²-acetyl-lysine. Chemical synthesis also provides a simple process for incorporating non-natural amino acids. With the recent development of enzymatic ligation and the invention of chemical ligation of peptide fragments, it is possible to make longer proteins. See, prg., PE Dawson and S. Β. H. Kent, Annu. Rev. Biochem, 69: 923 (2000). Chemical ligation of peptides and isomic chemical ligation are described in US Pat. No. No. 6,184,344, U.S. Pat. 2004/0138412, US Pat. 2003/0208046, WO 02/098902, and WO 03/042235, which are incorporated herein by reference. A general in vitro biosynthesis process in which a tRNA suppressor is chemically acylated with a desired non-natural amino acid added to an in vitro extract that can support protein biosynthesis is used to incorporate at a specific position rgeko 100 non-natural amino acids into various proteins of almost any size. See, eg, VW Cornish, D. Mendel and PG Schultz, Angew. Chem. Int. Ed. Engl., 1995, 34: 621 (1995); CJ Noren, SJ Anthony-Cahill, MC Griffith, PG Schultz, A general method for site-specific incorporation of non-natural amino acids into proteins, Science 244: 182-188 (1989); and, JD Bain, CG Glabe, TA. Dix, AR Chamberlin, ES Diala, Biosynthetic site-specific incorporation of a non-natural amino acids into a polypeptide, J. Am. Chem. Soc. 111: 8013-8014 (1989). A wide range of functional groups has been introduced into proteins for protein stability studies, protein folding, enzymatic mechanisms, and signal transduction.

An in vivo procedure, called selective pressure insertion, has been developed to exploit the promiscuity of wild-type synthesis. See, eg, N Budisa, C. Minks, S. Alefelder, W. Wenger, FM Dong, L. Moroder, and R. Huber, FASEB J, 13:41 (1999). An auxotropic strain in which the relevant metabolic pathway of cell supply with a particular natural amino acid is excluded is grown on a minimal medium containing limited concentrations of natural amino acids, while transcription of the target gene is suppressed. At the beginning of the stationary growth phase, the natural amino acid is depleted and replaced with an analogous non-natural amino acid. Induction of recombinant protein expression results in accumulation of protein containing no

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51998 Β natural analogue. For example, using this strategy, o, mi p-fluorophenylalanines are included in the proteins, which show two characteristic kgaks in the UV spectrum that can be easily identified, see, eg, C. Minks, R. Huber, L. Moroder, and N Budisa, Anal. Biochem. 284: 29 (2000); trifluoromethionine is used to replace methionine in bacteriophage T4 lysozymes to study their interaction with cytooligosaccharide ligands using^,9F NMR, see, eg, H. Duewel, E. Daub, V. Robinson, and JF Honek, Biochemistry, 36: 3404 (1997); and trifluoroleukin is incorporated into the leukin site, resulting in increased thermal and chemical stability of the leukin-zipper protein. See, eg, Y. Tang, G. Ghirlanda, IS A. Petka, T. Nakajima, JV. F. DeGrado and

DA Tirrell, Angew. Chem. Int. Ed. Engl., 40: 1494 (2001). Moreover, selenomethionine and telluromethionine are included in various recombinant proteins to facilitate phase solubility in X-ray crystallography. See, eg, WA Hendrickson, JR Horton, and DM Lemaster, EMBO J. 9: 1665 (1990); JO Boles, K. Lewinski, M. Rankle, JD Odom, B. Dunlap, L. Lebioda, and M. Hatada, Nat. Struct. Biol., 1: 283 (1994); N Budisa, B. Steipe, P. Demange, C. Eckerskorn, J. Kellermann and R. Huber, Eur. J. Biochem., 230: 788 (1995); and, N. Budisa, W. Kambrock, S. Steinbacher, A. Humm, L. Prade, T. Neuefeind, L. Moroder and R. Huber, J. Mol. Biol., 270: 616 (1997). Methionine analogs with a functional alkene or alkyne have also been successfully included, allowing for additional modifications of the protein with chemical agents. See, eg, JC van Hest and DA Tirrell, FEBS Lett., 428: 68 (1998); JC van Hest, KL Kiick and DA Tirrell, J. Am. Chem. Soc., 122: 1282 (2000); and, KL Kiick and DA Tirrell, Tetrahedron, 56: 9487 (2000) ·, US Pat. 6,586,207; U.S. Patent Publication No. 2002/0042097.

