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

CA 02625540 2014-01-28

ACCELERANTS TOR THE MODHjTCATION OF NON-NATURAL AMINO ACIDS AND NONNATURAL AMINO ACID POLYPEPTIDES

RELATED APPLICATIONS

FIET,» OF THE INVENTION

[0002] Accelerants for the modification of molecules containing a carbonyl moiety, including non-natural amino acids and agents containing a non-natural amino acids.

BACKGROUND OF THE INVENTION

[0003] Hie ability to incorporate non-genctically encoded amino acids (he., non-natural amino acids”) into proteins permits the introduction of chemical functional groups that conoid provide valuable alternatives to the naturally-occurring functional groups, such as the epsilon-NH_a of lysine, the sulfhydryl -SH of cysteine, the imino group of histidine, etc. Certain chemical functional groups are known to be inert to the functional groups found in the 20 common, genetically-encoded amino acids hut react cleanly and efficiently to form stable linkages with functional groups that can be incorporated onto non-natural amino acids.

(0004] Methods are now available to selectively introduce chemical functional groups that are not found in proteins, that are chemically inert to all of the functional groups found in the 20 common, genetically-encoded amino acids and that may be used to react efficiently and selectively with reagents comprising certain functional groups to form stable covalent linkages.

SUMMARY OF THE INVENTION

[0005] Described herein are methods, compositions, techniques and strategies comprising accelerants for the reaction of hydroxylamine-containmg compounds with carbonyl-containing compounds. The accelerants find use in the synthesis of oxime-containing compounds. The accelerants, in some embodiments, form bonds with foe carbonyl-containing compounds, and as such, these new compounds arc more reactive with hydroxylamine25 containing compounds. Described herein are chemical compounds that can modulate the reaction of hydroxylaxninecontaining compounds with, carbonyl-containing compounds. Also described herein are chemical compounds that can lower the activation barrier for the reaction of hydroxylamine-containmg compounds with carbonyl-containing compounds. Also described herein are' chemical compounds that, when included in a reaction comprising hydroxylamine-contaimng compounds and carbonyl-containing compounds, increase foe rate at which oxime30 containing compounds are formed. The bydroxylamine-, carbonyl-, and oxime-containing compounds can include non-natural amino acids, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides. The carbonyl-containing compounds include compounds comprising an aromatic ketone moiety. Such compounds comprising an aromatic ketone moiety include amino acids and polypeptides. By way of example only, paraaceijdphenylalanine or pAcF, is an amino acid that comprises an aromatic ketone moiety.

[0006] In one aspect are compounds that accelerate (referred to herein as accelerants) the rate of reaction between hydroxylamine-containing compounds with carbonyl-containing compounds to form oxime-containing compounds. In one embodimettf, foe hydroxylamine-containing compound is a non-natural amino acid, non-natural amino acid polypeptide or a modified non-natural amino acid polypeptide, and foe carbonyl-containing compound comprises a i

CA 02625540 2008-04-09

WO 2007/056448

PCT/US2006/043472 fesîrW if embodiment, the resulting oxime-containing compound comprises one of the aforementioned desired groups (i.e., a desired functionality). In a related aspect are the use of such compounds to accelerate the rate of reaction between a hydroxylamine-containing moiety on a non-natural amino acid, non-natural amino acid polypeptide or a modified non-natural amino acid polypeptide with a carbonyl-containing compound comprising a desired group (i.e., a desired functionality) to form an oxime-containing non-natural amino acid, nonnatural amino acid polypeptide or modified non-natural amino acid polypeptide comprising a desired group. In another related aspect are reaction mixtures containing an accelerant, a hydroxylamine-containing non-natural amino acid, non-natural amino acid polypeptide or modified non-natural amino acid polypeptide, and a carbonylcontaining compound comprising a desired group. In another related aspect are oxime-containing non-natural amino acids, non-natural amino acid polypeptides or modified non-natural amino acid polypeptides comprising a desired group, wherein such oxime-containing compounds are formed from the reaction of a hydroxylamine-containing nonnatural amino acid, non-natural amino acid polypeptide or modified non-natural amino acid polypeptide with a carbonyl-containing compound comprising a desired group in the presence of an accelerant. In one embodiment, the carbonyl group is not an aldehyde. In another embodiment, the carbonyl group is an aromatic ketone.

[0007] In another embodiment, the carbonyl-containing compound is a non-natural amino acid, non-natural amino acid polypeptide or a modified non-natural amino acid polypeptide, and the hydroxylamine-containing compound comprises a desired functionality. In a further embodiment, the oxime-containing compound comprises one of the aforementioned groups. In a related aspect are the use of such compounds to accelerate the rate of reaction between a carbonyl-containing moiety on a non-natural amino acid, non-natural amino acid polypeptide or a modified nonnatural amino acid polypeptide with a hydroxylamine-containing compound comprising a desired group to form an oxime-containing non-natural amino acid, non-natural amino acid polypeptide or modified non-natural amino acid polypeptide comprising a desired group. In another related aspect are reaction mixtures containing an accelerant, a carbonyl-containing non-natural amino acid, non-natural amino acid polypeptide or modified non-natural amino acid polypeptide, and a hydroxylamine-containing compound comprising a desired group. In another related aspect are oxime-containing non-natural amino acids, non-natural amino acid polypeptides or modified non-natural amino acid polypeptides comprising a desired group, wherein such oxime-containing compounds are formed from the reaction of a carbonyl-containing non-natural amino acid, non-natural amino acid polypeptide or modified nonnatural amino acid polypeptide with a hydroxylamine-containing compound comprising a desired group in the presence of an accelerant. In one embodiment, the carbonyl group is not an aldehyde. In another embodiment, the carbonyl group is an aromatic ketone.

[0008] In a further aspect are methods for optimizing the reaction of a carbonyl-containing compound and a hydroxylamine-containing compound to form an oxime-containing compound by selection of at least one appropriate accelerant. In one embodiment, such optimization comprises comparing the yield of the oximecontaining compound in the presence of different accelerants, different molar ratios of accelerants, or a combination of the foregoing. In a further embodiment the yield of the oxime-containing compound is monitored by chromatography. In another embodiment, such optimization comprises comparing the amount of side-products that result in the presence of different accelerants, different molar ratios of accelerants, or a combination of the foregoing. In a further embodiment the quantity of side products is monitored by chromatography. In further embodiments, such optimization includes changing additional reaction conditions, including by way of example only pH and temperature. In one embodiment, the carbonyl group is not an aldehyde. In another embodiment, the carbonyl group is an aromatic ketone.

CA 02625540 2008-04-09

WO 2007/056448 PCT/US2006/043472

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

[0010] In another aspect are hydroxylamine-substituted molecules for the production of derivatized non-natural amino acid polypeptides based upon an oxime bond in the presence of an accelerant described herein (although such a reaction may be less efficient in the absence of an accelerant described herein). In a further embodiment are hydroxylamine-substituted molecules used to derivatize carbonyl- or dicarbonyl-containing non-natural amino acid polypeptides via the formation of an oxime bond between the derivatizing molecule and the carbonyl- or dicarbonylcontaining non-natural amino acid polypeptide in the presence of an accelerant described herein (although such a reaction may be less efficient in the absence of an accelerant described herein). In further embodiments the aforementioned carbonyl- or dicarbonyl-containing non-natural amino acid polypeptides are keto-containing nonnatural amino acid polypeptides. In further or additional embodiments, the carbonyl- or dicarbonyl-containing nonnatural amino acids comprise sidechains selected from a ketone or an aldehyde. In further or additional embodiments, the hydroxylamine-substituted molecules comprise a desired functionality. In further or additional

CA 02625540 2008-04-09

WO 2007/056448

PCT/US2006/043472

-Eiyfei-oxyfaltiiitie'iisubstituted molecules are hydroxylamine-substituted polyethylene glycol (PEG) molecules. In a further embodiment, the sidechain of the non-natural amino acid has a chemistry orthogonal to those of the naturally-occurring amino acids that allows the non-natural amino acid to react selectively with the hydroxylamine-substituted molecules in the presence of an accelerant described herein (although such a reaction may be less efficient in the absence of an accelerant described herein). In a further embodiment, the sidechain of the non-natural amino acid comprises an electrophile-containing moiety that reacts selectively with the hydroxylaminecontaining molecule in the presence of an accelerant described herein (although such a reaction may he less efficient in the absence of an accelerant described herein); in a further embodiment, the electrophile-containing moiety on the sidechain of the non-natural amino acid can undergo nucleophilic attack to generate an oxime-derivatized protein in the presence of an accelerant described herein (although such a reaction may he less efficient in the absence of an accelerant described herein). In a further aspect related to the embodiments described in this paragraph are the modified non-natural amino acid polypeptides that result from the reaction of the derivatizing molecule with the non-natural amino acid polypeptides in the presence of an accelerant described herein (although such a reaction may be less efficient in the absence of an accelerant described herein). Further embodiments include any further modifications of the already modified non-natural amino acid polypeptides. In one embodiment, the carbonyl group is not an aldehyde. In another embodiment, the carbonyl group is an aromatic ketone.

[0011] In another aspect are carbonyl- or dicarbonyl-substituted molecules for the production of derivatized nonnatural amino acid polypeptides based upon an oxime bond, wherein the oxime bond is formed in the presence of an accelerant described herein (although such a reaction may he less efficient hi the absence of an accelerant described herein). In a further embodiment are carbonyl- or dicarbonyl-substituted molecules used to derivatize hydroxylamine-containing non-natural amino acid polypeptides via the formation of an oxime bond in the presence of an accelerant described herein (although such a reaction may be less efficient in the absence of an accelerant described herein). In a further embodiment the carbonyl- or dicarbonyl-substituted molecules are aldehydesubstituted molecules or ketone-substituted moieties. In further embodiments, the carbonyl- or dicarbonylsubstituted molecules comprise a desired functionality. In further or additional embodiments, the aldehydesubstituted molecules are aldehyde-substituted polyethylene glycol (PEG) molecules. In further or additional embodiments, the ketone-substituted molecules are ketone-substituted polyethylene glycol (PEG) molecules. In a further embodiment, the sidechain of the non-natural amino acid has a chemistry orthogonal to those of the naturally-occurring amino acids that allows the non-natural amino acid to react selectively with the carbonyl- or dicarbonyl-substituted molecules in the presence of an accelerant described herein (although such a reaction may be less efficient in the absence of an accelerant described herein). In a further embodiment, the sidechain of the nonnatural amino acid comprises a moiety (e.g., hydroxylamine group) that reacts selectively with the carbonyl- or dicarbonyl-containing molecule in the presence of an accelerant described herein (although such a reaction may be less efficient in the absence of an accelerant described herein); in a further embodiment, the nucleophilic moiety on the sidechain of the non-natural amino acid can undergo electrophilic attack to generate an oxime-derivatized protein in tire presence of an accelerant described herein (although such a reaction may be less efficient in the absence of an accelerant described herein). In a further aspect related to the embodiments described in this paragraph are the modified non-natural amino acid polypeptides that result from the reaction of the derivatizing molecule with the non-natural amino acid polypeptides in the presence of an accelerant described herein (although such a reaction may be less efficient in the absence of an accelerant described herein). Further embodiments include any further modifications of the already modified non-natural amino acid polypeptides. In one embodiment, the carbonyl group is not an aldehyde. In another embodiment, the carbonyl group is an aromatic ketone.

CA 02625540 2008-04-09

WO 2007/056448

PCT/US2006/043472 $ioÎ2®P -g Ocfeef a^peel’ afe-monb-, bi- and multi-functional linkers for the generation of derivatized non-natural amino acid polypeptides based upon an oxime bond, wherein the oxime bond is formed in the presence of an accelerant described herein (although such a reaction may be less efficient in the absence of an accelerant described herein). In one embodiment are molecular linkers (bi- and multi-functional) that can be used to connect carbonyl- or dicarbonyl-containing non-natural amino acid polypeptides to other molecules in the presence of an accelerant described herein (although such a reaction may be less efficient in the absence of an accelerant described herein). In another embodiment are molecular linkers (bi- and multi-functional) that can be used to connect hydroxylaminecontaining non-natural amino acid polypeptides to other molecules in the presence of an accelerant described herein (although such a reaction may be less efficient in the absence of an accelerant described herein). In another embodiment the carbonyl- or dicarbonyl-containing non-natural amino acid polypeptides comprise a ketone and/or an aldehyde sidechain. In an embodiment utilizing a hydroxylamine-containing non-natural amino acid polypeptide, the molecular linker contains a carbonyl or dicarbonyl group at one of its termini; in further embodiments, the carbonyl or dicarbonyl group is selected from an aldehyde group or a ketone group. In further or additional embodiments, the hydroxylamine-substituted linker molecules are hydroxylamine-substituted polyethylene glycol (PEG) linker molecules. In further or additional embodiments, the carbonyl- or dicarbonyl-substituted linker molecules are carbonyl- or dicarbonyl-substituted polyethylene glycol (PEG) linker molecules. Throughout, the phrase “other molecules” includes, by way of example only, proteins, other polymers (branched and unbranched), small molecules, and groups also identified as a “desired functionality.” In further or additional embodiments, the hydroxylamine-containing molecular linkers comprise the same or equivalent groups on all termini so that upon reaction with a carbonyl- or dicarbonyl-containing non-natural amino acid polypeptide in the presence of an accelerant described herein (although such a reaction may be less efficient in the absence of an accelerant described herein), the resulting product is the homo-multimerization of the carbonyl- or dicarbonyl-containing non-natural amino acid polypeptide. In further embodiments, the homo-multimerization is a homo-dimerization. In further or additional embodiments, the carbonyl- or dicarbonyl-containing molecular linkers comprise the same or equivalent groups on all termini so that upon reaction with a hydroxylamine-containing non-natural amino acid polypeptide in the presence of an accelerant described herein (although such a reaction may be less efficient in the absence of an accelerant described herein), the resulting product is the homo-multimerization of the hydroxylamine-containing non-natural amino acid polypeptide. In further embodiments, the homo-multimerization is a homo-dimerization. In a further embodiment, the sidechain of the non-natural amino acid has a chemistry orthogonal to those of the naturally-occurring amino acids that allows the non-natural amino acid to react selectively with the hydroxylaminesubstituted linker molecules in the presence of an accelerant described herein (although such a reaction may be less efficient in the absence of an accelerant described herein). In a further embodiment, the sidechain of the non-natural amino acid has a chemistry orthogonal to those of the naturally-occurring amino acids that allows the non-natural amino acid to react selectively with the carbonyl- or dicarbonyl-substituted linker molecules in the presence of an accelerant described herein (although such a reaction may be less efficient in the absence of an accelerant described herein). In a further embodiment, the sidechain of the non-natural amino acid comprises an electrophile-containing moiety that reacts selectively with the hydroxylamine-containing linker molecule in the presence of an accelerant described herein (although such a reaction may be less efficient in the absence of an accelerant described herein); in a further embodiment, the electrophile-containing moiety on the sidechain of the non-natural amino acid can undergo nucleophilic attack by the hydroxylamine-containing linker molecule to generate an oxime-derivatized protein in the presence of an accelerant described herein (although such a reaction may be less efficient in the absence of an accelerant described herein). In a further aspect related to the embodiments described in this

CA 02625540 2008-04-09

WO 2007/056448

PCT/US2006/043472 pLr&gfa’bëh’W non-natural amino acid polypeptides that result from the reaction of the linker molecule with the non-natural amino acid polypeptides. Further embodiments include any further modifications of the already linked (modified) non-natural amino acid polypeptides. In one embodiment, the carbonyl group is not an aldehyde. In another embodiment, the carbonyl group is an aromatic ketone.

[0013] In one aspect are methods to derivatize proteins via the condensation of carbonyl or dicarbonyl and hydroxylamine reactants in the presence of an accelerant described herein (although such a reaction may be less efficient in the absence of an accelerant described herein) to generate an oxime-based product. Included within this aspect are methods for the derivatization of proteins based upon the condensation of carbonyl- or dicarbonyl- and hydroxylamine- containing reactants to generate an oxime-derivatized protein adduct In additional or further embodiments are methods to derivatize keto-containing proteins with hydroxylamine-functionalized polyethylene glycol (PEG) molecules. In yet additional or further aspects, the hydroxylamine-substituted molecule can include proteins, other polymers (branched and unbranched), small molecules and groups also identified as a “desired functionality.” In one embodiment, the carbonyl group is not an aldehyde. In another embodiment, the carbonyl group is an aromatic ketone.

[0014] In another aspect are methods for the chemical synthesis of hydroxylamine-substituted molecules for the derivatization of keto-substituted proteins in the presence of an accelerant described herein (although such a reaction may be less efficient in the absence of an accelerant described herein). In one embodiment, the hydroxylaminesubstituted molecule can comprise peptides, other polymers (non-branched and branched) and small molecules. In one embodiment are methods for the preparation of hydroxylamine-substituted molecules suitable for the derivatization of carbonyl- or dicarbonyl-containing non-natural amino acid polypeptides in the presence of an accelerant described herein (although such a reaction may be less efficient in the absence of an accelerant described herein), including by way of example only, keto-containing non-natural amino acid polypeptides. In a further or additional embodiment, the non-natural amino acids are incorporated site-specifically during the in vivo translation of proteins. In a further or additional embodiment, the hydroxylamine-substituted molecules allow for the sitespecific derivatization of this carbonyl- or dicarbonyl-containing non-natural amino acid via nucleophilic attack of the carbonyl or dicarbonyl group to form an oxime-derivatized polypeptide in a site-specific fashion, wherein the oxime-derivatized polypeptide is formed in the presence of an accelerant described herein (although such a reaction may be less efficient in the absence of an accelerant described herein). In a further or additional embodiment, the method for the preparation of hydroxylamine-substituted molecules provides access to a wide variety of sitespecifically derivatized polypeptides. In a further or additional embodiment are methods for synthesizing hydroxylamine-functionalized polyethylene glycol (PEG) molecules.

[0015] In another aspect are methods for the chemical derivatization of carbonyl- or dicarbonyl-substituted nonnatural amino acid polypeptides, in the presence of an accelerant described herein (although such a reaction may be less efficient in the absence of an accelerant described herein), using a hydroxylamine-containing bi-functional linker. In one embodiment are methods for attaching a hydroxylamine-substituted linker to a carbonyl- or dicarbonyl-substituted protein via a condensation reaction to generate an oxime bond in the presence of an accelerant described herein (although such a reaction may be less efficient in the absence of an accelerant described herein). In further or additional embodiments, the carbonyl- or dicarbonyl-substituted non-natural amino acid is a keto-substituted non-natural amino acid. In further or additional embodiments, the non-natural amino acid polypeptides are derivatized site-specifically and/or with precise control of three-dimensional structure, using a hydroxylamine-containing bi-functional linker. In one embodiment, such methods are used to attach molecular linkers (mono- bi- and multi-functional) to carbonyl- or dicarbonyl-containing (including by way of example keto6

CA 02625540 2008-04-09

WO 2007/056448

PCT/US2006/043472 centàimg^’À^^aafu&i'aâjyb dciâ'polypeptides, wherein at least one of the linker termini contains a hydroxylamine group which can link to the carbonyl- or dicarbonyl-containing non-natural amino acid polypeptides via an oxime bond in the presence of an accelerant described herein (although such a reaction may be less efficient in the absence of an accelerant described herein). In a further or additional embodiment, these linkers are used to connect the carbonyl- or dicarbonyl-containing non-natural amino acid polypeptides to other molecules, including by way of example, proteins, other polymers (branched and non-branched), small molecules and groups also identified as a “desired functionality.” In one embodiment, the carbonyl group is not an aldehyde. In another embodiment, the carbonyl group is an aromatic ketone.

[0016] In some embodiments, the non-natural amino acid polypeptide is linked to a water soluble polymer. In some embodiments, the water soluble polymer comprises a polyethylene glycol) moiety. In some embodiments, the poly(ethylene glycol) molecule is a bifunctional polymer. In some embodiments, the bifunctional polymer is linked to a second polypeptide. In some embodiments, the second polypeptide is identical to the first polypeptide, in other embodiments, the second polypeptide is a different polypeptide. In some embodiments, the non-natural amino acid polypeptide comprises at least two amino acids linked to a water soluble polymer comprising a polyethylene glycol) moiety.

[0017] In some embodiments, the non-natural amino acid polypeptide comprises a substitution, addition or deletion that increases affinity of the non-natural amino acid polypeptide for a receptor. In some embodiments, the non-natural amino acid polypeptide comprises a substitution, addition, or deletion that increases die stability of the non-natural amino acid polypeptide. In some embodiments, the non-natural amino acid polypeptide comprises a substitution, addition, or deletion that increases the aqueous solubility of the non-natural amino acid polypeptide. In some embodiments, the non-natural amino acid polypeptide comprises a substitution, addition, or deletion that increases the solubility of the non-natural amino acid polypeptide produced in a host cell. In some embodiments, the non-natural amino acid polypeptide comprises a substitution, addition, or deletion that modulates protease resistance, serum half-life, immunogenicity, and/or expression relative to the amino-acid polypeptide without the substitution, addition or deletion.

[0018] In some embodiments, the non-natural amino acid polypeptide is an agonist, partial agonist, antagonist, partial antagonist, or inverse agonist. In some embodiments, the agonist, partial agonist, antagonist, partial antagonist, or inverse agonist comprises a non-natural amino acid is linked to a water soluble polymer. In some embodiments, the water polymer comprises a poly(ethylene glycol) moiety. In some embodiments, the polypeptide comprising a non-natural amino acid linked to a water soluble polymer prevents dimerization of the corresponding receptor. In some embodiments, the polypeptide comprising a non-natural amino acid linked to a water soluble polymer modulates binding of the polypeptide to a binding partner. In some embodiments, the polypeptide comprising a non-natural amino acid linked to a water soluble polymer modulates one or more properties or activities of the polypeptide.

[0019] Also described herein are methods of making a non-natural amino acid polypeptide linked to a water soluble polymer. In some embodiments, the method comprises contacting an isolated polypeptide comprising a nonnatural amino acid with a water soluble polymer comprising a moiety that reacts with the non-natural amino acid in the presence of an accelerant described herein (although such a reaction tnay.be less efficient in the absence of an accelerant described herein). In some embodiments, the non-natural amino acid incorporated into is reactive toward a water soluble polymer that is otherwise unreactive toward any of the 20 common amino acids. In some embodiments, the water polymer comprises a polyethylene glycol) moiety. The molecular weight of the polymer may be of a wide range, including but not limited to, between about 100 Da and about 100,000 Da or more. The

CA 02625540 2008-04-09

WO 2007/056448 PCT/US2006/043472 ’iHolecâlar'Siÿei^Sof àe’p'i^lhër i-riay be between about 100 Da-and about 100,000 Da, including but not limited to, 100,000 Da, 95,000 Da, 90,000 Da, 85,000 Da, 80,000 Da, 75,000 Da, 70,000 Da, 65,000 Da, 60,000 Da, 55,000 Da, 50,000 Da, 45,000 Da, 40,000 Da, 35,000 Da, 30,000 Da, 25,000 Da, 20,000 Da, 15,000 Da, 10,000 Da, 9,000 Da, 8,000 Da, 7,000 Da, 6,000 Da, 5,000 Da, 4,000 Da, 3,000 Da, 2,000 Da, 1,000 Da, 900 Da, 800 Da, 700 Da, 600 Da, 500 Da, 400 Da, 300 Da, 200 Da, and 100 Da. In some embodiments, the molecular weight of the polymer is between about 100 Da and about 50,000 Da. In some embodiments, the molecular weight of the polymer is between about 100 Da and about 40,000 Da. In some embodiments, the molecular weight of the polymer is between about 1,000 Da and about 40,000 Da. In some embodiments, the molecular weight of the polymer is between about 5,000 Da and about 40,000 Da. In some embodiments, the molecular weight of the polymer is between about 10,000 Da and about 40,000 Da. In some embodiments, the polyethylene glycol) molecule is a branched polymer. The molecular weight of the branched chain PEG may be between about 1,000 Da and about 100,000 Da, including but not limited to, 100,000 Da, 95,000 Da, 90,000 Da, 85,000 Da, 80,000 Da, 75,000 Da, 70,000 Da, 65,000 Da, 60,000 Da, 55,000 Da, 50,000 Da, 45,000 Da, 40,000 Da, 35,000 Da, 30,000 Da, 25,000 Da, 20,000 Da, 15,000 Da, 10,000 Da, 9,000 Da, 8,000 Da, 7,000 Da, 6,000 Da, 5,000 Da, 4,000 Da, 3,000 Da, 2,000 Da, and 1,000 Da. In some embodiments, the molecular weight of the branched chain PEG is between about 1,000 Da and about 50,000 Da. In some embodiments, the molecular weight of the branched chain PEG is between about 1,000 Da and about 40,000 Da. In some embodiments, the molecular weight of the branched chain PEG is between about 5,000 Da and about 40,000 Da. In some embodiments, the molecular weight of the branched chain PEG is between about 5,000 Da and about 20,000 Da.

[0020] Also described herein are compositions comprising a polypeptide comprising at least one of the non-natural amino acids described herein and a pharmaceutically acceptable carrier. In some embodiments, the non-natural amino acid is linked to a water soluble polymer. Also described herein are pharmaceutical compositions comprising a pharmaceutically acceptable carrier and a polypeptide, wherein at least one amino acid is substituted by a nonnatural amino acid. In some embodiments, the non-natural amino acid comprises a saccharide moiety. In some embodiments, the water soluble polymer is linked to the polypeptide via a saccharide moiety. Also described herein are prodrugs of the 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 the non-natural amino acids, non-natural amino acid polypeptides, and modified non-natural amino acid polypeptides; such metabolites may have a desired activity that complements or synergizes with the acivity of the non-natural amino acids, non-natural amino acid polypeptides, and modified non-natural amino acid polypeptides. Also described herein are the use of the non-natural amino acids, non-natural amino acid polypeptides, and modified non-natural amino acid polypeptides described herein to provide a desired metabolite to an organism, including a patient in need of such metabolite.

[0021] Also described herein are libraries of the non-natural amino acids described herein or libraries of the nonnatural amino acid polypeptides described herein, or libraries of the modified non-natural amino acid polypeptides described herein, or combination libraries thereof, wherein the members of the library comprise on oxime-linkage formed in the presence of an accelerant described herein (although such a reaction may be less efficient in the absence of an accelerant described herein).

[0022] Also described herein are methods for screening libraries described herein for a desired activity, or for using the arrays to screen the libraries described herein, or for other libraries of compounds and/or polypeptides and/or polynucleotides for a desired activity. Also described herein is the use of such activity data from library screening to develop and discover new therapeutic agents, as well as therapeutic agents themselves.

CA 02625540 2008-04-09

WO 2007/056448

PCT/US2006/043472 ^: [à020]ⁱ4^ÂB&-k^!èArib&à''hererh-aré-methods for accelerating the conjugation of small molecules, including by way of example the conjugation of a hydroxylamine group on one reagent with a carbonyl group on another reagent, wherein neither reagent is a non-natural amino acid. In other words, the use of accelerants described herein is not limited to the further functionalization of non-natural amino acids and non-natural amino acid polypeptides, but can also be used to facilitate the formation of oxime bonds between any two reagents. By way of example only, this embodiment includes the use of accelerants in the formation/building of dynamic libraries from hydroxylaminecontaining reagents and carbonyl-containing reagents. Of course, such dynamic libraries can include non-natural amino acids, but such dynamic libraries are not limited to the inclusion of non-natural amino acids.

[0024] Also described herein are methods of increasing therapeutic half-life, serum half-life or circulation time of a polypeptide that comprise substituting a non-natural amino acid for any one or more amino acids in a naturally occurring polypeptide, and/or adding a non-natural amino acid into a naturally occurring polypeptide, and/or linking the polypeptide to a water soluble polymer via an oxime bond formed in the presence of an accelerant described herein (although such a reaction may be less efficient in the absence of an accelerant described herein).

[0025] Also described herein are methods of treating a patient in need of such treatment with an effective amount of a pharmaceutical composition comprising a polypeptide comprising a non-natural amino acid comprising an oxime bond formed in the presence of an accelerant described herein (although such a reaction may be less efficient in tiie absence of an accelerant described herein) and a pharmaceutically acceptable carrier. In some embodiments, the non-natural amino acid is linked to a water soluble polymer.

[0026] In any of the aforementioned aspects or embodiments, the use of an accelerant includes the use of a single accelerant or multiple accelerants. Further, in any of the aforementioned aspects or embodiments the molar ratio of accelerant 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. Further, in any of the aforementioned aspects or embodiments the molar ratio of accelerant 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. Further, in any of the aforementioned aspects or embodiments the accelerant includes compounds that can be substantially removed in vacuo from the resulting oxime-containing compound. Further, in any of the aforementioned aspects or embodiments the accelerant includes compounds containing an amine moiety, a semicarbazide moiety, a hydrazine, or a hydrazide moiety.

[0027] Further, in any of the aforementioned aspects or embodiments, the accelerant is selected from the group consisting of bifunctional aromatic amines, oxoamine derivatives, and compounds having the following structures:

R_y h₂n

H χ,,ζΝ

HXV W HX'V

Aux n

Η_?Ν'

<img file="CA2625540C_D0001.tif" />

^Η2Ν^Νγ'^ΝΗ2 Η2Ν'^Ι'^Ιγ'^ΙΝΠ2 Η2Ν^χΝγ·^Ν^ H2N^Nty⁰^ H2N''^N)S/' ^H2ⁿ'\^Sk

ΝΗ

CA 02625540 2008-04-09

WO 2007/056448

PCT/US2006/043472 ' Éÿ’âÎtf â'électéS'from the group consisting of: L_x-H, L_x-alkyl, Lx-aryl, Lx-heteroaryl, Lx-alkenyl,

Lx-alkynyl, L_x-alkoxy, and Lx-alkylamine, where Lx is a bond, C(=O), C(=NH), C(=NH)-NH and SO, SO₂.

[0028] In a further embodiment, the accelerant is a bifunctional aromatic amine. In a further embodiment, the aromatic amine is selected from the group:

Bifuctionai aromatic amines:

<img file="CA2625540C_D0002.tif" />

[0029] In a further embodiment, the accelerant is an oxoamine derivative. In a further embodiment, the oxoamine derivative is selected from the group:

Oxoamine derivatives:

H H H H ^H H N 1 N JL 'V ΙΐΛ* Me'%'^Et Me'V Me'^N\A μΛΑ&lt;Η₂ Me' ri/V

Me'

[0030] In a further embodiment, the accelerant include compounds selected from the group consisting of:

w'Y

NH

Η H H

H₂N Υ Η₂Ν'^Νγ^Η H₂N

0

H,N' wherein R_x, R_y and R_z are selected from the group consisting of: L_x-H, L_x-alkyl, L_x-aryl, Lx-heteroaryl, L_x-alkenyl, Lx-alkynyl, L_x-alkoxy, and L_x-alkylamine, where L_x is a bond, C(=O), C(=NH), and C(=NH)-NH. Further, in any of the aforementioned aspects or embodiments, the accelerant is selected from the compounds presented in FIG. 5, FIG. 9, or FIG. 10, including by way of example any of compounds 6, 8, 10, 7, and 20 of Figure 5. In any of the aforementioned aspects or embodiments, the accelerant includes an agent that can form a hydrazone upon reaction with a carbonyl-containing group. Further, in any of the aforementioned aspects the accelerant activity depends on the rate of reaction with the ketone moiety and the stability of the resulting intermediate. Further, in any of the aforementioned aspects or embodiments, the pH of the reaction mixture comprising the accelerant, the carbonylcontaining 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. Note however, for any pH ranges described herein, the phrase “between about” in reference to a low and high pH value means that the “about” applies to both the low and the high pH value; by way of example only, “between about 3.0 and 10.0” is equivalent to “between about 3.0 and about 10.0.” Furthermore, unless stated otherwise, for any range presented herein, in which “about” is presented before a lower limit and not before an upper limit (or in the case where “about” is placed before an upper limit and not a lower limit), then that is understood to mean that the word “about” appears before both limits of the range.

[0031] Further, in any of the aforementioned aspects or embodiments, the term “accelerant” includes a compound having a least one of the following properties: (a) increase the rate of reaction between a carbonyl-containing compound and a hydroxylamine-containing compound to form an oxime-containing compound, where the increase in rate is relative to the reaction in the absence of the accelerant; (b) lower the activation energy of the reaction

CA 02625540 2008-04-09

WO 2007/056448

PCT/US2006/043472

TetweeyaicaihonylA&amp;iWmïî'gÎdtnpound and a hydroxylamine-containing compound to form an oxime-containing compound, where the decrease in activation energy is relative to the reaction in the absence of the accelerant; (c) increase the yield of an oxime-containing compound from the reaction of a carbonyl-containing compound with a hydroxylamine-containing compound, where the increase in yield is relative to the reaction in the absence of the accelerant; (d) lower the temperature at which a carbonyl-containing compound reacts with a hydroxylaminecontaining compound to form an oxime-containing compound, where the decrease in temperature is relative to the reaction in the absence of the accelerant; (e) decrease the time necessary to react a carbonyl-containing compound with a hydroxylamine-containing compound to form an oxime-containing compound, wherein the decrease in time is relative to the reaction in the absence of accelerant; (f) decrease the amount of reagents necessary to form an oxime-containing compound, wherein the decrease in amount of reagents is relative to the reaction in the absence of accelerant; (g) decrease the side products resulting from the reaction of a carbonyl-containing compound with a hydroxylamine-containing compound to form an oxime-containing compound, wherein the decrease in side products is relative to the reaction in the absence of accelerant; (h) does not irreversibly destroy the tertiary structure of a polypeptide undergoing an oxime-forming reaction in the presence of an accelerant (excepting, of course, where the purpose of the Reaction is to destroy such tertiary structure); (i) can be separated from an oxime-containing compound in vacuo; and (j) modulate the reaction of a carbonyl-containing compound with a hydroxylaminecontaining compound. In a further embodiments, the accelerant 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, or all of the aforementioned properties. In a further embodiment, the accelerant has none of the aforementioned properties.

[0032] It is to be understood that the methods and compositions described herein are not limited to the particular methodology, protocols, cell lines, constructs, and reagents described herein and as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the methods and compositions described herein, which will be limited only by the appended claims.

[0033] As used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural reference unless the context clearly indicates otherwise.

[0034] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which the inventions described herein belong. Although any methods, devices, and materials similar or equivalent to those described herein can be used in the practice or testing of the inventions described herein, the preferred methods, devices and materials are now described.

[0035] The terms “alkoxy,” “alkylamino” and “alkylthio” (or thioalkoxy) are used in their conventional sense, and refer to those alkyl groups attached to the remainder of the molecule via an oxygen atom, an amino group, or a sulfur atom, respectively.

[0036] The term “alkyl,” by itself or as part of another substituent, means, unless otherwise stated, a straight or branched chain, or cyclic hydrocarbon radical, or combination thereof, which may be fully saturated, mono- or polyunsaturated and can include di- and multivalent radicals, having the number of carbon atoms designated (i.e. C_rCio means one to ten carbons). Examples of saturated hydrocarbon radicals include, but are not limited to, groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl, (cyclohexyl)methyl, cyclopropylmethyl, homologs 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

CA 02625540 2008-04-09

WO 2007/056448 PCT/US2006/043472 groups-mSfi&amp;fc'buf to, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3(1,4-pentadienyl), ethynyl, 1- and 3-propynyl, 3-butynyl, and the higher homologs and isomers. The term “alkyl,” unless otherwise noted, is also meant to include those derivatives of alkyl defined in more detail below, such as “heteroalkyl.” Alkyl groups which are limited to hydrocarbon groups are termed “homoalkyl”·

[0037] The term “alkylene” by itself or as part of another substituent means a divalent radical derived from an alkane, as exemplified, but not limited, by the structures -CH₂CH₂- and —CH₂CH₂CH₂CH₂-, and further includes those groups described below as “heteroalkylene.” Typically, an alkyl (or alkylene) group will have from 1 to 24 carbon atoms, with those groups having 10 or fewer carbon atoms being a particular embodiment of the methods and compositions described herein. A “lower alkyl” or “lower alkylene” is a shorter chain alkyl or alkylene group, generally having eight or fewer carbon atoms.