The success of this method depends on the recognition of non-natural amino acid analogs with aminoacyl-tRNA syntheses, which, in general, require high selectivity to ensure the translationality of the protein. One way to expand the scope of this method is to reduce the specificity of the substrate for aminoacyl-tRNA synthesis, which has been achieved in a limited number of cases. For example, replacing Ala²⁹⁴ with Gly in Escherichia coli phenylalanyl-tRNA synthesis (PheRS) increases the size of the substrate that binds, and results in acylation of tRNAPhe with P-Cl-phenylalanine (pCl-Phe). M. Ibba, P. Kast, and H. Hennecke, Biochemistry, 33: 7107 (1994). The Escherichia coli strain that provides refuge for this mutant PheRS allows the insertion of p-Clfenylalanine or p-Br-phenylalanine in place of phenylalanine. See, eg, M. lbba and H.

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Hennecke, FEBS Lelt., 364: 272 (1995); N. Sharma, R. Furter, P. Kast and DA Tirrell, FEBS Lett., 467: 37 (2000). Similarly, the Phel30Ser mutation point near the amino acid binding site for Escherichia coli tyrosyl-tRNA synthesis has been shown to allow azatirosine to be incorporated more efficiently than tyrosine. See, F. NatapoTakaki, T. Iwama, S. Saito-Yano, K. Takaki, Y. Monden, M. Kitabatake, D. Soll and S. Nishimura, J. Biol. Chem., 275: 40324 (2000).

Another strategy for incorporating non-natural amino acids into proteins in vivo is to modify syntheses that have corrective mechanisms. These syntheses cannot discriminate and therefore activate amino acids that are structurally similar to related natural amino acids. This error was corrected at a separate position, which deacylates the misplaced amino acid from the tRNA to maintain protein translation ability. If the correct synthesis activity is not enabled, structural analogs that are incorrectly activated can avoid the editing function and be included. This approach has been demonstrated recently with valyl-tRNA synthesis (ValRS). See, V. Doring, H. D: Mootz, LA Nangle, TL Hendrickson, V. de Sgesu-Lagard, P. Schimmel, and P. Marliere, Science, 292: 501 (2001). ValRS may incorrectly aminoacylate tRNAVal with Cys, Thr, or aminobutyrate (Abu); these non-negative amino acids are subsequently hydrolyzed with domain editing. After random mutagenesis of the Escherichia coli chromosome, a mutated strain of Escherichia coli that has a mutation at the ValRS regulation site was selected. This edit-faulty ValRs incorrectly changes the tRNAVal with Cys. Because Abu sterically resembles Cys (the -SH group of Cys is replaced by -SNZ in Abu), the ValRS mutant also includes Abu in proteins when this mutated strain of Escherichia coli was grown in the presence of Abu. Mass spectrometry analysis showed that about 24% of the valine was replaced with Abu at each position of the valine in the whole protein.

Solid-phase synthesis and semi-synthetic methods have also enabled the synthesis of a number of proteins containing non-natural amino acids. For example, see the following publications and cited references, which are listed as follows: Crick, FHC, Barrett, L. Brenner, S. Watts-Tobin, R. General nature of the genetic code for proteins. Nature, 192: 1227-1232 (1961); Hofmann, K., Bohn, H. Studies on polypeptides. XXXVI. The effect of pyrazole-imidazole replacements on the S-protein

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51998 Β activating potency of an S-peptide fragment, J. Am Chem, 88 (24): 5914-5919 (1966); Kaiser, ET Synthetic approaches to biologically active peptides and proteins including enzymes, Acc Chem Res, 22: 47-54 (1989); Nakatsuka, T., Sasaki, T. Kaiser, ET Peptide segment coupling catalyzed by the semisynthetic enzyme thiosubtilisin, J Am Chem Soc, 109: 3808-3810 (1987); Schnolzer, M., Kent, S. Yu. Constructing proleins by dovetailing unprotected synthetic peptides: backboneengineered HIV protease, Science, 256 (5054): 221-225 (1992); Chaiken, lM Semisynthetic peptides and proteins, CRC Crit Rev Biochem, 11 (3): 255-301 (1981); Offord, RE Protein engineering by chemical means? Protein Eng., 1 (3): 151-157 (1987); and, Jackson, DY, Burnier, J, Quan, C., Stanley, M., Tom, J., Wells, JA A Designed Peptide Ligase for Total Synthesis of Ribonuclease A with Non-natural Catalytic Residues, Science, 266 (5183): 243 (1994).

Chemical modification has been used to introduce a number of non-natural side chains, including cofactors, spin labels, and oligonucleotides, into proteins in vitro. See, eg, Sogeu, DR, Schultz, PG Generation of a hybrid sequence-specific single-stranded deoxyribonuclease, Science, 238 (4832): 1401-1403 (1987); Kaiser,

ET, Lawrence DS, Rokita, SE The Chemical modification of enzymatic specificity, Annu Rev Biochem, 54: 565-595 (1985); Kaiser, ET, Lawrence, DS Chemical mutation of active siles enzyme, Science, 226 (4674): 505-511 (1984); Neel, KE, Nanci A, Koshland, DE Properties of thiol-subtilisin, J Biol. Chem, 243 (24): 63926401 (1968); Polgar, L. et ML Bender. A new enzyme containing a synthetically formed active site. Thiol-subtilisin. J. Am Chem Soc, 88: 3153-3154 (1966); and, Pollack, SJ, Nakayama, G. Schultz, PG Introduction of nucleophiles and spectroscopic probes into antibody combining sites, Science, 242 (4881): 1038-1040 (1988).

Alternatively, biosynthesis methods using chemically modified aminoacyltRNAs have been used to incorporate several biophysical probes into in vitro synthesized proteins. See the following publications and indicated references, which are cited within them: Brunner, J. New Photolabeling and crosslinking methods, Annu. Rev Biochem, 62: 483-514 (1993); and, Krieg, UC, Walter, P., Hohnson, AE Photocrosslinking of the signal seguence of nascent preprolactin of the 54-kilodalton polypeptide of the

114

51998Β signal recognilion particle, Proc. Natl. Acad. Sci, 83 (22): 8604-8608 (1986).

It has previously been shown that non-natural amino acids can be incorporated into proteins at a specific position in vitro by adding chemically aminoacylated suppressor tRNAs for protein synthesis reactions programmed with a gene containing the desired yantam non-sense mutation. Using these approaches, a large number of common twenty amino acids can be substituted with close structural homologues, eg, fluorophenylalanine to phenylalanine, using auxotropic strains for a particular amino acid. See, eg, Noren, CJ, Anthony-Cahill, Griffith, MC, Schultz, PG A general method for site-specific incorporation of non-natural amino acids into proteins, Science, 244: 182-188 (1989); MW Nowak, et al., Science 268: 439-42 (1995); Bain, JD, Glabe, CG, Dix, TA, Chamberlin, AR, Diala, ES Biosynthetic site-specific Incorporation of a non-natural amino acid into a polypeptide, J. Am Chem Soc, 111: 8013-8014 (1989); N. Budisa et al., FASEB J. 13: 41-51 (1999); Ellman, JA, Mendel, D., Anthony-Cahill, S., Noren, CJ, Schultz,

PG Biosynthetic method for introducing non-natural amino acids site-specifically into proteins, Methods in Enz., Vol. 202, 301-336 (1992); and, Mendel, D., Cornish, V. FF. &amp; Schultz, PG Site-Directed Mutagenesis with an Expanded Genetic Code, Annu Rev Biophys. Biomol Struct. 24, 435-62 (1995).

The following patents are cited for in vivo methods for incorporating non-natural amino acids into proteins and other polypeptides, and for methods for obtaining the corresponding syntheses / tRNAs: US Pat. 7,045,337 and 7,083,970.

For example, a tRNA suppressor recognizing the stop codon UAG ii was prepared and was chemically aminoacylated with a non-natural amino acid. The usual position-directed mutagenesis was used to introduce the TAG stop codon, to the position of interest in the protein gene. See, eg, Sayers, JR, Schmidt, W. Eckstein,

F. 5'-3 'Exonucleases in phosphorothioate-based olignoucleotide-directed mutagensis, Nucleinic acids Res, 16 (3): 791-802 (1988). When an acylated suppressive tRNA and a mutated gene are combined in an in vitro transcription / translation system, a non-natural amino acid is involved in response to the UAG codon that yields a protein containing this

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51998 Β amino acid at the specified position. Experiments with [³H] -Phe and α-hydroxy acid experiments showed that only the desired amino acid was included at the position specified by the UAG codon and that this amino acid was not included at any other position in the protein. See, eg, Noren, et al, supra; Kobayashi et al., (2003) Nalure Structural Biology 10 (b): 425-432; and, Ellman, JA, Mendel, D., Schultz, PG Site-specific incorporation of novel backbone structures inlo proteins, Science, 255 (5041): 197-200 (1992).

The tRNA can be aminoacylated with the desired amino acid by any method or technique, including but not limited to, chemical or enzymatic aminoacylation.

Aminoacylation can be achieved by aminoacyl tRNA syntheses or by other enzyme molecules, including, but not limited to, ribozymes. The term ribozyme can be replaced with catalytic RNA. Cech et al. (Cech, 1987, Science, 236: 1532-1539; McCorkle et al., 1987, Concepts Biochem. 64: 221-226) have shown the presence of natural RNAs that can act as catalysts (ribozymes). However, although these natural RNA catalysts have only been shown to act on ribonucleic acid substrates for cleavage and excision, recent developments in the artificial evolution of ribozymes have extended the potential catalysis to a variety of chemical reactions. Studies have revealed RNA molecules that can catalyze aminoacyl-RNA bonds at their own (2 ') 3'-terminations (Illangakekare et al., 1995 Science 267: 643-647), and RNA molecules that can transfer amino acids from a single RNA molecule to another (Lohse et al., 1996, Nature 381: 442-444).