[0038] The term “amino acid” refers to naturally occurring and non-natural amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally encoded amino acids are the 20 common amino acids (alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine) and pyrrolysine and selenocysteine. Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an a carbon that is bound to a 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 structure as a naturally occurring amino acid.

[0039] Amino acids may be referred to herein by either their commonly known three letter symbols or by the oneletter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.

[0040] An “amino terminus modification group” refers to any molecule that can be attached to the amino terminus of a polypeptide. Similarly, a “carboxy terminus modification group” refers to any molecule that can be attached to the carboxy terminus of a polypeptide. Terminus modification groups include but are not limited to various water soluble polymers, peptides or proteins such as serum albumin, or other moieties that increase serum half-life of peptides.

[0041] By “antibody fragment” is meant any form of an antibody other than the full-length form. Antibody fragments herein include antibodies that are smaller components that exist within full-length antibodies, and antibodies that have been engineered. Antibody fragments include but are not limited to Fv, Fc, Fab, and (Fab')₂, single chain Fv (scFv), diabodies, triabodies, tetrabodies, bifunctional hybrid antibodies, CDR1, CDR2, CDR3, combinations of CDR’s, variable regions, framework regions, constant regions, heavy chains, light chains, and variable regions, and alternative scaffold non-antibody molecules, 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 a single chain Fv (scFv), comprised of the variable regions of the immunoglobulin heavy and light chain, covalently connected by a peptide linker (S-z Hu et al., 1996, Cancer Research, 56, 3055-3061). These small (Mr 25,000) proteins generally retain specificity and affinity for antigen in a single polypeptide and can provide a convenient building block for larger, antigen-specific molecules. Unless specifically noted otherwise, statements and claims that use the term “antibody” or “antibodies” specifically includes “antibody fragment” and “antibody fragments.”

[0042] The term “aryl” means, unless otherwise stated, a polyunsaturated, aromatic, hydrocarbon substituent which can be a single ring or multiple rings (including but not limited to from 1 to 3 rings) which are fused together

CA 02625540 2014-01-28 or linked covalently. The term heteroaryl refers to aiyl groups (or rings) that contain from one to four heteroatoms selected from N, 0, and S, wherein the nitrogen and sulfur atoms are optionally oxidized, and the nitrogen atomfs) are optionally quatemized. A heteroaryl group can be attached to the remainder of the molecule through a hoteroatom. NorUimiting examples of aryl and heteroaryl groups include phenyl, 1-naphihyl, 2-naphthyl, 45 biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pynolyl, 3-pyrazolyl, 2-imldazotyI, 4-imidazolyi, pyrazinyl, 2-oxazolyI, 4oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazotyl, 4-thiazolyl, 5* thiazolyl, 2-finyl, 3-foryl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyd, 4-pyridyI, 2-pyriroidyI, 4-pyrimidyl, 5benzothiazolyl, purinyl, 2-bsnzfaudazolyl, 5-indolyl, l-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyi, 3quinolyl, and 6-qeinolyl. Substituents for each of the above noted aryl and heteroaryl ring systems are selected from the group of acceptable substituents described below.

(0043] For brevity, die 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 shove. Thus, the term “aralkyl” or alkaiyl is meant to include those radicals in which an aryl group is attached to an alkyl group (including but not limited to, benzyl, phenethyl, pyridyhnethyl and the like) including those alkyl groups in which a carbon atom (induding but not limited to, a methylene group) has been replaced by, for example, an oxygen atom (including but not limited to, phenoxymethyi, 2-pyridyloxymethyl, 3-(I-naphthyloxy)propyl, and the like), (0044] A “bifimctional polymer” refers to a polymer comprising two discrete functional groups that are capable of reacting specifically with other moieties (including but not limited to, amino acid side groups) to form covalent or non-covalent linkages. 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 a second biological component, may be used to form a conjugate that includes the first biologically active component, the bifunctional linker and fire second biologically active component. Many procedures and linker molecules for attachment of varions compounds to peptides are known. See, e.g., European Patent Application No. 188,256; U.S. Patent Nos. 4,671,958, 4,659,839, 4,414,148, 4,©9,784; 4,680,338; and 4,569,789. Amulti25 functional polymer” refers to a polymer comprising two or more discrete functional groups that are capable of reacting specifically with other moieties (including but not limited to, amino acid side groups) to form covalent or non-covalent linkages, Λ bi-fUnctional polymer or multi-functional polymer may be any desired length or molecular weight, and may be selected to provide a particular desired spacing or conformation between one or more molecules linked to a compound and molecules it hinds to or the compound.

(0045] The terra biologically active molecule”, biologically active moiety” or biologically active agent” when used herein means any substance which can affect any physical or biochemical properties of a biological system, pathway, molecule, or interaction relating to an organism, including but not limited to, viruses, bacteria, bacteriophage, transposon, prion, insects, fungi, plants, animals, and humans. In particular, as used herein, biologically active molecules include but are not limited to any substance intended for diagnosis, cure, mitigation, treatment, or prevention of disease in humans or other animals, or to otherwise enhance physical or mental wellbeing of humans or animals. Examples of biologically active molecules include^ but are not limited to, peptides, proteins, enzymes, small molecule drugs, hard drugs, soft drugs, carbohydrates, inorganic atoms or molecules, dyes, lipids, nucleosides, radionuclides, oligonucleotides, toxins, cells, viruses, liposomes, microparticles and micelles. Classes of biologically active agents that are suitable for use with the methods and conçositions described herein include, hut are not limited to, drugs, prodrugs, radionuclides, imaging agents, polymers, antibiotics, fongicides, anti-viral agents, anti-inflammatory agents, anti-tumor agents, cardiovascular agents, anti-anxiety agents, hormones, growth firctors, steroidal agents, microbially derived toxins, and file like.

CA 02625540 2008-04-09

WO 2007/056448

PCT/US2006/043472

at least two polypeptides which interact with each other and result in the transformation of unfolded or improperly folded polypeptides to native, properly folded polypeptides.

[0047] A “comparison window,” as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison can be conducted, including but not limited to, by the local homology algorithm of Smith and Waterman (1970) Adv. Appl. Math. 2:482c, by the homology alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443, by the search for similarity method of Pearson and Lipman (1988) Proc. Nat’l. Acad. Sci. USA 85:2444, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by manual alignment and visual inspection (see, e.g., Ausubel etal., Current Protocols in Molecular Biology (1995 supplement)).

[0048] One example of an algorithm that is suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1997) Nuc. Acids Res. 25:3389-3402, and Altschul et al. (1990) J. Mol. Biol. 215:403-410, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) or 10, M=5, N=-4 and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1992) Proc. Natl. Acad. Sci. USA 89:10915) alignments (B) of 50, expectation (E) of 10, M=5, N=-4, and a comparison of both strands. The BLAST algorithm is typically performed with the “low complexity” filter turned off.

[0049] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5787). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, less than about 0.01, and in another embodiment less than about

0.001.

[0050] The term “conservatively modified variants” applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, “conservatively modified variants” refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are “silent variations,” which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid. One of ordinary skill will recognize that each codon in a nucleic acid (except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally

CA 02625540 2008-04-09

WO 2007/056448

PCT/US2006/043472

<img file="CA2625540C_D0003.tif" />

each described sequence.

10051] As to amino acid sequences, one of ordinary skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a “conservatively modified variant” where the alteration results in the deletion of an amino acid, addition of an amino acid, or substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the methods and compositions described herein.

[0052] The following eight groups each contain amino acids that are conservative substitutions for one another:

1)

3)

4)

5)

6)

7)

8)

Alanine (A), Glycine (G);

Aspartic acid (D), Glutamic acid (E);

Asparagine (N), Glutamine (Q);

Arginine (R), Lysine (K);

Isoleucine (I), Leucine (L), Methionine (M), Valine (V); Phenylalanine (F), Tyrosine (Y), Tryptophan (W); Serine (S), Threonine (T); and

Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins:Structures and Molecular Properties ÇW H Freeman &amp; Co.; 2nd edition (December 1993)

[0053] The terms “cycloalkyl” and “heterocycloalkyl”, by themselves or in combination with other terms, represent, unless otherwise stated, cyclic versions of “alkyl” and “heteroalkyl”, respectively. Thus, a cycloalkyl or heterocycloalkyl include saturated, partially unsaturated and fully unsaturated ring linkages. Additionally, for heterocycloalkyl, a heteroatom can occupy the position at which the heterocycle is attached to the remainder of the molecule. Examples of cycloalkyl include, but are not limited to, cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3cyclohexenyl, cycloheptyl, and the like. Examples of heterocycloalkyl include, but are not limited to, 1-(1,2,5,6tetrahydropyridyl), 1-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl, 1-piperazinyl, 2-piperazinyl, and the like. Additionally, the term encompasses bicyclic and tricyclic ring structures. Similarly, the term “heterocycloalkylene” by itself or as part of another substituent means a divalent radical derived from heterocycloalkyl, and the term “cycloalkylene” by itself or as part of another substituent means a divalent radical derived from cycloalkyl.

[0054] “Denaturing agent” or “dénaturant,” as used herein, is defined as any compound or material which will cause a reversible unfolding of a protein. The strength of a denaturing agent or dénaturant will be determined both by the properties and the concentration of the particular denaturing agent or dénaturant. Suitable denaturing agents or dénaturants may be chaotropes, detergents, organic, water miscible solvents, phospholipids, or a combination of two or more such agents. Suitable chaotropes include, but are not limited to, 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), Sarkosyl, mild non-ionic detergents (e.g., digitonin), mild cationic detergents such as N-&gt;2,3-(Dioleyoxy)-propyl-N,N,N-trimethylammonium, mild ionic detergents (e.g. sodium cholate or sodium deoxycholate) or zwitterionic detergents including, but not limited to, sulfobetaines (Zwittergent), 3-(3-chlolamidopropyl)dimethylammonio-l-propane sulfate (CHAPS), and 3-(3chlolamidopropyl)dimethylammonio-2-hydroxy-l-propane sulfonate (CHAPSO). Organic, water miscible solvents

CA 02625540 2008-04-09

WO 2007/056448

PCT/US2006/043472

Süeîî OâelO&amp;é] lbWr Mtainoï.® (especially C₂ - C₄ alkanols such as ethanol or isopropanol), or lower alkandiols (especially C₂ - C₄ alkandiols such as ethylene-glycol) may be used as dénaturants. Phospholipids useful in the methods and compositions described herein may be naturally occurring phospholipids such as phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, and phosphatidylinositol or synthetic phospholipid derivatives or variants such as dihexanoylphosphatidylcholine or diheptanoylphosphatidylcholine. [0055] The term “desired functionality,” as used herein refers to any one or all of the following groups: a label; a dye; a polymer; a water-soluble polymer; a derivative of polyethylene glycol; a photocrosslinker; a cytotoxic compound; a drug; an affinity label; a radionuclide; a derivative of biotin; a quantum dot; a nanotransmitter; a radiotransmitter; a photoaffinity label; a reactive compound; a resin; a second protein or polypeptide or polypeptide analog; an antibody or antibody fragment; a metal chelator; a cofactor; a fatty acid; a carbohydrate; a polynucleotide; a DNA; a RNA; an antisense polynucleotide; a saccharide, a water-soluble dendrimer, a cyclodextrin, a biomaterial; a nanoparticle; a spin label; a fluorophore, a metal-containing moiety; a radioactive moiety; a novel functional group; a group that covalently or noncovalently interacts with other molecules; a photocaged moiety; an actinic radiation excitable moiety; a ligand; a photoisomerizable moiety; biotin; a biotin analogue; a moiety incorporating a heavy atom; a chemically cleavable group; a photocleavable group; an elongated side chain; a carbon-linked sugar; a redox-active agent; an amino thioacid; a toxic moiety; an isotopically labeled moiety; a biophysical probe; a phosphorescent group; a chemiluminescent group; an electron dense group; a magnetic group; an intercalating group; a chromophore; an energy transfer agent; a biologically active agent; a detectable label; a small molecule; an inhibitory ribonucleic acid, and any combination of the above.

[0056] The term “dicarbonyl” as used herein refers to a group containing at least two moieties selected from the group consisting of -C(O)-, -S(O)-, -S(O)₂-, and -C(S)-, including, but not limited to, 1,2-dicarbonyl groups, a 1,3dicarbonyl groups, and 1,4-dicarbonyl groups, and groups containing a least one ketone group, and/or at least one aldehyde groups, 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, ketoacids, ketoesters, and ketothioesters. In addition, such groups may be part of linear, branched, or cyclic molecules. The two moieties in the dicarbonyl group may be the same or different, and may include substituents that would produce, by way of example only, an ester, a ketone, an aldehyde, a thioester, or an amide, at either of the two moieties.

[0057] The term “effective amount” as used herein refers to that amount of the (modified) non-natural amino acid polypeptide being administered which will relieve to some extent one or more of the symptoms of the disease, condition or disorder being treated. Compositions containing the (modified) non-natural amino acid polypeptide described herein can be administered for prophylactic, enhancing, and/or therapeutic treatments.

[0058] The terms “enhance” or “enhancing” means to increase or prolong either in degree, amount, potency or duration a desired effect. Thus, in regard to enhancing the effect of therapeutic agents, the term “enhancing” refers to the ability to increase or prolong, either in potency or duration, the effect of other therapeutic agents on a system. An “enhancing-effective amount,” as used herein, refers to an amount adequate to enhance the effect of another therapeutic agent in a desired system. When used in a patient, amounts effective for this use will depend on the severity and course of the disease, disorder or condition, previous therapy, the patient's healüi status and response to the drugs, and the judgment of the treating physician.

[0059] As used herein, the term “eukaryote” refers to organisms belonging to the phylogenetic domain Eucarya such as animals (including but not limited to, mammals, insects, reptiles, birds, etc.), ciliates, plants (including but not limited to, monocots, dicots, algae, etc.), fungi, yeasts, flagellates, microsporidia, protists, etc.

CA 02625540 2008-04-09

WO 2007/056448

PCT/US2006/043472 hfe “tëtïHS '“ïüheti'foàl “group”, “active moiety”, “activating group”, “leaving group”, “reactive site”, “chemically reactive group” and “chemically reactive moiety” are used in the art and herein to refer to distinct, definable portions or units of a molecule. The terms are somewhat synonymous in the chemical arts and are used herein to indicate the portions of molecules that perform some function or activity and are reactive with other molecules.

[0061] The term “halogen” includes fluorine, chlorine, iodine, and bromine.

[0062] The term “heteroalkyl,” by itself or in combination with another term, means, unless otherwise stated, a stable straight or branched chain, or cyclic hydrocarbon radical, or combinations thereof, consisting of the stated number of carbon atoms and at least one heteroatom selected fiom the group consisting of Ο, N, Si and S, and wherein the nitrogen and sulfur atoms may optionally be oxidized and the nitrogen heteroatom may optionally be quatemized. The heteroatom(s) Ο, N and S and Si may be placed at any interior position of the heteroalkyl group or at the position at which the alkyl group is attached to the remainder of the molecule. Examples include, but are not limited to, -CH₂-CH₂-O-CH₃, -CH₂-CH₂-NH-CH₃, -CH₂-CH₂-N(CH₃)-CH₃, -CH₂-S-CH₂-CH₃, -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 may be consecutive, such as, for example, -CH₂-NH-OCH₃ and -CH₂-O-Si(CH₃)₃. Similarly, the term “heteroalkylene” by itself or as part of another substituent means a divalent radical derived fiom heteroalkyl, as exemplified, but not limited by, -CH₂-CH₂-S-CH₂-CH₂- and -CH₂-S-CH₂-CH₂-NH-CH₂-. For heteroalkylene groups, the same or different heteroatoms can also occupy either or both of the chain termini (including but not limited to, alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, aminooxyalkylene, and the like). Still further, for alkylene and heteroalkylene linking groups, no orientation of the linking group is implied by 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-.

[0063] The terms “identical” or percent “identity,” in the context 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 residues or nucleotides that are the same (i.e., about 60% identity, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% identity over a specified region), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms (or other algorithms available to persons of ordinary skill in the art) or by manual alignment and visual inspection. This definition also refers to the complement of a test sequence. The identity can exist over a region that is at least about 50 amino acids or nucleotides in length, or over a region that is 75-100 amino acids or nucleotides in length, or, where not specified, across the entire sequence of a polynucleotide or polypeptide.

[0064] For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.

[0065] The term “isolated,” when applied to a nucleic acid or protein, denotes that the nucleic acid or protein is free of at least some of the cellular components with which it is associated in the natural state, or that the nucleic acid or protein has been concentrated to a level greater than the concentration of its in vivo or in vitro production. It can be in a homogeneous state. Isolated substances can be in either a dry or semi-dry state, or in solution, including

CA 02625540 2008-04-09

WO 2007/056448

PCT/US2006/043472 solution. It can be a component of a pharmaceutical composition that comprises additional pharmaceutically acceptable carriers and/or excipients. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. A protein which is the predominant species present in a preparation is substantially purified. In particular, an isolated gene is separated from open reading frames which flank the gene and encode a protein other than the gene of interest. The term “purified” denotes that a nucleic acid or protein gives rise to substantially one band in an electrophoretic gel. Particularly, it 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 greater pure.