U.S. Patent Application Publication 2003/0228593, which is incorporated herein by reference, describes methods for constructing ribozymes and their use and aminoacylation of tRNA with naturally encoded and non-natural amino acids. Forms of substrate-immobilized from enzyme molecules that can aminoacylate tRNA, including, but not limited to, ribozymes, can provide efficient affinity purification of aminoacylated products. Examples of suitable substrates include agarose, sepharose, and magnetic beads. The production and use of a substrate immobilized from ribozymes for aminoacylation is described in Chemistry and Biology

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2003, 10: 1077-1084 and disclosing U.S. Patent Application 2003/0228593.

Chemical aminoacylation processes include those introduced by Hecht et al. (Hecht, SM Acc. Chem. Res. 1992, 25, 545; Heckler, TG; Roesser, JR; Hi, C .; Chang, P .; Hecht, SM Biochemislry 1988, 27, 7254; Hecht, SM; Alford, B.

L .; Kuroda, X .; Kitano, SJ Biol. Chem 1978, 253, 4517) and by Schultz, Chamberlin, Dougherty and others (Cornish, VW; Mendel, D .; Schultz, PG Angew. Chem. Int. Ed. Engl. 1995, 34, 621; Roberlson, SA; Ellman , JA; Schultz, PG J Am. Chem. Soc. 1991, 113, 2722; Noren, CJ, Anthony Cahill, S. J; Griffith,

MC; Schultz, PG Science 1989, 244, 182; Bain, JD; Glabe, CG; Dix, TA; Chamberlin, ARJ Am. Chem. Soc. 1989, 111, 8013; Bain, JD et al. Nature 1992, 356, 537; Gallivan, JP; Lester, Η. A .; Dougherty, DA Chem. Biol. 1997, 4, 740; Turcatti, et al. J. Biol. Chem. 1996, 271, 19991; No-wak, MW et al. Science, 1995, 268, 439; Saks, Μ. E. et al. J. Biol. Chem. 1996, 271, 23169; Hohsaka, T et al. J. Am. Chem. Soc. 1999, 121, 34), to avoid the use of sinters in aminoacylation. Such methods or other chemical aminoacylation methods can be used for the aminoacylation of tRNA molecules described herein.

Methods for generating catalytic RNA may include generating separate groups of randomized ribozyme sequences, performing group-directed evolution, searching for groups for the desired aminoacylation activity, and selecting the sequences of those ribozymes that exhibit the desired aminoacylation activity.

Ribozymes may contain motifs and / or regions that facilitate acylation activity, such as the GGU motif and the U-rich region. For example, it has been shown that rich regions can facilitate the recognition of amino acid substrates, and the GGU motif can form base pairs with a 3 'terminus of tRNA. In combination, GGU and the motif and β-rich region facilitate the simultaneous recognition of both amino acids and tRNA simultaneously, and therefore fac facilitate aminoacylation of the 3 'end of the tRNA.

Ribozymes can be generated in vitro by selection using partially randomized r24mini conjugates with tRNA ^AsncccG, followed by systematic engineering on the consensus sequence found in the active clones. Sample

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51998 Β The ribozyme obtained by this process is called the Fx3 ribozyme and is described in US Pat. 2003/0228593, acts as a versatile catalyst for the synthesis of various aminoacyl-tRNAs loaded with related non-natural amino acids.

Aminoacylate tRNA ribozymes can be immobilized on a substrate to allow efficient affinity purification of aminoacylated tRNAs. Examples of suitable substrates include, but are not limited to, agarose, sepharose, and magnetic beads. Ribozymes can be immobilized on resins using the chemical structure of RNA, just as 3'-cis-diol on ribose RNA can be oxidized with a periodate to give the appropriate dialdehyde to facilitate immobilization of RNA on the resin. Various types of resins can be used including cheap hydrazide resins where the reductive amination makes the interaction between the resin and the ribozyme as an inverse link. Aminoacyl-tRNA synthesis can be significantly facilitated with this on-column aminoacylation technique. Kourouklis et al. Methods 2005; 36: 239-4 describes a column-based aminoacylation system.

Isolation of aminoacylated tRNAs can be accomplished in a variety of ways. One suitable method is to elute aminoacylated tRNA from a column with a buffer such as sodium acetate solution with 10 mM EDTA, a buffer containing 50 mM N- (2-hydroxyethyl) piperazine-N '- (3-propanesulfonic acid), 12.5 mM KCl, pH 7.0, 10 mM EDTA, or simply EDTA buffered water (pH 7.0).

Aminoacylated tRNAs can be added to translation reactions to include amino acids with which the tRNA is aminoacylated at a position of choice in the polypeptide obtained by the translation reaction. Examples of translation systems in which the aminoacylated tRNAs described herein may be used include, but are not limited to, cell lysates. Cell lysates provide the reaction components required for in vitro translation of polypeptides from mRNA input. Examples of such reaction components include, but are not limited to, ribosome proteins, rRNA, amino acids, tRNA, GTP, ATP, translation initiators and elongation factors, and additional translation-related factors. Additionally, translation systems can be batch translations or sectorization translations. Batch translation systems combine reaction components in one compartment while sectorization translation systems separate

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51998 Β translation reaction components of reaction products that may inhibit translation efficiency. Such translation systems are commercially available.

Furthermore, a paired transcription / translation system may be used. paired transcription / translation systems allow both, transcription of the DNA input into the corresponding mRNA, which in turn is translated with the reaction components. An example of a commercially available paired transcription / translation is the Rapid Translation System (RTS, Roche Inc.). The system comprises a mixture containing E. coli lysate for the administration of translation components such as ribosomes and translation factors. Additionally, RNA polymerase is involved in transcribing the input DNA into the mRNA template for use in translation. RTS can use the sectorization of the reaction components using a membrane inserted between the reaction compartments, including the supply / waste compartment and the transcription / translation compartment.

Aminoacylation of tRNA can be performed with other agents, including but not limited to, transferases, polymerases, catalytic antibodies, multifunctional proteins, and the like.

Stephan and Scientist 2005 Ocl 10; pages 30-33 describe additional methods for incorporating non-natural amino acids into proteins. Lou et al. in Mol Cell. 2001 Oct; 8 (4): 759-69 describes a process in which a protein is chemically ligated into a synthetic peptide containing non-natural amino acids (protein expression ligation).

Microinjection techniques are also used to incorporate non-natural amino acids into proteins. See, eg, MW Nowak, PC Keateu, JR Sampson, Μ. E. Saks, CG Labarca, SK Silverman, IK G. Zhong, J. Thorson, JN Abelson, N. Davidson, PG Schultz, DA Dougherty and Η. A. Lester, Science, 268: 439 (1995); and DA Dougherty, Curr. Opin. Chem. Biol., 4: 645 (2000). The xenopus oocyte was coinjected with two RNA species obtained in vitro: mRNA encoding the target protein with the UAG stop codon at the amino acid position of interest and the yantami suppressor tRNA aminoacylated with the desired non-natural amino acid. The oocyte translation mechanism then introduces the non-natural amino acid to the position specified by the UAG. This procedure enabled in vivo studies of the structure-function of integral membranes

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51998 Β proteins, which are generally not susceptible to in vitro expression systems. Examples include the insertion of fluorescent amino acids into the tachykinin neurokinin-2 receptor to measure distance for energy transfer by fluorescence resonance, see, eg, G. Turcatti, K. Nemet, MD Edgerton, U. Meseth, F. Talabot, M. Peitsch, J. Knowles, H. Vogel and A. Chollet, J. Biol. Chem., 271: 19991 (1996); insertion of a biotinylated amino acid to identify surface-exposed residues in the ion channel, see, eg, JP Gallivan, Η. A. Lester and DA Dougherty, Chem. Biol., 4: 739 (1997); for the use of cage tyrosine analogs to monitor conformational changes in the ion channel in real time, see, eg, JC Miller, SK Silverman, PM England, DA Dougherty and HA Lesler, Neuron, 20: 619 (1998); and to use alpha hydroxy amino acids to alter the backbone of the ion channel to examine their gateway mechanisms. See, eg, PM England, Y. Zhang, DA Dougherty and Η. A. Lester, Cell, 96:89 (1999); T. Lu, AY Ting, J. Mainland, LY Jan, PG Schultz, and J. Yang, Nat. Neurosci., 4: 239 (2001).

The ability to incorporate non-natural amino acids directly into proteins in vivo provides a wide range of advantages, including, by way of example only, high yields of mutated proteins, technological simplicity, potential for studying mutated proteins in cells or possibly in living organisms mutated proteins for therapeutic treatments and diagnostic use. The ability to incorporate unnatural amino acids of different sizes, acidity, nucleophilicity, hydrophobicity, and other properties into proteins can greatly expand our ability to rationally and systematically manipulate protein structures, both to test protein function and to create new proteins or organisms with new properties .

In one attempt to incorporate para-F-Phe, a yantamine suppressor tRNAPheCUA / phenylalanyl-tRNA at a specific position, a synthetic pair in pF-Phe resistance, the Phe auxotropic strain of Escherichia coli, was used. See, eg, R. Furter, Protein Sci., 7: 419 (1998).