[0066] The term “linkage” or “linker” is used herein to refer to groups or bonds that normally are formed as the result of a chemical reaction and typically are covalent linkages or bonds (the process of creating such a linkage or linker is referred to herein as linking/linked or coupling/coupled, as well as other synonyms recognized by those in the art). Hydrolytically stable linkages means that the linkages are substantially stable in water and do not react with water at useful pH values, including but not limited to, under physiological conditions for an extended period of time, perhaps even indefinitely. Hydrolytically unstable or degradable linkages means that the linkages are degradable in water or in aqueous solutions, including for example, blood. Enzymatically unstable or degradable linkages means that the linkage can be degraded by one or more enzymes. As understood in the art, PEG and related polymers may include degradable linkages in the polymer backbone or in the linker group between the polymer backbone and one or more of the terminal functional groups of the polymer molecule. For example, ester linkages formed by the reaction of PEG carboxylic acids or activated PEG carboxylic acids with alcohol groups on a biologically active agent generally hydrolyze under physiological conditions to release the agent. Other hydrolytically degradable linkages include but are not limited to carbonate linkages; imine linkages resulted from reaction of an amine and an aldehyde; phosphate ester linkages formed by reacting an alcohol with a phosphate group; hydrazone linkages which are reaction product of a hydrazide and an aldehyde; acetal linkages that are the reaction product of an aldehyde and an alcohol; orthoester linkages that are the reaction product of a formate and an alcohol; peptide linkages formed by an amine group, including but not limited to, at an end of a polymer such as PEG, and a carboxyl group of a peptide; and oligonucleotide linkages formed by a phosphoramidite group, including but not limited to, at the end of a polymer, and a 5' hydroxyl group of an oligonucleotide.

[0067] As used herein, the term “medium” or “media” includes any culture medium, solution, solid, semi-solid, or rigid support that may support or contain any host cell, including bacterial host cells, yeast host cells, insect host cells, plant host cells, eukaryotic host cells, mammalian host cells, CHO cells, prokaryotic host cells, E. coli, or Pseudomonas host cells, and cell contents. Thus, the term may encompass medium in which the host cell has been grown, e.g., medium into winch the polypeptide has been secreted, including medium either before or after a proliferation step. The term also may encompass buffers or reagents that contain host cell lysates, such as in the case where the polypeptide is produced intracellularly and Hie host cells are lysed or disrupted to release the polypeptide. [0068] A “metabolite” of a (modified) non-natural amino acid polypeptide disclosed herein is a derivative of that (modified) non-natural amino acid polypeptide that is 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 that is formed when the (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 reactions catalyzed by enzymes) by which a particular substance is changed by an organism. Further information on metabolism may be obtained from The Pharmacological Basis of Therapeutics, 9th Edition, McGraw-Hill (1996). Metabolites of the (modified) non-natural amino acid polypeptide disclosed herein can be

CA 02625540 2008-04-09

WO 2007/056448 PCT/US2006/043472 ileûtiMfeByfeÿ'’ aàfeiâMàti'oïï^::of (modified) nôn-natural amino acid polypeptide to a host and analysis of tissue samples from the host, or by incubation of (modified) non-natural amino acid polypeptide with hepatic cells in vitro and analysis of the resulting compounds.

[0069] The term “modified,” as used herein refers to the presence of a post-translational modification on a polypeptide. The form “(modified)” term means that the polypeptides being discussed are optionally modified, that is, the polypeptides under discussion can be modified or unmodified.

[0070] As used herein, the term “modulated serum half-life” means the positive or negative change in circulating half-life of a modified polypeptide relative to its non-modified form. Serum half-life is measured by taking blood samples at various time points after administration of the polypeptide, and determining the concentration of that molecule in each sample. Correlation of the serum concentration with time allows calculation of the serum half-life. Increased serum half-life desirably has at least about two-fold, but a smaller increase may be useful, for example where it enables a satisfactory dosing regimen or avoids a toxic effect. In some embodiments, the increase is at least about three-fold, at least about five-fold, or at least about ten-fold.

[0071] The term “modulated therapeutic half-life” as used herein means the positive or negative change in the half-life of therapeutically effective amount of a modified polypeptide, relative' to its non-modified form. Therapeutic half-life is measured by measuring pharmacokinetic and/or pharmacodynamic properties of the polypeptide at various time points after administration. Increased therapeutic half-life desirably enables a particular beneficial dosing regimen, a particular beneficial total dose, or avoids an undesired effect. In some embodiments, the increased therapeutic half-life results from increased potency, increased or decreased binding of the modified molecule to its target, increased or decreased breakdown of the molecule by enzymes such as proteases, or an increase or decrease in another parameter or mechanism of action of the non-modified molecule.

[0072] As used herein, the term “non-eukaryote” refers to non-eukaryotic organisms. For example, a noneukaryotic organism can belong to the Eubacteria (including but not limited to, Escherichia coli, Thermus thermophilus, Bacillus stearothermophilus, Pseudomonas fluorescens, Pseudomonas aeruginosa, Pseudomonas putida etc.) phylogenetic domain, or the Archaea (including hut not limited to, Methanococcus jannaschii, Methanobacterium thermoautotrophicum, Halobacterium such as Haloferax volcanii and Halobacterium species NRC-1, Archaeoglobus fulgidus, Pyrococcus furiosus, Pyrococcus horikoshii, Aeuropyrum pernix, etc.) phylogenetic domain.

[0073] A “non-natural amino acid” refers to an amino acid that is not one of the 20 common amino acids or pyrrolysine or selenocysteine; other terms that may be used synonymously with the term “non-natural amino acid” is “non-naturally encoded amino acid,” “unnatural amino acid,” “non-naturally-occurring amino acid,” and variously hyphenated and non-hyphenated versions thereof. The term “non-natural amino acid” includes, but is not limited to, amino acids that occur naturally by modification of a naturally encoded amino acid (including but not limited to, the 20 common amino acids or pyrrolysine and selenocysteine) but are not themselves incorporated into a growing polypeptide chain by the translation complex. Examples of naturally-occurring amino acids that are not naturallyencoded include, but are not limited to, A-acetylglucosaminyl-L-serine, W-acetylglucosaminyl-L-threonine, and Ophosphotyrosine.

[0074] The term “nucleic acid” refers to deoxyribonucleotides, deoxyribonucleosides, ribonucleosides or ribonucleotides and polymers thereof in either single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides which have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless specifically limited otherwise, the term also refers oligonucleotide analogs incuding PNA (peptidonucleic acid),

CA 02625540 2008-04-09

WO 2007/056448

PCT/US2006/043472 ahalbgs^ofy9s&amp;t-1)864-ih'Mhtïsônse technology (phosphorothioates, phosphoroamidates, and the like). Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (including but not limited to, degenerate codon substitutions) and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (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)).

[0075] “Oxidizing agent,” as used hereinwith respect to protein refolding, is defined as any compound or material which is capable of removing an electron from a compound being oxidized. Suitable oxidizing agents include, but are not limited to, oxidized glutathione, cystine, cystamine, oxidized dithiothreitol, oxidized erythreitol, and oxygen. A wide variety of oxidizing agents are suitable for use in the methods and compositions described herein.

[0076] 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 exemplary embodiments are listed, for example, in commercial supplier catalogs, such as Shearwater Corporation's catalog “Polyethylene Glycol and Derivatives for Biomedical Applications” (2001).

[0077] The terms “polypeptide,” “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. That is, a description directed to a polypeptide applies equally to a description of a peptide and a description of a protein, and vice versa. The terms apply to naturally occurring amino acid polymers as well as amino acid polymers 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.

[0078] The term “post-translationally modified” refers to any modification of a natural or non-natural amino acid that occurs to such an amino acid after it has been incorporated into a polypeptide chain. The term encompasses, by way of example only, co-translational in vivo modifications, co-translational in vitro modifications (such as in a cellfree translation system), post-translational in vivo modifications, and post-translational in vitro modifications.

[0079] A “prodrug” refers to an agent that is converted into the parent drug in vivo. Prodrags are often useful because, in some situations, they may be easier to administer than the parent drug. They may, for instance, be bioavailable by oral administration whereas the parent is not. The prodrug may also have improved solubility in pharmaceutical compositions over the parent drug. A pro-drug includes a pharmacologically inactive, or reducedactivity, derivative of an active drug. Prodrugs may be designed to modulate the amount of a drag or biologically active molecule that reaches a desired site of action through the manipulation of the properties of a drug, such as physiochemical, biopharmaceutical, or pharmacokinetic properties. Prodrugs are converted into active drag within the body through enzymatic or non-enzymatic reactions. Prodrugs may provide improved physiochemical properties such as better solubility, enhanced delivery characteristics, such as specifically targeting a particular cell, tissue, organ or ligand, and improved therapeutic value of the drug.

[0080] In prophylactic applications, compositions containing the (modified) non-natural amino acid polypeptide are administered to a patient susceptible to or otherwise at risk of a particular disease, disorder or condition. Such an amount is defined to be a “prophylactically effective amount.” In this use, the precise amounts also depend on the patient's state of health, weight, and the like. It is considered well within the skill of the art for one to determine such prophylactically effective amounts by routine experimentation (e.g., a dose escalation clinical trial).

CA 02625540 2014-01-28

[flOS'l] The term protected foftrs to the presence of a “protecting group” or moiety that prevents reaction of the chemically reactive functional group under certain reaction conditions. The protecting group will vary depending on foe type of chemically reactive group being protected. For example, if the chemically reactive group is an amine or a hydrazide, the protecting group can be selected from the group of tert-bufyfoxycarbonyl (t-Boo) and 9fluorenylmefooxycafbonyl (Fmoe). If the chemically reactive group is a thiol, the protecting group can be orfoopyridyldisulfide. If tire chemically reactive group is a carboxylic add, such as butanoic or propionic acid, or a hydroxyl group, the protecting group can be benzyl or an alkyl group such as methyl, ethyl, or teri-butyL Other protecting groups known in the ait may also be used in or with the methods and compositions described herein, including photolabile groups such as Nvoc and MeNvoc,

[0082] By way of example only, blocking/protecting groups may be selected from:

H £ H^⁰· h₂ %

Bn

<img file="CA2625540C_D0004.tif" />

<img file="CA2625540C_D0005.tif" />

H,\ CH, (H_aC)sC-^ (H₃C)_sC^SK t-butyl TBDMS (HaCJaSi

<img file="CA2625540C_D0006.tif" />

(CH₃)₃C&lt;^O'T^ o

Boc

HsCO

H₂

CpMBn (CeHsJsC— trityl

O

<img file="CA2625540C_D0007.tif" />

acetyl

<img file="CA2625540C_D0008.tif" />

[0083] Other protecting groups are described in Greene and Wufs, Protective Groups in Organic Synthesis, 3rd Bd„ John Wiley &amp; Sons, New York NY, 1999.

[0084] A recombinant host cell or host cell refers to a cell that includes an exogenous polynucleotide, regardless of the method used for insertion, for example, direct uptake, transduction, f-msting, or other methods known in the art to create recombinant host cells. The exogenous polynucleotide may be maintained as a nonintegrated vector, for example, a plasmid, or alternatively, may be integrated into the host genome.

[8085] Reducing agent,” as used heroin with respect to protein refolding, is defined as any compound or material which maintains snl&amp;ydryl groups in foe reduced state and reduces intra- or intramolecular disulfide bonds. Suitable reducing agents include, but an not limited to, tiithiothreitol (DTI), 2-mercaptoefoanoI, dithioeiythritol, cysteine, cysteamine (2-aminocfoancthiol), and reduced glutathione. A wide variety of reducing agents are suitable for use in the methods and compositions described herein.

[00861 Refolding,” as used herein describes any process, reaction or method which transforms disulfide bond containing polypeptides from an improperly folded or unfolded state to a native or properly folded conformation with respect to disulfide bonds.

CA 02625540 2008-04-09

WO 2007/056448

PCT/US2006/043472

J Çfig-pfyhs'e ‘yhefitively (or specifically) hybridizes to” refers to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under stringent hybridization conditions when that sequence is present in a complex mixture (including but not limited to, total cellular or library DNA or RNA).

[0088] The phrase “stringent hybridization conditions” refers to hybridization of sequences of DNA, RNA, or PNA, other nucleic acid mimes, or combinations thereof under conditions of low ionic strength and high temperature as is known in the art. Typically, under stringent conditions a probe will hybridize to its target subsequence in a complex mixture of nucleic acid (including but not limited to, total cellular or library DNA or RNA) but does not hybridize to other sequences in the complex mixture. Stringent conditions are sequencedependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Laboratory Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Probes, “Overview of principles of hybridization and the strategy of nucleic acid assays” (1993)- Generally, stringent conditions are selected to be about 5-10° C lower than thermal melting point (T_m) for the specific sequence at a defined ionic strength pH. The T_m is the temperature (under defined ionic strength, pH, and nucleic concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at T_m, 50% of the probes are occupied at equilibrium). Stringent conditions may be those in which 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 probes (including but not limited to, 10 to 50 nucleotides) and at least about 60° C for long probes (including but not limited to, greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. For selective or specific hybridization, a positive signal may be at least two times background, optionally 10 times background hybridization. Exemplary stringent hybridization conditions can he as following: 50% formamide, 5X SSC, and 1% SDS, incubating at 42°C, or 5X SSC, 1% SDS, incubating at 65°C, with wash in 0.2X SSC, and 0.1% SDS at 65°C. Such washes can be performed for 5,15, 30, 60,120, or more minutes.

[0089] The term “subject” as used herein, refers to an animal, in some embodiments a mammal, and in other embodiment a human, who is the object of treatment, observation or experiment.

[0090] The term “substantially purified” refers to a polypeptide that may be substantially or essentially free of components that normally accompany or interact with the protein as found in its naturally occurring environment, i.e. a native cell, or host cell in the case of recombinantly produced polypeptide. A polypeptide that may be substantially free of cellular material includes preparations of protein 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% (by dry weight) of contaminating protein. When the polypeptide or variant thereof is recombinantly produced by the host cells, the protein may be present at about 30%, about 25%, about 20%, about 15%, about 10%, about 5%, about 4%, about 3%, about 2%, or about 1% or less of the dry weight of the cells. When the polypeptide or variant thereof is recombinantly produced by the host cells, the protein may be present in the culture medium at about 5g/L, about 4g/L, about 3g/L, about 2g/L, about lg/L, about 750mg/L, about 500mg/L, about 250mg/L, about 100mg/L, about 50mg/L, about 10mg/L, or about lmg/L or less of the dry weight of the cells. Thus, “substantially purified” polypeptide as produced 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%, specifically, a purity level of at least about 75%, 80%, 85%, and more specifically, a purity level of at least about 90%, a purity level of at least

CA 02625540 2008-04-09

WO 2007/056448

PCT/US2006/043472

Soht’^fejk-’jpfrKfy fWe^b^kt'leist about 99% or greater as determined by appropriate methods such as SDS/PAGE analysis, RP-HPLC, SEC, and capillary electrophoresis.

[0091] The term “substituents” includes but is not limited to “non-interfering substituents.” “Non-interfering substituents” are those groups that yield stable compounds. Suitable non-interfering substituents or radicals include, but are not limited to, halo, C_rC₁₀ alkyl, C₂-Cio alkenyl, C₂-C_l0 alkynyl, C_rC₁₀ alkoxy, C₅-C_t2 aralkyl, C₃-C₁₂cycloalkyl, C₄-Ci₂ cycloalkenyl, phenyl, substituted phenyl, toluoyl, xylenyl, biphenyl, C₂-Ci₂ alkoxyalkyl, C₅-C₁₂alkoxyaryl, C5-C.12 aryloxyalkyl, C7-C12 oxyaryl, Ci-C₆ alkylsulfmyl, C_rCi₀ alkylsulfonyl, -(CH₂)_m-O-(Ci-Ci₀ alkyl) wherein m is from 1 to 8, aryl, substituted aryl, substituted alkoxy, fluoroalkyl, heterocyclic radical, substituted heterocyclic radical, nitroalkyl, -NO₂, -CN, -NRC(0)-(C_rCio alkyl), -C(O)-(Ci-Ci₀ alkyl), C₂-Cio alkthioalkyl, C(0)0-(C_rCio alkyl), -OH, -SO₂, =S, -COOH, -NR₂, carbonyl, -C(O)-(C,-C₁₀ alkyl)-CF3, -C(O)-CF3, -C(O)NR2, (Ci-C.o aryl)-S-(C₆-C₁₀ aryl), -C(O)-(C6-C₁₀ aryl), -(CH₂)_m-O-(CH₂)_m-O-(C_rCi₀ alkyl) wherein each m is from 1 to 8, -C(O)NR₂, -C(S)NR₂, -SO₂NR_2j -NRC(O)NR₂, -NRC(S)NR₂, salts thereof, 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 by their conventional chemical formulas, written from left to right, they equally encompass the chemically identical substituents that would result from writing the structure from right to left, for example, -CH₂O- is equivalent to -OCH₂-.

[0092] Substituents for alkyl and heteroalkyl radicals (including those groups often referred to as alkylene, alkenyl, heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl) can be one or more of a variety of groups selected from, but not limited to: -OR, =0, =NR, =N-0R, -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, -NRC(NR₂)=NR, -S(O)R, -S(O)₂R, -S(O)₂NR₂, -NRSO₂R, -CN and -NO₂ in a number ranging from zero to (2m’+l), 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 but not limited to, 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, -NR₂ is meant to include, but not be limited to, 1-pyrrolidinyl and 4-morpholinyl. From the above discussion of substituents, one of ordinary skill in the art will understand that the term “alkyl” is meant to include groups including carbon atoms bound to groups other than hydrogen groups, such as haloalkyl (including hut not limited to, -CF₃ and -CH₂CF₃) and acyl (including but not limited to, -C(O)CH₃, -C(O)CF₃, -C(O)CH₂OCH₃, and the like).

[0093] Similar to the substituents described for the alkyl radical, substituents for aryl and heteroaryl groups are varied and are selected from, but are not limited to-OR, =0, =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, -NR-C(NR₂)=NR, -S(O)R, -S(O)₂R, S(O)₂NR₂, -NRSO₂R, -CN, —NO₂, -R, -N₃, -CH(Ph)₂, fluoro(Ci-C4)alkoxy, and fluoro(Ci-C4)alkyl, in a number ranging from zero to the total number of open valences on the aromatic ring system; and where each R group in the preceding list is independently selected from hydrogen, alkyl, heteroalkyl, aryl and heteroaryl.

[0094] In therapeutic applications, compositions containing the (modified) non-natural amino acid polypeptide are administered to a patient already suffering from a disease, condition or disorder, in an amount sufficient to cure or at least partially arrest the symptoms of the disease, disorder or condition. Such an amount is defined to he a “therapeutically effective amount,” and will depend on the severity and course of the disease, disorder or condition, previous therapy, the patient's health status and response to the drugs, and the judgment of the treating physician. It

CA 02625540 2014-01-28 is considered wen within the skill of fl» art for one to determine such therapeutically effective amounts by routine experimentation (e.g., a dose escalation clinical trial).

[0095] The term “treating is used to refer to either prophylactic and/or therapeutic treatments.

[00961 Ah used herein, the tens “water soluble polymer” refers to any polymer that is soluble in aqueous solvents. Linkage of water soluble polymers to a polypeptide can 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 association characteristics such as aggregation and multimet formation, altered receptor binding, altered binding to oae or more binding partners, and altered receptor dimerization or multteerâation. The water soluble polymer may or may not have its own biological activity. Suitable polymers include, but are not limited to, polyethylene glycol, polyethylene glycol propionaldéhyde, mono C1-C10 alkoxy or aryloxy derivatives thereof (described in U.S. Patent No. 5,252,714), monomethoxy-polyethyiene glycol, polyvinyl pyrrolidcne, polyvinyl alcohol, polyatnino acids, divinylether maleic anhydride, A^Hydroxypropyty-metbacrylamide, dextran, dextran derivatives including dextran aulfete, polypropylene glycol, polypropylene oxide/ethylene oxide copolymer, polyoxyethylated polyol, heparin, heparin fragments, polysaccharides, oligosaccharides, glycans, cellulose and cellulose derivatives, including but not limited to methylcellulose and carhoxymethyl cellulose, starch and starch derivatives, polypeptides, polyalkylene glycol and derivatives thereof, copolymers of polyalkylene glycols and derivatives thereof, polyvinyl ethyl ethers, and alpha-beta-poly[(2-hydroxyethyl)-OL-aspartainide, and fee like, or mixtures thereof. Bxamples of such water soluble polymers include but are not limited to polyethylene glycol and serum albumin. In some embodiments, the water polymer comprises a polyethylene glycol) moiety. The molecular weight of fee polymer may be of a wide range, including but not limited io, between about 100 Da and about 100,000 Da or more. The mokcutar weight of the polymer may be between about 100 Da and about 100,000 Da, including but not limited to. 100,000 Da, 95,000 Da, 90,000 Da, 85,000 Da. 80,000 Da, 75,000 Da, 70,000 Da, 65,000 Da, 60,000 Da, 55,000 Da, 50,000 Da, 45,000 Da, 40,000 Da, 35,000 Da, 30,000 Da, 25,000 Da, 20,000 Da, 15,000 Da, 10,000 Da, 9,000 Da, 8,000 Da, 7,000 Da, 6,000 Da, 5,000 Da, 4,000 Da, 3,000 Da, 2,000 Da, 1,000 Da, 900 Da, 800 Da, 700 Da, 600 Da, 500 Da, 400 Da, 300 Da, 200 Da, and 100 Da. hi some embodiments, the molecular weight of the polymer is between about 100 Da and about 50,000 Da. In some embodiments, the molecular weight of fee polymer is between about 100 Da and about 40,000 Da. In some embodiments, fee molecular weight of the polymer is between about 1,000 Da and about 40,000 Da. In some embodiments, the molecular weight of the polymer is between about 5,000 Da and about 40,000 Da. In some embodiments, the molecular weight of fee polymer is between about 10,000 Da and about 40,000 Da. In some embodiments, the polyethylene glycol) molecule is a branched polymer. The molecular weight of fee branched chain PEO may be between about 1,000 Da and about 100,000 Da, including but not limited to, 100,000 Da, 95,000 Da, 90,000 Da, 85,000 Da, 80,000 Da, 75,000 Da, 70,000 Da, 65,000 Da, 60,000 Da, 55,000 Da, 50,000 Da, 45,000 Da, 40,000 Da, 35,000Da, 30,000 Da, 25,000 Da, 20,000 Da, 15,000 Da, 10,000 Da, 9,000 Da, 8,000 Da, 7,000 Da, 6,000 Da, 5,000 Da, 4,000 Da, 3,000 Da, 2,000 Da, and 1,000 Da. In some embodiments, fee molecular weight of the branched chain PEG is between about 1,000 Da and about 50,000 Da. hi some embodiments, the molecular weight of fee branched chain PBG is between about 1,000 Da and about 40,000 Da. hi some embodiments, the molecular weight of the branched chain-PEGis between about 5,000 Da and about 40,000 Da. In some embodiments, the molecular weight of the branched chain PEG is between about 5,000 Da and about 20,000 Da.

[00971 Unless otherwise indicated, conventional methods of mass spectroscopy, NMR, ΗΡΙΧζ protein chemistry, biochemistry, recombinant DNA techniques and pharmacology, within fee skill of fee art are employed.

CA 02625540 2008-04-09

WO 2007/056448

<img file="CA2625540C_D0009.tif" />

PCT/US2006/043472 ng, flout not limited to non-natural amino acids, (modified) non-natural amino acid .

polypeptides and reagents for producing either of the aforementioned compounds) presented herein include isotopically-labelled compounds, which are identical to those recited in the various formulas and structures presented herein, but for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number usually found in nature. Examples of isotopes that can be incorporated into the present compounds include isotopes of hydrogen, carbon, nitrogen, oxygen, fluorine and chlorine, such as ²H, ³H, ¹³C, ¹⁴C, ¹⁵N, ^1SO, ¹⁷O, ³⁵S, ¹⁸F, ³⁶C1, respectively. Certain isotopically-labelled compounds described herein, for example those into which radioactive isotopes such as ³H and ¹⁴C are incorporated, are useful in drug and/or substrate tissue distribution assays. Further, substitution with isotopes such as deuterium, i.e., ²H, can afford certain therapeutic advantages resulting from greater metabolic stability, for example increased in vivo halflife or reduced dosage requirements.

[0099] Some of the compounds herein (including, but not limited to non-natural amino acids, (modified) nonnatural amino acid polypeptides and reagents for producing either of the aforementioned compounds) have asymmetric carbon atoms and can therefore exist as enantiomers or diastereomers. Diasteromeric mixtures can be separated into their individual diastereomers on the basis of their physical chemical differences by methods known, for example, by chromatography and/or fractional crystallization. Enantiomers can be separated by converting the enantiomeric mixture into a diastereomeric mixture by reaction with an appropriate optically active compound (e.g., alcohol), separating the diastereomers and converting (e.g., hydrolyzing) the individual diastereomers to the corresponding pure enantiomers. All such isomers, including diastereomers, enantiomers, and mixtures thereof are considered as part of the compositions described herein.

[00100] In additional or further embodiments, the compounds described herein (including, but not limited to nonnatural amino acids, (modified) non-natural amino acid polypeptides and reagents for producing either of the aforementioned compounds) are used in the form of pro-drugs. In additional or further embodiments, the compounds described herein (including, but not limited to non-natural amino acids, (modified) non-natural amino acid polypeptides and reagents for producing either of the aforementioned compounds) are metabolized upon administration to an organism in need to produce a metabolite that is then used to produce a desired effect, including a desired therapeutic effect. In a further or additional embodiments are active metabolites of non-natural amino acids and (modified) non-natural amino acid polypeptides.

[00101] The methods and formulations described herein include the use of A-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 scope of the non-natural amino acids and (modified) non-natural amino acid polypeptides presented herein. In addition, the non-natural amino acids and (modified) nonnatural amino acid polypeptides described herein can exist in unsolvated as well as solvated forms with pharmaceutically acceptable solvents such as water, ethanol, and the like. The solvated forms of the non-natural amino acids and (modified) non-natural amino acid polypeptides presented herein are also considered to be disclosed herein.

[00102] Those of ordinary skill in the art will recognize that some of the compounds herein (including, but not limited to non-natural amino acids, (modified) non-natural amino acid polypeptides and reagents for producing either of the aforementioned compounds) can exist in several tautomeric forms. All such tautomeric forms are considered as part of the compositions described herein. Also, for example all enol-keto forms of any compounds (including, but not limited to non-natural amino acids, (modified) non-natural amino acid polypeptides and reagents

CA 02625540 2014-01-28 for producing cither of the aforementioned compounds) herein ^are considered at pact of thecomporitions described herein, (001(3] Some of the compounds herein (includiog, hut not limited to uon-natursl unno adds, (modified) nonnatnral ammo acid polypeptides and reagenti for producing eifter of the aforementioned compounds) are acidic and may form a salt with a phamtaceutically acceptable cation. Some of die compounds herein (melnding, bid not limited to non-natural amino acids, (modified) non-natural Βπΰηο acid polypeptides and reagents for producing either of the aforementioned compounds) can be basic and accordingly, may form a salt with a pharmaceutically acceptable anion. AU such salts, including dl-salts are within the scope of the conçositions described herein and they can be prepared by conventional methods. For example, salts can be prepared by contacting the acidic and basic entities, in either an aqueous, non-aqueous or partially aqueous medium. The salts are recovered by using at leaat ana of the following techniques: filtration, precipitation with a non-solvent followed by filtration, evaporation of the solvent, or, in the case of aqueous solutions, lyophilization.

(00104] Salts, for example, include: (1) acid addition salts, formed with inorganic acids such as hydrochloric add, hydrebromic add, sulfitric acid, nitric acid, phosphoric add, and foe like; or formed with organic acids and) as acetic add, propionic add, hexanoic acid, cyclopentanepropicmSe add, glycolic add, pyruvic add, lactic add, malonic acid, succinic acid, malic add, maleic acid, fomaric add, tartaric add, citric acid, benzoic add, 3-(4hydmxybenzoyQbenstic add, cinnamic acid, mandelic acid, medwnetulfonic add, ethanosulfonic aoid, 14efoanedisutfoidc acid, 2-hydtnxyethanesulfonjc add, benzenesutfonic add, ZnaphflalanMulfonic add, 4mefhylbicyclo-[2.2.2]oct-2-ene-l-c«boxylic acid, ghicoheptonic add, 4,4'-n»foylenebis-(3-hydroxy-2-ene-l carboxylic acid), 3-phanylpropionic acid, triinethylacetic acid, tertiary butyboetic acid, lauryl sulfuric acid, gluconic acid, glutamic add, hydroxynaphthoic acid, salicylic add, stearic acid, muconic acid, and foe like; (2) salts formed when an add» proton present in foe parent compound either is replaced by a metal ion, e.g., an alkali metal ion, an alkaline earth ion, or an altnniuum ion; or coordinates with an organ» base. Acceptable organic bases include efoanolamine, diethanolamine, triethanolamine, tromethamine, hf-mefoylglucamine, and foe like. Acceptable inorgame bases include aluminum hydroxide, calcium hydroxide, potassium hydroxide, sodium carbonate, sodium hydroxide, and foe like.

(00105] It should be understood that a reference to a salt includes tie solvent addition forms or crystal forms thereof, particularly solvates or polymorphs. Solvates contain either stoichiometric or non-etdehirenetric «nanti of a solvent, and are often formed during foe process of crystallization. Hydrates are formed when foe sohrentds water, or aleobolatos are formed when die solvent is alcohol. Polymorphs include foe different crystal packing arrangements of foe same elemental composition of a compound. Polymorphs usually have different X-ray diffraction patterns, infrared spectra, malting points, density, lrardness, crystal shape, optical end electrical properties, stability, and solubility. Various factors such as foe recrystallization solvent, rate of crystallization, and storage tenqterature may cause a single crystal form to dominate.

{00106] Further publications and patents for the purpose of describing and disclosing constructs and methodologies which might be used in connection with the presently described inventions include: WO 2006/069246 published 6/29/2006; WO 2007/079130 published 7/12/2007; WO 2007/070659 published 6/21/2007; US 2006-0217532 Al published 9/28/2006; US 2006-0194256 Al published 8/31/2006; and US 2006-0217289 Al published 9/28/2006.

All of the aforesaid and the publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the inventions described herein are not entitled to antedate such disclosure by virtue of prior invention or for any other reason.

CA 02625540 2008-04-09

WO 2007/056448 PCT/US2006/043472

BRIEF DESCRIPTION OF THE FIGURES

[00107] A better understanding of the features and advantages of the present methods and compositions may be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of our methods, compositions, devices and apparatuses are utilized, and the accompanying drawings of which:

[00108] FIG. 1 presents a schematic representation of the relationship of certain aspects of the methods, compositions, strategies and techniques described herein.

[00109] FIG. 2 presents a non-limiting example of a SDS-PAGE analysis of one step dimerization reactions of scFv 108 using 2 K homobifunctional hydroxylamine PEG linker with different molar ratios : 1 ) scFvdinker = 1.6:1, with acetic hydrazide; 2) scFv:linker = 2:1, with acetic hydrazide; 3 ) scFvriinker = 2.4:1, with acetic hydrazide; 4) scFv:linker = 2:1, without acetic hydrazide; 5) scFvrlinker = 2:1, with acetic hydrazide without PEG linker.

[00110] FIG. 3 presents a non-limiting example of a SDS-PAGE analysis of scFv-pAcF and 30 K mono hydroxylamine PEG conjugation 1) the standard of 100% starting scFv-pAcF; 2) the standard of 20% starting scFvpAcF; 3) the standard of 10% starting scFv-pAcF; 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.

[00111] FIG. 4 presents a non-limiting example of a SDS-PAGE analysis of scFv-pAcF and 30 K mono hydroxylamine PEG conjugation with different concentration 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, no acetic hydrazide; 5) the standard of 10% scFv-pAcF; 6) the standard of 20% scFv-pAcF; 7) the standard of 100% scFv-pAcF.

[00112] FIG. 5 presents non-limiting examples of accelerants that can be used in the methods, reactions and syntheses described herein.

[00113] FIG. 6 presents a non-limiting example of an SDS-PAGE analysis comparing the formation of oxime in the presence of different accelerants; the lane number corresponds to the accelerant number in FIG. 5 and the last lane is a control reaction without accelerant.

[00114] FIG. 7 presents a non-limiting example of an SDS-PAGE analysis of the conjugation of hGH-pAcF with 30 K monohydroxylamine PEG with accelerants 7 and 20: 1) hGH-pAcF:PEG = 1:2 with accelerant 7; 2) hGH-pAcF PEG = 1:2 with accelerant 20; 3) hGH-pAcF:PEG = 1:2 no accelerant; 4) hGH-pAcF:PEG = 1:5 no accelerant. [00115] FIG. 8 presents a non-limiting example of an LCMS analysis of hGH incubated with different concentrations of accelerant acetic hydrazide: A) total LCMS trace; B) Mass spectrum of hGH without accelerant; C) Mass spectrum of hGH with 200 mM accelerant acetic hydrazide.

[00116] FIG. 9 presents non-limiting examples of accelerants that can be used in the methods, reactions and syntheses described herein.

[00117] FIG. 10a presents a non-limiting reaction of a model ketone with a model hydroxylamine in the presence of an accelerant to form a model oxime; FIG. 10b presents non-limiting examples of accelerants that can be used in the methods, reactions and syntheses described herein.

[00118] FIG. 11 presents a non-limiting set of oxime yields for a model reaction conducted in the absence and in the presence of various accelerants described herein.

CA 02625540 2014-01-28

DETAILED DESCRIPTION OF THE INVENTION

I. Introduction

[00119] Recently, an entirely new technology in the protein sciences has been reported, which promisos to overcome many of the limitations associated with site-specific modifications of proteins. Specifically, new components have been added to the protein biosynthetic machinery of the prokaryote Escherichia colt (£. ooii) (e.g, L. Wang, et (2001), Science 292:498-500) and the eukaryote Sacchronryces cerevlsiae (£ cerevisiaa) (e.g, J. Chin et at., Science 301564-7 (2003)), which has enabled the incorporation of non-natural amino acids to proteins in vivo. A number of new amino acids with novel chemical, physical or biological properties, including photoaffinity labels and photoisomerizable amino acids, photocrosslinking amino acids (see, e.g. Chin, J.W, et al. (2002) Proc, Natl, Acad, Sci, U.S.A, 99:11020-11024; and Chin, J.W., et al. (2002) I Am, Chem. Soc. 124:902627); keto amino adds, and glycosylated amino acids have been incorporated efficiently and with high fidelity into proteins in E. coll and in yeast in response to the amber codon, TAG, using this methodology. See, ecg., I. W. Chin ei al., (2002), Journal of the American Chemical Sorietv 124:9026-9027;

J. W. Chin, &amp; P. G. Schultz, (2002), ChemBioChem 3(11):1135-1137; J.

W. Chin, et al., (2002), PNAS United States of America 99;11020-11024;

and, L. Wang, &amp; P. G. Schultz, (2002), Chem, Comm.. 1:1-11. These studies have demonstrated that it is possible to selectively and routinely introduce chemical functional groups that are not found in proteins, that are chemically inert to all of fi» functional groups found in the 20 common, genetically-encoded amino adds (he., “natural” amino acids) and that may be used to react efficiently and selectively to form stable covalent linkages.

[00120] Chemical functional groups not found in the natural amino acids include carbonyl groups, such as ketones and aldehydes, and hydroxylamine groups. A hydroxylamine moiety reacts with a carbonyl group such as ketone and aldehyde to form a relatively stable oxime; this pairing (hydroxylamine with a carbonyl group) thus provides a means for further functionalizing non-natural amino acid polypeptides. One non-limiting example of such a paving is shown below:

<img file="CA2625540C_D0010.tif" />

protein

[00121] For example, when either a hydroxylamine moiety or a carbonyl group is incorporated into a non-natural amino add polypeptide and reacted with a reagent that contains that other member of fi» pair, fire non-natural anrino acid polypeptide can be functionalized with fi» reagent via the formation of an oxime group. Though compatible for protein functionalization reactions, the standard oxime formation reaction may be made more efficient which would allow, for example, for the use of lower amounts of reactants and reduce foe time to rection completion. Therefore, foe development of an accelerant is highly desirable.