It may also be possible to achieve the expression of the non-natural amino acid polypeptides described herein using an in-vitro (non-cell) translatome system. Translatome systems can be cellular or non-cellular, and can be prokaryotic or

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51998 Uk eukaryotic. Cell translation systems include, but are not limited to, whole cell compositions such as permeable cells or cell cultures where the desired nucleic acid sequence may be transcribed into mRNA and translated mRNA. Non-cell translatome systems are commercially available in many different types and the systems are well known. Examples of non-cell systems include, but are not limited to, prokaryotic lysates such as Escherichia coli lysates, and eukaryotic lysates such as wheat germ extracts, insect cell lysates, rabbit reticulocyte lysates, rabbit oocyte lysates, and human cell lysates. Eukaryotic extracts or lysates may be preferred when the resulting protein is glycosylated, phosphorylated, or otherwise modified because many such modifications are only possible in eukaryotic systems. Some of these extracts and lysates are commercially available (Promega; Madizon, HOs.; Stratagene; La Jolla, Calif; Amersham; Arlington Heighis, III.; GIBCO / BRL; Grand Island, (.Υ.). Membrane extracts, like canine pancreatic extracts, contain microsomal membranes, which are available to be effective in translating secretory proteins. In these systems, which may include either mRNA as a template (in-vitro translation) or DNA as a template (combined in-vitro transcription and translation), in vitro synthesis is directed with ribosomes. Significant effort has been applied to develop a cell-free protein expression system. See, eg, Kim, DM, and JR Swaitz, Biotechnology and Bioengineering, 74. 309-316 (2001); Kim, DM and JR Swcirtz, Biotechnology Letters, 22, 1537-1542, (2000); Kim, DM, and JR Swartz, Biolechnology Progress, 16, 385390, (2000); Kim, DM, andJ.R. Swarlz, Biotechnology and Bioengineering, 66, 180188, (1999); Patnaik. R. and JR Swartz, Biotechnics 24, 862-868, (1998); US Patent no. 6,337,191; U.S. Patent Publication No. 2002/0081660; WO 00/55353; WO 90/05785. Another approach that can be applied to the expression of non-natural amino acid polypeptides involves mRNA-peptide fusion techniques. See, eg, R. Roberts iJ. Szostak, Proc. Natl Acad. Sci. (USA) 94: 12297-12302 (1997); A. Frankel, et al., Chemistry &amp; Biology 10: 1043-1050 (2003). In this approach, the mRNA template is bound to puromycin, and is translated into a peptide on the ribosome. If one or more tRNA molecules have been modified, non-natural amino acids may also be included in the peptide. After the last mRNA codon is read, puromycin occupies 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 easily detected from the mRNA. sequence. This way, one can browse the polypeptide libraries not

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51998 Β natural amino acids containing one or more non-natural amino acids to identify polypeptides having the desired properties. Recently, in vitro translations of ribosomes with purified components that allow the synthesis of peptides substituted with non-natural amino acids have been reported. See, eg, A. Forster et al., Proc. NatlAcad. Sci. (USA) 100: 6353 (2003).

Reconstituted translation systems can also be used. Mixtures of purified translation factors have also been used successfully to translate mRNA into protein as well as combinations of lysates or lysates enriched with purified translation factors such as initiation Factor-1 (IF-1), IF-2, IF-3 (α or β), elongation Factor T (EF-Tu), or termination Factors. Non-cell systems can also be paired with transcription / translation systems whereby DNA is introduced into the system, transcribed into mRNA. and mRNA translated as described in Current Protocols in Molecular Biology (FM Ausubel et al. editors, Wiley Interscience, 1993). RNA transcribed in the eukaryotic transcription system may be in the form of heteronuclear RNA (hnRNA) or 5'-terminal coatings (7-methyl guanosine) and 3'-terminal poly A mature tail mRNAs, which may be an advantage in certain translation systems . For example, cover mRNAs were translated with high efficiency into the reticulocyte lysate system.

EXAMPLES

Example 1; Improved oxime formation in the presence of an accelerator A carbonyl-containing compound (non-natural amino acid polypeptide) is shown in Scheme 1A. The keto group of the amino acid para-acetylphenylalanine included in the protein reacts with the hydroxylamine-containing compound to form a relatively stable oxime. This reaction is highly specific. In the presence of an accelerator, a faster reaction was observed (Scheme 1B).

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<img file="RS51998B_D0096.tif" />

protein

Scheme 18

Example 2: Verification of accelerator potential using 30 K PEG hGH-pAcF conjugation Human growth hormone (hGH) with substituted para-acetylphenylalanine for tyrosine at position 35 was used to verify a panel of 20 compounds (Figure 5). hGH was buffered to hGH reaction buffer (20 mM NaOAc, 20 mg / ml glycine, 5 mg / ml mannitol, 1 mM EDTA, pH 4.0) using a PD 10 column, and concentrated to 10 mg / ml using Centrocon (10 K MWCO ) concentrator. hGE1 (10 μΐ) was mixed with 3.6 μΐ mono hydroxylamine 30 K PEG (2.5 mM), 3 μΐ potential accelerator solution (200 tM in hGH reaction buffer) and buffered to give a final volume of 30 μΐ. The molar ratio for hGH: PEG was 1: 2. The reaction mixtures were incubated at 28 ° C for 16 hours and 36 hours and analyzed by SDS-PAGE (Figure 6). Each zone of gels shown in Figure 6 is labeled with the test compound as shown in Figure 5. The last zone in each gel was the control reaction (without accelerator). Compounds 6, 7, 8, 10, and 20 were accelerators. The two panel compounds, compounds 7 and 20 (acetic hydrazide) (shown in Figure 5), were accelerators and were further evaluated under similar reaction conditions with a higher concentration of hGH protein (8 mg / ml). The reaction mixtures were incubated at 28 ° C for 16 hours, and the results of SDS123

51998 Β

PAGE analyzes are shown in Figure 7. Zone 1 was a reaction mixture with hGH: PEG with a molar ratio of 1: 2 and a 50 mM accelerator of compound 7. Zone 2 was a reaction mixture with hGH: PEG with a molar ratio of 1: 2 and 50 mM accelerator of compound 20. Zone 3 was the reaction mixture for hGH: PEG with a molar ratio of 1: 2 without accelerator. Zone 4 was the reaction mixture for hGH: PEG with a molar deviation of 1: 5 without accelerator. After 16 hours, both compounds were shown to catalyze the reaction.

Example 3: LCMS analysis for hGH after accelerator incubation In hGH reaction buffer, wild-type hGH (5.8 mg / ml) was incubated at different concentrations of acetic hydrazide accelerator (200 mM, 100 mM, 50 tM, 25 tM, 12.5 tM, 6.25 tM and 0 were stirred at 28 ° C for 48 hours The accelerator was removed by dialysis (10 k MWCO) The resulting protein solutions were analyzed by LCMS (Figure 8).] Figure 10Α shows the total LCMS trace.Figure 8B shows the mass spectrum for hGH without accelerator. Figure 8C shows the mass spectrum for hGH with a 200 mM acetic hydrazide accelerator.

The protein conjugation accelerator preferably does not exhibit any degraded effect on the protein, such as fragmentation, precipitation, and undesirable covalent modifications. No fragmentation was observed using accelerators 7 and 20 (shown in Figure 5) based on SDS-PAGE analysis, and protein precipitation under all conditions used with both scFv and hGH. For example, no covalent modification was observed with LCMS after 48 hours of incubation of wild-type hGH with up to 200 mM accelerator 20 (acetic hydrazide). The measured weight molecules for hGH in all conditions are the same and correspond to the theoretical value of 22256.

Examples 4; One-step dimerization for scFv-pAcF A single chain Fv (scFv) 108 protein substituted with para-acetylphenylalanine at position 259 (scFv 108 259-pAcF) was used for the next conjugation assay. scFv 108 259-pAcF in the prepared solution was buffered to the reaction buffer (150 mM NaCl, 20 mM NaOAc, 5 mM EDTA, pH 4.0) using a PD 10 column. The protein solution was concentrated to 0.5 mM, mixed with

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51998 Β hydroxylamine homobifunctional 2 K. PEG linker 2.5 mM in the prepared solution and supplemented with acetic hydrazide as accelerator. The final reaction mixture contained 147 μΜ of homobifunctional 2 K PEG linker, the appropriate concentration of pAcF-substituted scFv, and 47 mM acetic hydrazide. The reaction mixtures were incubated at 28 ° C and analyzed at various time points (6 hours, 20 hours, 44 hours) with SDS-PAGE (Figure 2).

To obtain a more efficient and simple one-step reaction with a homobifunctional 2 K PEG linker, accelerators were used to facilitate the dimerization process. Without an accelerator, very little smoke product could be detected by SDS-PAGE analysis after 20 hours. Zone 4 for 6-hour, 20-hour, and 44-hour gels shows reaction mixtures for scFv: PEG linker and a molar ratio of 2.0: 1 without acetic hydrazide accelerator. On the other hand, in the presence of 47 mM acetic hydrazide, the dimetic product appeared after 6 hours. As shown in Figure 2, different molar proportions of protein and linker, 1: 6: 1, 2.0: 1, and 2.4: 1, were tested to review the best conjugation conditions. Zone 1 for 6-hour, 20-hour, and 44-hour gels shows reaction mixtures for scFv: PEG linker and a molar ratio of 1.6: 1 with acetic hydrazide. Zone 2 for 6-hour, 20-hour, and 44-hour gels shows reaction mixtures for scFv: linker and a molar ratio of 2.0: 1 with acetic hydrazide. Zone 3 for 6-hour, 20-hour, and 44-hour gels shows reaction mixtures with scFv: linker and a 2.4: 1 mole molar with acetic hydrazide. As a control, scFv 108 was incubated with 47 mM acetic hydrazide without homobifunctional PEG linker for 44 hours. No dimer formation was observed. Zone 5 for 6-hour, 20-hour, and 44-hour gels shows reaction mixtures of scFv and acetic hydrazide accelerator without PEG linker. This result indicates that the accelerator, acetic hydrazide, does not facilitate the formation of intermolecular disulfide bonds between scFv. In the presence of a homobifunctional PEG linker, the dimer was obtained by forming an oxime between the PEG linker and the protein.