If, Overview

[QQ122] Figure 1 is one embodiment of foe compositions, methods and techniques that are described herein. A csrbonyl-eontaining compound is selected for reaction with a hydroxylamine-cantaining compound to finro an oxime-containing compound. The carbonyl-containing compound includes non-natural amino acids, polypeptides, oligonucleotides, polymers (including by way of example only polyethylene glycol), reagents, linker groups, groups comprising further functionality, and combinations thereof; the present disclosure provides numerous examples of groups comprising further functionality. The hydroxylamine-containing compound includes non-natural amino

CA 02625540 2008-04-09

WO 2007/056448 PCT/US2006/043472 acid’s,ttÿbfefaebÆés, polymers (including by way of example only polyethylene glycol), reagents, linker groups, groups comprising further functionality, and combinations thereof; the present disclosure provides numerous examples of groups comprising further functionality. The oxime-containing compound includes nonnatural amino acids, polypeptides, oligonucleotides, polymers (including by way of example only polyethylene glycol), reagents, linker groups, groups comprising further functionality, and combinations thereof; the present disclosure provides numerous examples of groups comprising further functionality. To the reaction mixture of the hydroxylamine-containing compound and the carbonyl-containing compound is added an accelerant, wherein the accelerant has at least one of the following properties: (a) increase the rate of reaction between a carbonylcontaining compound and a hydroxylamine-containing compound to form an oxime-containing compound, where the increase in rate is relative to the reaction in the absence of the accelerant; (b) lower the activation energy of the reaction between a carbonyl-containing compound and a hydroxylamine-containing compound to form an oximecontaining compound, where the decrease in activation energy is relative to the reaction in the absence of the accelerant; (c) increase the yield of an oxime-containing compound from the reaction of a carbonyl-containing compound with a hydroxylamine-containing compound, where the increase in yield is relative to the reaction in the absence of the accelerant; (d) lower the temperature at which a carbonyl-containing compound reacts with a hydroxylamine-containing compound to form an oxime-containing compound, where the decrease in temperature is relative to the reaction in the absence of the accelerant; (e) decrease the time necessary to react a carbonylcontaining compound with a hydroxylamine-containing compound to form an oxime-containing compound, wherein the decrease in time is relative to the reaction in the absence of accelerant; (f) decrease the amount of reagents necessary to form an oxime group on a non-natural amino acid polypeptide, wherein the decrease in amount of reagents is relative to the reaction in the absence of accelerant; (g) decrease the side products resulting from the reaction of a carbonyl-containing compound with a hydroxylamine-containing compound to form an oximecontaining compound, wherein the decrease in side products is relative to the reaction in the absence of accelerant; (h) does not irreversibly destroy the tertiary structure of a polypeptide undergoing an oxime-forming reaction in the presence of an accelerant (excepting, of course, where the purpose of the reaction is to destroy such tertiary structure); (i) can be separated from an oxime-containing compound in vacuo', and (j) modulate the reaction of a carbonyl-containing compound with a hydroxylamine-containing compound. In a further embodiment, the accelerant has none of the aforementioned properties. Optionally, a variety of accelerants described herein are tested and an accelerant is selected based on its possession of at least one of the aforementioned properties. Optionally, the reaction characteristics (e.g., yield of oxime-containing compound) can be further optimized by at least one of the following: (a) varying the amount of accelerant, (b) varying the amount of carbonyl-containing compound, (c) varying the amount of hydroxylamine-containing compound, (d) varying the temperature of the reaction, (e) varying the pH of the reaction, and (f) varying the solvent in the reaction mixture. Optionally, additional accelerants are tested at an optimized reaction condition, or the selection and optimization steps are reversed, or the selection and optimization steps are repeated in an iterative fashion. The carbonyl-containing compound and the hydroxylaminecontaining compound are reacted in the presence of the accelerant to form the oxime-containing compound. Optionally the progress of the reaction is monitored by a detection means, including by way of example only, chromatography. The oxime-containing compound resulting from the reaction of a carbonyl-containing compound and a hydroxylamine-containing compound in the presence of an accelerant can be optionally isolated, purified and characterized. The accelerant 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-based bioseparation, electrophoresis, precipitation of the oxime-containing compound, distillation, or a combination thereof. Thus, in one

CA 02625540 2008-04-09

WO 2007/056448 PCT/US2006/043472

IF* ** 8 B il* î|^wîi Ü' / H II il II ^n,,P^l ’Il emDOd!m§Ht!'QeseribM'^::&amp;eipeifi, The accelerants can be removed in vacuo from the oxime-containing material; however, in other embodiments described herein, the accelerants can be removed using any (or any combination) of the aforementioned methods. Isolation and purification signifies the removal of at least some non-oxime containing compound from the materials in the reaction mixture.

[00123] At one level, described herein are the tools (methods, compositions, techniques) for creating and using a polypeptide comprising at least one non-natural amino acid or modified non-natural amino acid with an oxime group formed in the presence of an accelerant (although such a reaction may be less efficient in the absence of an accelerant described herein). Such non-natural amino acids may contain further functionality, including but not limited to, a desired functionality.

[00124] Also described herein are non-natural amino acids that have or can be modified to contain an oxime moiety formed in the presence of an accelerant (although such a reaction may be less efficient in the absence of an accelerant described herein). Included with this aspect are methods for producing, purifying, characterizing and using such non-natural amino acids. In another aspect described herein are methods, strategies and techniques for incorporating at least one such non-natural amino acids into a polypeptide. Also included with this aspect are methods for producing, purifying, characterizing and using such polypeptides containing at least one such nonnatural amino acids. Also included with this aspect are compositions of and methods for producing, purifying, characterizing and using polynucleotides (including DNA and RNA) that can be used to produce, at least in part, a polypeptide containing at least one non-natural amino acid that can react, in the presence of an accelerant (although such a reaction may be less efficient in the absence of an accelerant described herein) to form an oxime-containing non-natural amino acid polypeptide, including such a polypeptide that has been modified. Also included with this aspect are compositions of and methods for producing, purifying, characterizing and using cells that can express such polynucleotides that can be used to produce, at least in part, a polypeptide containing at least one non-natural amino acid.

[00125] Also included within the scope of the methods, compositions, strategies and techniques described herein are accelerants for reacting a reagent with a non-natural amino acid (containing a carbonyl or dicarbonyl group, hydroxylamine group, or protected forms thereof) that is part of a polypeptide so as to produce any of the aforementioned post-translational modifications. In general, the resulting post-translationally modified non-natural amino acid polypeptide will contain at least one oxime group; the resulting modified oxime-containing non-natural amino acid polypeptide may undergo subsequent modification reactions. Also included with this aspect are methods for selecting, producing, optimizing, purifying, characterizing and using such accelerants that can be used with any such post-translational modifications of such non-natural amino acid(s).

[00126] The non-natural amino acid containing polypeptide can 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 containing an oxime group (or protected or masked forms thereof), wherein at least one oxime group was produced in the presence of the accelerants described herein, and further, such an oxime-containing non-natural amino acid polypeptide may optionally contain at least non-natural amino acid polypeptide containing one carbonyl or dicarbonyl group, hydroxylamine group, or protected forms thereof. The non-natural amino acids can be the same or different, for example, there can be 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14,15,16, 17, 18, 19, 20, or more different sites in the protein that comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more different non-natural amino acids. In certain embodiments, at least one, but fewer than all, of a particular amino acid present in a naturally occurring version of the protein is substituted with a non-natural amino acid.

CA 02625540 2008-04-09

WO 2007/056448

PCT/US2006/043472 iï G il à’cid methods and compositions described herein provides having a wide variety of functional groups (provided that at least one conjugate is chemically linked to a non-natural amino acid via an oxime group formed in the presence of the accelerants described herein), substituents or moieties, with other substances including but not limited to a desired functionality.

[00128] In another aspect of the compositions, methods, techniques and strategies described herein are methods for studying or using any of the aforementioned (modified) non-natural amino acid polypeptides. Included within this aspect, by way of example only, are therapeutic, diagnostic, assay-based, industrial, cosmetic, plant biology, environmental, energy-production, and/or military uses which would benefit from a polypeptide comprising a (modified) non-natural amino acid polypeptide or protein.

III. Post-Translational Modifications of Non-Natural Amino Acid Components of a Polypeptide in the Presence of at Least One Accelerant

[00129] Methods, compositions, techniques and strategies have been developed to site-specifically incorporate nonnatural amino acids during the in vivo translation of proteins. By incorporating a non-natural amino acid with a sidechain chemistry that is orthogonal to those of the naturally-occurring amino acids, this technology makes possible the site-specific derivatization of recombinant proteins. As a result, a major advantage of the methods, compositions, techniques and strategies described herein is that derivatized proteins can now be prepared as defined homogeneous products. However, the methods, compositions, reaction mixtures, techniques and strategies described herein involving an accelerant are not limited to non-natural amino acid polypeptides formed by in vivo protein translation techniques, but includes non-natural amino acid polypeptides formed by any technique, including by way of example only expressed protein ligation, chemical synthesis, ribozyme-based techniques (see, e.g., section herein entitled “Expression in Alternate Systems”). For convenience, the phrase “post-translational modification,” when directed to the use of an accelerant to form an oxime bond on a non-natural amino acid polypeptide, includes nonnatural amino acid polypeptides formed by any technique, including any in vivo and in vitro techniques, such as those described herein and known to those of ordinary skill in the art.

[00130] The ability to incorporate non-natural amino acids into recombinant proteins broadly expands the chemistries which may be implemented for derivatization. More specifically, protein derivatization to form an oxime bond on a non-natural amino acid portion of a polypeptide offers several advantages. First, the naturally occurring amino acids generally do not form oxime bonds and thus reagents designed to form oxime bonds will react site-specifically with the non-natural amino acid component of the polypeptide (assuming of course that the non-natural amino acid and the corresponding reagent have been designed to form an oxime bond), thus the ability to site-selectively derivatize proteins provides a single homogeneous product as opposed to the mixtures of derivatized proteins produced using prior art technology. Second, oxime adducts are stable under biological conditions, suggesting that proteins derivatized by oxime exchange are valid candidates for therapeutic applications. Third, the stability of the resulting oxime bond can be manipulated based on the identity (i.e., the functional groups and/or structure) of the non-natural amino acid to which the oxime bond has been formed. Thus, in some embodiments, the oxime bond to the non-natural amino acid polypeptide has a decomposition half life 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 other embodiments more than 1 week. In yet other 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 at least 1 day in a pH between about 2 and 8; in other embodiments, from a pH between about 2 to 6; in other embodiment, in a pH between about 2 to 4. In other embodiments, using the strategies, methods, compositions and techniques

CA 02625540 2008-04-09

WO 2007/056448 PCT/US2006/043472 in the art will be able to synthesize an oxime bond-to a non-natural amino acid polypeptide with a decomposition half-life tuned to the needs of that skilled artisan (e.g., for a therapeutic use such as sustained release, or a diagnostic use, or an industrial use or a military use).

[00131] The formation of an oxime-containing non-natural amino acid or non-natural amino acid polypeptide from the reaction of (a) a carbonyl-containing non-natural amino acid or carbonyl-containing non-natural amino acid polypeptide and a hydroxylamine-containing reagent, or (b) a hydroxylamine-containing non-natural amino acid or hydroxylamine-containing non-natural amino acid polypeptide and a carbonyl-containing reagent, can be enhanced by addition of an accelerant to the reaction mixture. An accelerant is a compound that has at least one of the following properties: (a) increase the rate of reaction between a carbonyl-containing compound and a hydroxylamine-containing compound to form an oxime-containing compound, where the increase in rate is relative to the reaction in the absence of the accelerant; (b) lower the activation energy of the reaction between a carbonylcontaining compound and a hydroxylamine-containing compound to form an oxime-containing compound, where the decrease in activation energy is relative to the reaction in the absence of the accelerant; (c) increase the yield of an oxime-containing compound from the reaction of a carbonyl-containing compound with a hydroxylaminecontaining compound, where the increase in yield is relative to the reaction in the absence of the accelerant; (d) lower the temperature at which a carbonyl-containing compound reacts with a hydroxylamine-containing compound to form an oxime-containing compound, where the decrease in temperature is relative to the reaction in the absence of the accelerant; (e) decrease the time necessary to react a carbonyl-containing compound with a hydroxylaminecontaining compound to form an oxime-containing compound, wherein the decrease in time is relative to the reaction in the absence of accelerant; (f) decrease the amount of reagents necessary to form an oxime group on a non-natural amino acid polypeptide, wherein the decrease in amount of reagents is relative to the reaction in the absence of accelerant; (g) decrease the side products resulting from the reaction of a carbonyl-containing compound with a hydroxylamine-containing compound to form an oxime-containing compound, wherein the decrease in side products is relative to the reaction in the absence of accelerant; (h) does not irreversibly destroy the tertiary structure of a polypeptide undergoing an oxime-forming reaction in the presence of an accelerant (excepting, of course, where the purpose of the reaction is to destroy such tertiary structure); (i) can be separated from an oxime-containing compound in vacuo·, and (j) modulate the reaction of a carbonyl-containing compound with a hydroxylaminecontaining compound. In further embodiments, the accelerant 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, or all of the aforementioned properties. In a further embodiment, the accelerant has none of the aforementioned properties.

[00132] The use of an accelerant includes the use of a single accelerant or multiple accelerants. In addition, the molar ratio of accelerant 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. Further, the molar ratio of accelerant 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. Further, the accelerant includes compounds that can be substantially removed in vacuo from the resulting oxime-containing compound. Further, the accelerant includes compounds containing a diamine moiety, a semicarbazide moiety, a hydrazine, or a hydrazide moiety.

CA 02625540 2008-04-09

WO 2007/056448

PCT/US2006/043472

IS icLOJ™ aforementioned aspects or embodiments, the accelerant is selected from the group consisting of bifunctional aromatic amines, oxoamine derivatives, and compounds having the following structures:

H

HoN' h₂n-v yy h₂n'Y h

<img file="CA2625540C_D0011.tif" />

h₂nV²

NH

H H hk x-N

H₂N Y - Η₂Ν'Ύ' ⁰ 0

H₂N'^NY° o

^ΝΎ UN Y 0 0

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

[00134] In a further embodiment, the accelerant is a bifunctional aromatic amine. In a further embodiment, the aromatic amine is selected from the group:

Bifuctionai aromatic amines:

<img file="CA2625540C_D0012.tif" />

[00135] In a further embodiment, the accelerant is an oxoamine derivative. In a further embodiment, the oxoamine derivative is selected from the group:

Oxoamine derivatives:

H H

Me O Me O

Me'

H H Hu nJL nJU •^NO'^Et Me'^N'O'^Bn Me'^N0^&gt;&lt;NH₂ Me' '0 N

[00136] Further, the accelerant include compounds selected from the group consisting of:

H₂IC^NY^NH2

NH h₂n^SY^nh’

H h H R h₂nⁿY h2n'ⁿY^h ή/L h Λ oo wherein R_x, R_y and R_z are selected from the group consisting of: L_x-H, L_x-alkyl, L_x-aryl, Lx-heteroaryl, L_x-alkenyl, Lx-alkynyl, L_x-alkoxy, and L_x-alkylamine, where L_x is a bond, C(=O), C(=NH), and C(=NH)-NH. Further, in any of the aforementioned aspects or embodiments, the accelerant is selected from the compounds presented in FIG. 5, FIG. 9, or FIG. 10, including by way of example any of compounds 6, 8, 10, 7, and 20 of Figure 5. In any of the aforementioned aspects or embodiments, the accelerant includes an agent that can form a hydrazone upon reaction with a carbonyl-containing group. Further, in any of the aforementioned aspects the accelerant activity depends on the rate of reaction with the ketone moiety and the stability of the resulting intermediate. Further, in any of the aforementioned aspects or embodiments, the pH of the reaction mixture comprising the accelerant, the carbonylcontaining 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;

CA 02625540 2008-04-09

WO 2007/056448

PCT/US2006/043472 between aboul fe)·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.

[00137] 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 but not limited to, antibodies and antibody fragments), and including but not limited to, the study of protein structure and function. See, e.g., Dougherty, (2000) Unnatural Amino Acids as Probes of Protein Structure and Function, Current Opinion in Chemical Biology, 4:645-652. Other uses for the non-natural amino acid polypeptides described above include, by way of example only, assay-based, cosmetic, plant biology, environmental, energy-production, and/or military uses. However, the non-natural amino acid polypeptides described above can undergo further modifications so as to incorporate new or modified functionalities, including manipulating therapeutic effectiveness of the polypeptide, improving the safety profile of the polypeptide, adjusting the pharmacokinetics, pharmacologies and/or pharmacodynamics of the polypeptide (e.g., increasing water solubility, bioavailability, increasing serum half-life, increasing therapeutic half-life, modulating immunogenicity, modulating biological activity, or extending the circulation time), providing additional functionality to the polypeptide, incorporating a tag, label or detectable signal into the polypeptide, easing the isolation properties of the polypeptide, and any combination of the aforementioned modifications.

[00138] The methods, compositions, strategies and techniques described herein are not limited to a particular type, class or family of polypeptides or proteins. Indeed, virtually any polypeptides may include at least one non-natural amino acids described herein. By way of example only, the polypeptide can 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, C-X-C chemokine, T39765, NAP-2, ENA-78, gro-a, gro-b, gro-c, IP-10, GCP-2, NAP-4, SDF-1, PF4, MIG, calcitonin, ckit ligand, cytokine, CC chemokine, monocyte chemoattractant protein-1, monocyte chemoattractant protein-2, monocyte chemoattractant protein-3, monocyte inflammatory protein-1 alpha, monocyte inflammatory protein-i beta, RANTES, 1309, R83915, R91733, HCC1, 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, four-helical bundle 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 albumin, ICAM-1, ICAM-1 receptor, LFA-1, LFA-1 receptor, insulin, insulin-like growth factor (IGF), IGF-I, IGF-II, interferon (IFN), IFNalpha, IFN-beta, IFN-gamma, any inteferon-like molecule 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, kératinocyte growth factor (KGF), lactoferrin, leukemia inhibitory factor, luciferase, neurturin, neutrophil inhibitory factor (NIF), oncostatin M, osteogenic protein, oncogene product, paracitonin, parathyroid hormone, PD-ECSF, PDGF, peptide hormone, pleiotropin, protein A, protein G, pth, pyrogenic exotoxin A, pyrogenic exotoxin B, pyrogenic exotoxin C, pyy, 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, SEC1, SEC2, SEC3, SED, SEE, steroid hormone receptor, superoxide dismutase, toxic shock syndrome toxin, thymosin alpha 1, tissue plasminogen activator, tumor growth factor (TGF), tumor necrosis factor, tumor necrosis factor alpha, tumor necrosis factor beta, tumor necrosis factor receptor (TNFR), VLA-4 protein, VCAM-1

CA 02625540 2008-04-09

WO 2007/056448 PCT/US2006/043472 pro'teluf vtfsfcilltff ehcJo’thëîiaÎ 'gfowth 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 growth hormone supergene family.

[00139] Such modifications include the incorporation of further functionality onto the non-natural amino acid component of the polypeptide, including but not limited to, a desired functionality.

[00140] Thus, by way of example only, a non-natural amino acid polypeptide containing any one of the following amino acids may be further modified in the presence of the accelerants described herein using the methods and compositions described herein;

r₃

<img file="CA2625540C_D0013.tif" />

H R₄ (a) ⁰ (I) wherein:

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

B is optional, and when present is a linker selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O)_kwhere 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(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’)=N-N(R’)-, -C(R’)=N-N=, -C(R’)₂-N=N, and -C(R’)2-N(R’)-N(R’)-, where each R’ is independently H, alkyl, or substituted alkyl;

<img file="CA2625540C_D0014.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, two R” optionally form a heterocycloalkyl;

Ri is optional, and when present, is H, an amino protecting group, resin; and

CA 02625540 2008-04-09

PCT/US2006/043472

WO 2007/056448 is OH, an ester protecting group, resin;

each of R₃ and Rfis independently H, halogen, lower alkyl, or substituted lower alkyl, or R₃ and R4 or two R₃groups optionally form a cycloalkyl or a heterocycloalkyl;

or the -A-B-J-R groups together form a bicyclic or tricyclic cycloalkyl or heterocycloalkyl comprising at least one carbonyl group, including a dicarbonyl group, protected carbonyl group, including a protected dicarbonyl group, or masked carbonyl group, including a masked dicarbonyl group;

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

<img file="CA2625540C_D0015.tif" />

wherein:

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

Ri is optional, and when present, is H, an amino protecting group, resin; and R₂ is optional, and when present, is OH, an ester protecting group, resin; and each R_ais 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’, where each R’ is independently H, alkyl, or substituted alkyl.

<img file="CA2625540C_D0016.tif" />

wherein:

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

B is optional, and when present is a linker selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O)_kwhere 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(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’)-,

CA 02625540 2008-04-09

WO 2007/056448

PCT/US2006/043472 (R^3^^(5)_kN(R’)-, -N(R’)-N=, -C(R’)=N-, -C(R’)=N-N(R’)-, -C(R’)=N-N= -C(R’)₂-N=N, and -C(R’)₂-N(R’)-N(R’)-, where 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;

Ri is optional, and when present, is H, an amino protecting group, resin; and

R₂ is optional, and when present, is OH, an ester protecting group, resin;

each of R₃ and R4 is independently H, halogen, lower alkyl, or substituted lower alkyl, or R₃ and R4 or two R₃groups optionally form a cycloalkyl or a heterocycloalkyl;

each of R₆ 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 R^ or R₇ is L-X, where X is a selected from the group consisting of a desired functionality; and L is optional, and when present is a linker selected from the group consisting of alkylene, substituted alkylene, alkenylene, substituted alkenylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -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(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’)=N-N(R’)-, -C(R’)=N-N=, -C(R’)₂-N=N-, and -C(R’)₂-N(R’)-N(R’)-, where eachR’ is independently H, alkyl, or substituted alkyl;

(d)

<img file="CA2625540C_D0017.tif" />

(xxx);

wherein;

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

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

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

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

X! is C, S, or S(O); and L is alkylene, substituted alkylene, N(R’)(alkylene) orN(R’)(substituted alkylene), where each R’ is independently H, alkyl, or substituted alkyl; or

CA 02625540 2008-04-09

WO 2007/056448

PCT/US2006/043472 (XXXX), (e) wherein:

<img file="CA2625540C_D0018.tif" />

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

(b) (b) v/W¹

M is -C(R₃)-, ^(a) ’

V-^c-^cC?‘^b)

R4 R4 &lt;.,v

C=C—$ (b) (b) (a) 7 to (b) σνν»

V^e; (a) 5

-s—ξ (b) to (b) uw¹ (b) to

C—C—5 (b) / (a)

C=C—5 (b) (b)

A (b) (b)

A νΑΛΡ (a) uxaa (a) , or

S-C—S (b)

I * σνν&gt;

(a) , where (a) indicates bonding to the A group and (b) indicates bonding to respective carbonyl groups, R₃ and R4 are independently chosen from H, halogen, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl, or R₃ and R4 or two R₃ groups or two R4 groups optionally form a cycloalkyl or a 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;

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

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

[00141] In one aspect of the methods and compositions described herein are compositions that include 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 have been posttranslationally modified. The post-translationally-modified non-natural amino acids can be the same or different, including hut not limited to, there can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more different sites in the protein that comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, or more different post-translationally-modified non-natural amino acids. In another aspect, a composition includes a protein with at least one, but fewer than all, of a particular amino acid present in the protein is substituted with the posttranslationally-modified non-natural amino acid. For a given protein with more than one post-translationallymodified non-natural amino acids, the post-translationally-modified non-natural amino acids can be identical or

CA 02625540 2008-04-09

WO 2007/056448

PCT/US2006/043472 ‘ iî ii e .ο ii;

•nbf'ïhiïitêd to, the protein can include two or more different types of post-translationallymodified non-natural amino acids, or can include two of the same post-translationally-modified non-natural amino acid). For a given protein with more than two post-translationally-modified non-natural amino acids, the posttranslationally-modified non-natural amino acids can be the same, different or a combination of a multiple posttranslationally-modified non-natural amino acid of the same kind with at least one different post-translationallymodified non-natural amino acid.

A. Methods for Post-Translationally Modifying Non-Natural Amino Acid Polypeptides in the Presence of at Least One Accelerant: Reactions of Carbonyl-Containing Non-Natural Amino Acids with Hydroxylamine-Containing Reagents

[00142] The sidechains of the naturally occurring amino acids lack highly electrophilic sites. Therefore, the incorporation of an non-natural amino acid with an electrophile-containing sidechain, including, by way of example only, an amino acid containing a carbonyl or dicarbonyl group such as a ketone, makes possible the site-specific derivatization of this sidechain via nucleophilic attack of the carbonyl or dicarbonyl group. In the instance where the attacking nucleophile is a hydroxylamine, an oxime-derivatized protein will be generated. The methods for derivatizing and/or further modifying may be conducted with a polypeptide that has been purified prior to the derivatization step or after the derivatization step. Further, the derivatization step can occur under mildly acidic to slightly basic conditions, including by way of example, between a pH of about 2-8, or between a pH of about 4-8. [00143] The formation of an oxime-containing non-natural amino acid or non-natural amino acid polypeptide from the reaction of a carbonyl-containing non-natural amino acid or carbonyl-containing non-natural amino acid polypeptide and a hydroxylamine-containing reagent can be enhanced by addition of an accelerant to the reaction mixture. An accelerant is a compound that has at least one of the following properties: (a) increase the rate of reaction between a carbonyl-containing compound and a hydroxylamine-containing compound to form an oximecontaining compound, where the increase in rate is relative to the reaction in the absence of the accelerant; (b) lower the activation energy of the reaction between a carbonyl-containing compound and a hydroxylamine-containing compound to form an oxime-containing compound, where the decrease in activation energy is relative to the reaction in the absence of the accelerant; (c) increase the yield of an oxime-containing compound from the reaction of a carbonyl-containing compound with a hydroxylamine-containing compound, where the increase in yield is relative to the reaction in the absence of the accelerant; (d) lower the temperature at which a carbonyl-containing compound reacts with a hydroxylamine-containing compound to form an oxime-containing compound, where the decrease in temperature is relative to the reaction in the absence of the accelerant; (e) decrease the time necessary to react a carbonyl-containing compound with a hydroxylamine-containing compound to form an oxime-containing compound, wherein the decrease in time is relative to the reaction in the absence of accelerant; (f) decrease the amount of reagents necessary to form an oxime group on a non-natural amino acid polypeptide, wherein the decrease in amount of reagents is relative to the reaction in the absence of accelerant; (g) decrease the side products resulting from the reaction of a carbonyl-containing compound with a hydroxylamine-containing compound to form an oxime-containing compound, wherein the decrease in side products is relative to the reaction in the absence of accelerant; (h) does not irreversibly destroy the tertiary structure of a polypeptide undergoing an oxime-forming reaction in the presence of an accelerant (excepting, of course, where the purpose of the reaction is to destroy such tertiary structure); (i) can be separated from an oxime-containing compound in vacuo·, and (j) modulate the reaction of a carbonyl-containing compound with a hydroxylamine-containing compound. In further embodiments, the accelerant 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

CA 02625540 2008-04-09

WO 2007/056448

PCT/US2006/043472 àbieÀgnSfcfaleÎ-^rôpeteëlj'^gÉtÎ’oÉ' the aforementioned properties, nine of the aforementioned properties, or all of the aforementioned properties. In a further embodiment, the accelerant has none of the aforementioned properties. [00144] The use of an accelerant includes the use of a single accelerant or multiple accelerants. In addition, the molar ratio of accelerant 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. Further, the molar ratio of accelerant 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. Further, die accelerant includes compounds that can be substantially removed in vacuo ftom the resulting oxime-containing compound. Further, the accelerant includes compounds containing a diamine moiety, a semicarbazide moiety, a hydrazine, or a hydrazide moiety. [00145] Further, in any of the aforementioned aspects or embodiments, the accelerant is selected from the group consisting of bifunctional aromatic amines, oxoamine derivatives, and compounds having the following structures:

R_y

RxN.

H,N

H

-Ik

U H H h _u

H₂N^NV W ^H2^N Y ^H2 x H XI n

<img file="CA2625540C_D0019.tif" />

H

JSL _yNH, u _u NN NO N / u

H₂N γ H₂N Y H₂N^NV ^x H2N^z Y°^x h2K Ύ H₂N _zs_xmu , n ü U Α/'Ά Of wherein 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, Lx-alkenyl, L_x-alkynyl, L_K-alkoxy, and L_x-alkylamine, where L_x is a bond, C(=O), C(=NH), C(=NH)-NH and SO, SO₂.

[00146] In a further embodiment, the accelerant is a bifunctional aromatic amine. In a further embodiment, the aromatic amine is selected from the group:

Bifuctional aromatic amines:

<img file="CA2625540C_D0020.tif" />

[00147] In a further embodiment, the accelerant is an oxoamine derivative. In a further embodiment, the oxoamine derivative is selected from the group:

Oxoamine derivatives:

q Η ^υ H ji _Me'Vⁿ Me^'&lt;A ΜΥ^Ν(ΑνΗ₂[00148] Further, the accelerant include compounds selected from the group consisting of:

Η H ,, .-H

Me O Me O s'

H/γ’ uV’ ηΥυ

NH 0 q¹

H H R, !₂r^NY Υχ _ΗζΝΧ

CA 02625540 2008-04-09

WO 2007/056448

PCT/US2006/043472 the group consisting of: L_x-H, L_x-alkyl, Lx-aryl, Lx-heteroaryl, Lx-alkenyl, Lx-alkynyl, Lx-alkoxy, and L_x-alkylamme, where Lx is a bond, C(=O), C(=NH), and C(=NH)-NH. Further, in any of the aforementioned aspects or embodiments, the accelerant is selected from the compounds presented in FIG. 5, FIG. 9, or FIG. 10, including by way of example any of compounds 6, 8, 10, 7, and 20 of Figure 5. In any of the aforementioned aspects or embodiments, the accelerant includes an agent that can form a hydrazone upon reaction with a carbonyl-containing group. Further, in any of the aforementioned aspects the accelerant activity depends on the rate of reaction with the ketone moiety and the stability of the resulting intermediate. Further, in any of the aforementioned aspects or embodiments, the pH of the reaction mixture comprising the accelerant, the carbonylcontaining 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.

[00149] A protein-derivatizing method based upon the reaction of a carbonyl- or dicarbonyl-containing protein with a hydroxylamine-substituted molecule has distinct advantages. First, hydroxylamines undergo condensation with carbonyl- or dicarbonyl-containing compounds in a pH between about 2 and 8 (and in further embodiments in a pH between about 4 and 8) to generate oxime adducts. Under these conditions, the sidechains of the naturally occurring amino acids are unreactive. Second, such selective chemistry makes possible the site-specific derivatization of recombinant proteins: derivatized proteins can now be prepared as defined homogeneous products. Third, the mild conditions needed to effect the reaction of the hydroxylamines described herein with the carbonyl- or dicarbonylcontaining polypeptides described herein generally do not irreversibly destroy the tertiary structure of the polypeptide (excepting, of course, where the purpose of the reaction is to destroy such tertiary structure). Finally, although the hydroxylamine group amino appears to be metabolized by E. coli, the condensation of hydroxylamines with carbonyl- or dicarbonyl-containing molecules generates oxime adducts which are stable under biological conditions.

[00150] By way of example only, the following non-natural amino acids are the type of carbonyl- or dicarbonylcontaining amino acids that are reactive with the hydroxylamine-containing reagents described herein to form an oxime-containing non-natural amino acid or polypeptide in the presence of an accelerant described herein (although such a reaction may be less efficient in the absence of an accelerant described herein):

r₃

<img file="CA2625540C_D0021.tif" />

° (I) wherein:

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

CA 02625540 2008-04-09

WO 2007/056448

PCT/US2006/043472

Efi§ ’o^SriaV’anff^vhen'pffesSrit is a linker selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O)_kwhere 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(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’)=N-N(R’)-, -C(R’)=N-N=, -C(R’)₂-N=N, and -C(R’)₂-N(R’)-N(R’)-, where each R’ is independently H, alkyl, or substituted alkyl;

<img file="CA2625540C_D0022.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, two R” optionally form a heterocycloalkyl;

Ri is optional, and when present, is H, an amino protecting group, resin; and R₂ is optional, and when present, is OH, an ester protecting group, resin;

each of R₃ and R) is independently H, halogen, lower alkyl, or substituted lower alkyl, or R₃ and R4 or two R₃groups optionally form a cycloalkyl or a heterocycloalkyl;

or the -A-B-J-R groups together form a bicyclic or tricyclic cycloalkyl or heterocycloalkyl comprising at least one carbonyl group, including a dicarbonyl group, protected carbonyl group, including a protected dicarbonyl group, or masked carbonyl group, including a masked dicarbonyl group;

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

[00151] By way of example only, for the aforementioned purposes, compounds of Formula (I) include compounds

R·].

<img file="CA2625540C_D0023.tif" />

having the structure: ^υ (III);

wherein:

R is alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; Ri is optional, and when present, is H, an amino protecting group, R₂ is optional, and when present, is OH, an ester protecting group, resin; and resin; and

CA 02625540 2008-04-09

WO 2007/056448 PCT/US2006/043472 ''eâte^RÎOiltdepenâtehtfy'beiïeëted 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’, where each R’ is independently H, alkyl, or substituted alkyl.

[00152] By way of example only, for the aforementioned purposes, compounds of Formula (I) include compounds 0 0

R ,H N

<img file="CA2625540C_D0024.tif" />

/^XN A L

<img file="CA2625540C_D0025.tif" />

'C(O)R_; (xxx);

having the structure: wherein;

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

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

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

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

Xi is C, S, or S(O); and L is a bond, alkylene, substituted alkylene, N(R’)(alkylene) or N(R’)(substituted alkylene), where each R’ is independently H, alkyl, or substituted alkyl.

[001531 By way of further example only, for the aforementioned purposes, compounds of Formula (I) include compounds having the structure of Formula (XXXX):

R.

R₃

R₃\

Ύ ν^Μγ°

T₃.

R,

R?

(XXXX), wherein:

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

CA 02625540 2008-04-09

WO 2007/056448

PCT/US2006/043472 (13)

M is -C(R₃)-, +/^c\ (a) $ r₃

C— \

Rt R4 !(b) (b) +/ (a) $ ,C=C—(b)

R, (b)

-ΛΛ/¹ (b) vfW' (b) t/W

¢) (a) V Rt (b) v/W» +/^c\ (a) ? R» —^s~§ (b)

I Λ .c—c—s r/ ^x \ ?

Rt X (a) (b) /

Rt ,C-=C—S (b) oR₃ (b) (b) t/w* (a) uv\a (a) ,or

S-C—ξ (b) σν\ρ (a) , where (a) indicates bonding to the A group and (b) indicates bonding to respective carbonyl groups, R₃ and R4 are independently chosen from H, halogen, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl, or R₃ and R4 or two R₃ groups or two R4 groups optionally form a cycloalkyl or a 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;

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

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

[00154] The types of polypeptides that comprise such carbonyl- or dicarbonyl-containing non-natural amino acids is practically unlimited as long as the carbonyl- or dicarbonyl-containing non-natural amino acid is located on the polypeptide so that the hydroxylamine reagent can react with the carbonyl or dicarbonyl group and not create a resulting modified non-natural amino acid that destroys the tertiary structure of the polypeptide (excepting, of course, if such destruction is the purpose of the reaction).