Example 5: Conjugation of mono hydroxylamine 30 K PEG and scFv-pAcF scFv 108 259-pAcF 0.5 mM prepared solution in the above reaction buffer (10 μΐ) was mixed with different amounts of mono hydroxylamine 30 K PEG 2.5 mM prepared solution, 2.2 μΐ of 200 mM prepared solution

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51998 Β acetic hydrazide and reaction buffer. Reaction mixtures with a final reaction volume of 22 μΐ have different molar ratios of scFv: PEG (1: 3 or 1: 5). The acceleration of the action of acetic hydrazide on the conjugation of mono hydroxylamine 30 K PEG and scFv was estimated for protein and PEG for molar ratios of 1: 3 and 1: 5 with and without accelerators. The reaction mixtures were incubated at 28 ° C and analyzed at various time points (20 hours, 44 hours) with SDS-PAGE (Figure 3). Zones 1, 2, and 3 show 100%, 20%, and 10% for the corresponding scFv-pAcF,. Zone 4 for 20 hour and 44 hour gels shows reaction mixtures with scFv.PEG for molar ratio of 1: 3 with 20 mM acetic hydrazide. Zone 5 for 20 hour and 44 hour gels shows reaction mixtures with scFv: PEG and molar ratio of 1: 3 without accelerator. Zone 6 for 20 hour and 44 hour gels shows reaction mixtures with scFv: PEG and a molar ratio of 1: 5 with 20 mM acetic hydrazide. Zone 7 for 20 hour and 44 hour gels shows reaction mixtures with scFv: PEG and a molar ratio of 1: 5 without accelerator. The conjugation results show that acetic hydrazide accelerates the conjugation reaction. The reaction with 1: 3 moles of protein: PEG with accelerator proceeded faster than the reaction with 1: 5 ratio of protein: PEG without accelerator.

Similarly, the relative conjugation efficiencies of different concentrations of acetic hydrazide accelerator (5 mM, 20 mM, 80 mM) with scFv: 30 K PEG mono hydroxylamine with a molar weight of 1: 2 were compared (Figure 4). The reaction mixtures were incubated at 28 ° C. Zone 1 shows the reaction mixture with 5 mM acetic hydrazide (scFv: 30 K PEG mono hydroxylamine in a molar ratio of 1: 2). Zone 2 shows the reaction mixture with 20 mM acetic hydrazide (scFv: 30 K PEG mono hydroxylamine in a molar ratio of 1: 2). Zone 3 shows the reaction mixture with 80 mM acetic hydrazide (scFv: 30 K PEG mono hydroxylamine in a molar ratio of 1: 2). Zone 4 shows the reaction mixture without acetic hydrazide (scFv: 30 K PEG mono hydroxylamine in a molar ratio of 1: 5). Zones 5, 6, and 7 show 10%, 20%, and 100% of the initial corresponding scFv-pAcF. SDS-PAGE analysis shows that the higher the concentration of the accelerator, the faster the conjugation. Conjugation of scFv: PEG with a molar ratio of 1: 2 in the presence of an 80 mM accelerator is faster than conjugation of scFv: PEG with a molar ratio of 1: 5 without accelerator.

Example 6: Study of small molecules for accelerated oxime formation

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51998 A Acetophenone (0.5 mM) was reacted with ethylhydroxylamine (1 mM) in buffered aqueous solution at a pH of about 4.0; a series of accelerators (20 mM) were added to this reaction mixture to determine the effect of the accelerator type on the rate and yield of oxime formation (see Figure 10 (a)). The accelerators tested in this reaction model are shown in Figure 10 (b). Aliquots were taken from the reaction mixture and analyzed by high performance liquid chromatography after 2 hours, 5 hours, 9 hours, and 24 hours. Additionally, for each of these samples, the ketone absorption peak was compared to the oxime absorption peak at 260 nm using UV / Vis spectroscopy. Figure 11 shows the results after 2 hours and 9 hours of reaction. As can be seen, all accelerators increase the rate of oxime formation; however, accelerators 1 and 7 (shown in Figure 10 (b)) give the highest yield, with accelerator 1 giving the highest yield comes a longer reaction time. Without binding to a particular theory, the activity of the accelerator appears to depend on both the rate of reaction with the ketone and the stability of the hydrazone intermediate.