[00155] By way of example only, the following hydroxylamine-containing reagents are the type of hydroxylaminecontaining reagents that are reactive with the carbonyl- or dicarbonyl-containing non-natural amino acids described herein to form an oxime-containing non-natural amino acid in the presence of an accelerant described herein (although such a reaction may be less efficient in the absence of an accelerant described herein):

x•1

Jn (XIX) wherein:

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 heteroaryl, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl, -(alkylene or substituted alkylene)-ON(R”)₂, -(alkylene or substituted alkylene)-C(O)SR”, -(alkylene or substituted alkylene)-S-S-(aryl or substituted aryl), -C(O)R”, -C(O)₂R”, or -C(O)N(R”)₂, wherein each R” is independently hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, alkaryl, substituted alkaryl, aralkyl, or substituted aralkyl;

or each X is independently selected from the group consisting of a desired functionality;

CA 02625540 2008-04-09

WO 2007/056448

PCT/US2006/043472 '’e^5lPmd&amp;pentf&amp;n0y^:feledt®d 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(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’)=N-N(R’)-, C(R’)=N-N=, -C(R’)₂-N=N-, and -C(R’)₂-N(R’)-N(R’)-;

L[ is optional, and when present, is -C(R’)_p-NR’-C(O)O-(alkylene or substituted alkylene)- where p is 0, 1, or 2;

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

W is -N(R₈)₂, where each R_g is independently H or an amino protecting group; and n is 1 to 3; provided that LL_rW together provide at least one hydroxylamine group capable of reacting with a carbonyl (including a dicarbonyl) group on a non-natural amino acid or a (modified) non-natural amino acid poylpeptide.

[00156] In an illustrative embodiment, a hydroxylamine-derivatized reagent is added to a buffered solution (pH 2-8) of a carbonyl-containing non-natural amino acid polypeptide and an accelerant. The resulting oxime-containing nonnatural amino acid polypeptide is purified by HPLC, FPLC or size-exclusion chromatography.

[00157] In a further or alternative illustrative embodiment, the molar ratio of a compound of Formula (I) to a 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.

[00158] In one embodiment, multiple linker chemistries can react site-specifically with a carbonyl- or dicarbonylsubstituted non-natural amino acid polypeptide to form an oxime bond in the presence of an accelerant described herein (although such a reaction may be less efficient in the absence of an accelerant described herein). In one embodiment, the linker methods described herein utilize linkers containing the hydroxylamine functionality on at least one linker termini (mono, hi- or multi-functional). The condensation of a hydroxylamine-derivatized linker with a keto-substituted protein generates a stable oxime bond. Bi- and/or multi-functional linkers (e.g., hydroxylamine with one, or more, other linking chemistries) allow the site-specific connection of different molecules (e.g., other proteins, polymers or small molecules) to the non-natural amino acid polypeptide, while mono-functional linkers (hydroxylamine-substituted on all termini) facilitate the site-specific dimer- or oligomerization of the non-natural amino acid polypeptide. By combining this linker strategy with the in vivo translation technology described herein, it becomes possible to specify the three-dimensional structures of chemically-elaborated proteins.

B. Methods for Post-Translationally Modifying Non-Natural Amino Acid Polypeptides in the Presence of at Least One Accelerant: Reactions of Hydroxylamine-Containing Non-Natural Amino Acids with Carbonyl-Containing Reagents

[00159] The post-translational modification techniques and compositions described above may also be used with hydroxylamine-containing non-natural amino acids reacting with carbonyl- or dicarbonyl-containing reagents to produce modified oxime-containing non-natural amino acid polypeptides in the presence of an accelerant described herein (although such a reaction may be less efficient in the absence of an accelerant described herein).

[00160] The formation of an oxime-containing non-natural amino acid or non-natural amino acid polypeptide from the reaction of a hydroxylamine-containing non-natural amino acid or hydroxylamine-containing non-natural amino

CA 02625540 2008-04-09

WO 2007/056448 PCT/US2006/043472 aôid‘T4l|^ejSWWdM''’eâ:frôhÿjl-i'ontaiiimg reagent can be enhanced by addition of an accelerant to the reaction mixture. An accelerant is a compound that has at least one of the following properties: (a) increase the rate of reaction between a carbonyl-containing compound and a hydroxylamine-containing compound to form an oximecontaining compound, where the increase in rate is relative to the reaction in the absence of the accelerant; (b) lower the activation energy of the reaction between a carbonyl-containing compound and a hydroxylamine-containing compound to form an oxime-containing compound, where the decrease in activation energy is relative to the reaction in the absence of the accelerant; (c) increase the yield of an oxime-containing compound from the reaction of a carbonyl-containing compound with a hydroxylamine-containing compound, where the increase in yield is relative to the reaction in the absence of the accelerant; (d) lower the temperature at which a carbonyl-containing compound reacts with a hydroxylamine-containing compound to form an oxime-containing compound, where the decrease in temperature is relative to the reaction in the absence of the accelerant; (e) decrease the time necessary to react a carbonyl-containing compound with a hydroxylamine-containing compound to form an oxime-containing compound, wherein the decrease in time is relative to the reaction in the absence of accelerant; (f) decrease the amount of reagents necessary to form an oxime group on a non-natural amino acid polypeptide, wherein the decrease in amount of reagents is relative to the reaction in the absence of accelerant; (g) decrease the side products resulting from the reaction of a carbonyl-containing compound with a hydroxylamine-containing compound to form an oxime-containing compound, wherein the decrease in side products is relative to the reaction in the absence of accelerant; (h) does not irreversibly destroy the tertiary structure of a polypeptide undergoing an oxime-forming reaction in the presence of an accelerant (excepting, of course, where the purpose of the reaction is to destroy such tertiary structure); (i) can be separated from an oxime-containing compound in vacuo·, and (j) modulate the reaction of a carbonyl-containing compound with a hydroxylamine-containing compound. In further embodiments, the accelerant 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, or all of the aforementioned properties. In a further embodiment, the accelerant has none of the aforementioned properties. [00161] The use of an accelerant includes the use of a single accelerant or multiple accelerants. In addition, the molar ratio of accelerant 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. Further, the molar ratio of accelerant 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. Further, the accelerant includes compounds that can be substantially removed in vacuo from the resulting oxime-containing compound. Further, the accelerant includes compounds containing a diamine moiety, a semicarbazide moiety, a hydrazine, or a hydrazide moiety. [00162] Further, in any of the aforementioned aspects or embodiments, the accelerant is selected from the group consisting of bifunctional aromatic amines, oxoamine derivatives, and compounds having the following structures:

CA 02625540 2008-04-09

WO 2007/056448

PCT/US2006/043472

R«^Rx-n'ⁿ'r_z

H h₂n

O

<img file="CA2625540C_D0026.tif" />

H H LI u u m-^N\^^NH2 U m-^N^-^NH2 n J h₂n y h₂n y h₂nⁿV H₂F

NH 0 A wherein R_x, R_y and R₂ are selected from tile group consisting of: L_x-H, L_x-alkyl, L_x-aryl, Lx-heteroaryl, Lx-alkenyl, Lx-alkynyl, L_x-alkoxy, and L_x-alkylamine, where L_x is a bond, C(=O), C(=NH), C(=NH)-NH and SO, SO₂.

[00163] In a further embodiment, the accelerant is a bifunctional aromatic amine. In a further embodiment, the aromatic amine is selected from the group:

Bifuctional aromatic amines:

<img file="CA2625540C_D0027.tif" />

[00164] In a further embodiment, the accelerant is an oxoamine derivative. In a further embodiment, the oxoamine derivative is selected fiom the group:

Oxoamine derivatives:

H H

Me O Me O

Me' '^N'O'^Et Me^O^Bn Me^'cA·' Μθ^Ν\ΑνΗ₂ Me^xN'O^x^N^/

[00165] Further, the accelerant include compounds selected fiom the group consisting of:

H,N

H,N

H

Λ \ h 2

T

H,N

H,N

2« Ϊ H₂N-’'R_x wherein 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. Further, in any of the aforementioned aspects or embodiments, the accelerant is selected from the compounds presented in FIG. 5, FIG. 9, or FIG. 10, including by way of example any of compounds 6, 8, 10, 7, and 20 of Figure 5. In any of the aforementioned aspects or embodiments, the accelerant includes an agent that can form a hydrazone upon reaction with a carbonyl-containing group. Further, in any of the aforementioned aspects the accelerant activity depends on the rate of reaction with the ketone moiety and the stability of the resulting intermediate. Further, in any of the aforementioned aspects or embodiments, the pH of the reaction mixture comprising the accelerant, the carbonylcontaining 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.

CA 02625540 2008-04-09

WO 2007/056448 PCT/US2006/043472

[Ôoi6ê]‘^!Â^prtifein-deïlVaïii2ïkg^!' ihethod based upon the reaction of a hydroxylamine-containing protein with a carbonyl- or dicarbonyl-substituted molecule has distinct advantages. First, hydroxylamines undergo condensation with carbonyl- or dicarbonyl-containing compounds in a pH between about 2 to 8 (and in further embodiments in a pH between about 4 to 8) to generate oxime adducts. Under these conditions, the sidechains of the naturally occurring amino acids are unreactive. Second, such selective chemistry makes possible the site-specific derivatization of recombinant proteins: derivatized proteins can now be prepared as defined homogeneous products. Third, the mild conditions needed to effect the reaction 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 (excepting, of course, where the purpose of the reaction is to destroy such tertiary structure). Finally, although the hydroxylamine group amino appears to be metabolized by E. coli, the condensation of carbonyl- or dicarbonyl-containing reagents with hydroxylamine-containing amino acids generates oxime adducts which are stable under biological conditions.

[00167] By way of example only, the following non-natural amino acids are the type of hydroxylamine-containing amino acids that are reactive with the carbonyl- or dicarbonyl-containing reagents described herein to form an oxime-containing non-natural amino acid or polypeptide in the presence of an accelerant described herein (although such a reaction may be less efficient in the absence of an accelerant described herein):

<img file="CA2625540C_D0028.tif" />

K (xiv), wherein:

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

B is optional, and when present is a linker selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O)_kwhere 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(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’)=N-N(R’)-, -C(R’)=N-N=, -C(R’)₂-N=N, and -C(R’)₂-N(R’)-N(R’)-, where each R’ is independently H, alkyl, or substituted alkyl;

Kis-NH₂;

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

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

CA 02625540 2008-04-09

WO 2007/056448

PCT/US2006/043472

<img file="CA2625540C_D0029.tif" />

groups optionally form a cycloalkyl or a heterocycloalkyl.

[00168] The types of polypeptides that comprise such hydroxylamine-containing non-natural amino acids is practically unlimited as long as the hydroxylamine-containing non-natural amino acid is located on the polypeptide so that the carbonyl- or dicarbonyl-containing reagent can react with the hydroxylamine group and not create a resulting modified non-natural amino acid that destroys the tertiary structure of the polypeptide (excepting, of course, if such destruction is the purpose of the reaction).

[00169] By way of example only, the following carbonyl- or dicarbonyl-containing reagents are the type of carbonyl- or dicarbonyl-containing reagents that are reactive with the hydroxylamine-containing non-natural amino acids described herein to form an oxime-containing non-natural amino acid or polypeptide in the presence of an accelerant described herein (although such a reaction may be less efficient in the absence of an accelerant described herein):

l₁-w (xix) wherein:

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 heteroaryl, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl, -(alkylene or substituted alkylene)-ON(R”)₂, -(alkylene or substituted alkylene)-C(O)SR”, -(alkylene or substituted alkylene)-S-S-(aryl or substituted aryl), -C(O)R”, -C(O)₂R”, or -C(O)N(R”)₂, wherein each R” is independently hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, alkaryl, substituted alkaryl, aralkyl, or substituted aralkyl;

or each X is independently selected from the group consisting of a 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)j,(alkylene or substituted alkylene)-, -C(O)-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(aIkylene 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’)=N-N(R’)-, C(R’)=N-N=, -C(R’)₂-N=N-, and -C(R’)₂-N(R’)-N(R’)-;

Li is optional, and when present, is -C(R’)_p-NR’-C(O)O-(alkylene or substituted alkylene)- where p is 0,1, or 2;

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

W is -J-R, where

CA 02625540 2008-04-09

WO 2007/056448

PCT/US2006/043472

<img file="CA2625540C_D0030.tif" />

<img file="CA2625540C_D0031.tif" />

R

<img file="CA2625540C_D0032.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, two R” optionally form a heterocycloalkyl; and n is 1 to 3;

provided that L-L_rW together provide at least one carbonyl group (including a dicarbonyl group) capable of reacting with an hydroxylamine group on a non-natural amino acid or a (modified) non-natural amino acid poylpeptide.

[00170] In an illustrative embodiment, a carbonyl-derivatized reagent is added to a buffered solution (pH 2-8) of a hydroxylamine-containing non-natural amino acid polypeptide and an accelerant. The resulting oxime-containing non-natural amino acid polypeptide is purified by HPLC, FPLC or size-exclusion chromatography,

[00171] In a further or alternative illustrative embodiment, the molar ratio of a compound of Formula (XIV) to a 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.

[00172] In one embodiment, multiple linker chemistries can react site-specifically with a hydroxylamine-substituted non-natural amino acid polypeptide. In one embodiment, the linker methods described herein utilize linkers containing the carbonyl or dicarbonyl functionality on at least one linker termini (mono, bi- or multi-functional). The condensation of a carbonyl- or dicarbonyl-derivatized linker with a hydroxylamine-substituted protein generates a stable oxime bond. Bi- and/or multi-functional linkers (e.g., carbonyl or dicarbonyl with one, or more, other linking chemistries) allow the site-specific connection of different molecules (e.g., other proteins, polymers or small molecules) to the non-natural amino acid polypeptide, while mono-functional linkers (carbonyl- or dicarbonylsubstituted on all termini) facilitate the site-specific dimer- or oligomerization of the non-natural amino acid polypeptide. By combining this linker strategy with the in vivo translation technology described herein, it becomes possible to specify the three-dimensional structures of chemically-elaborated proteins.

C. Example of Adding Functionality in the Presence of at Least One Accelerant: Macromolecular Polymers Coupled to Non-Natural Amino Acid Polypeptides

[00173] Various modifications to the non-natural amino acid polypeptides described herein can be effected using the compositions, methods, techniques and strategies described herein. These modifications include the incorporation of further functionality onto the non-natural amino acid component of the polypeptide via an oxime bond formed in the presence of an accelerant described herein (although such a reaction may be less efficient in the absence of an accelerant described herein), including but not limited to, a desired functionality. As an illustrative, non-limiting example of the compositions, methods, techniques and strategies described herein, the following description will focus on adding macromolecular polymers to the non-natural amino acid polypeptide with the understanding that the compositions, methods, techniques and strategies described thereto are also applicable (with appropriate modifications, if necessary and for which one of ordinary skill in the art could make with the disclosures herein) to adding other functionalities, including but not limited to those listed above.

CA 02625540 2008-04-09

WO 2007/056448

PCT/US2006/043472

[OOÏ74J’ TO^,Tôrtaâtioia-'iF'ifu^abromolecular polymer coupled via an oxime bond to a non-natural amino acid polypeptide from (a) the reaction of a hydroxylamine-containing non-natural amino acid polypeptide and a carbonyl-containing reagent, or (b) the reaction of a carbonyl-containing non-natural amino acid polypeptide and a hydroxylamine-containing reagent, can be enhanced by addition of an accelerant to the reaction mixture. Such an accelerant is a compound that has at least one of the following properties: (a) increase the rate of reaction between a carbonyl-containing compound and a hydroxylamine-containing compound to form an oxime-containing compound, where the increase in rate is relative to the reaction in the absence of the accelerant; (b) lower the activation energy of the reaction between a carbonyl-containing compound and a hydroxylamine-containing compound to form an oxime-containing compound, where the decrease in activation energy is relative to the reaction in the absence of the accelerant; (c) increase the yield of an oxime-containing compound from the reaction of a carbonyl-containing compound with a hydroxylamine-containing compound, where the increase in yield is relative to the reaction in the absence of the accelerant; (d) lower the temperature at which a carbonyl-containing compound reacts with a hydroxylamine-containing compound to form an oxime-containing compound, where the decrease in temperature is relative to the reaction in the absence of the accelerant; (e) decrease the time necessary to react a carbonylcontaining compound with a hydroxylamine-containing compound to form an oxime-containing compound, wherein the decrease in time is relative to the reaction in the absence of accelerant; (f) decrease the amount of reagents necessary to form an oxime group on a non-natural amino acid polypeptide, wherein the decrease in amount of reagents is relative to the reaction in the absence of accelerant; (g) decrease the side products resulting from the reaction of a carbonyl-containing compound with a hydroxylamine-containing compound to form an oximecontaining compound, wherein the decrease in side products is relative to the reaction in the absence of accelerant; (h) does not irreversibly destroy the tertiary structure of a polypeptide undergoing an oxime-forming reaction in the presence of an accelerant (excepting, of course, where the purpose of the reaction is to destroy such tertiary structure); (i) can be separated from an oxime-containing compound in vacuo·, and (j) modulate the reaction of a carbonyl-containing compound with a hydroxylamine-containing compound. In further embodiments, the accelerant 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 propeifies, eight of the aforementioned properties, nine of the aforementioned properties, or all of the aforementioned properties. In a further embodiment, the accelerant has none of the aforementioned properties. [00175] The use of an accelerant includes the use of a single accelerant or multiple accelerants. In addition, the molar ratio of accelerant 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. Further, the molar ratio of accelerant 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. Further, the accelerant includes compounds that can be substantially removed in vacuo from the resulting oxime-containing compound. Further, the accelerant includes compounds containing a diamine moiety, a semicarbazide moiety, a hydrazine, or a hydrazide moiety. [00176] Further, in any of the aforementioned aspects or embodiments, the accelerant is selected from the group consisting of bifunctional aromatic amines, oxoamine derivatives, and compounds having the following structures:

CA 02625540 2008-04-09

WO 2007/056448

PCT/US2006/043472 ‘RL· ^Rxnⁿ'r_z

H h₂n

Η₂Ν^Νγ^Η

Η,Ν’

<img file="CA2625540C_D0033.tif" />

η₂ν'Υ^Η2

NH h₂n'Y^H2 h₂n

Η H

Ύ^Ν0

Η₂Ν^ζΝγ°' η₂νΎ 0 0

H₂N' wherein R_x, R_y and R_z are selected from the group consisting of: L_x-H, L_x-alkyl, L_x-aryl, Lx-heteroaryl, Lx-alkenyl, Lx-alkynyl, Lx-alkoxy, and Lx-alkylamine, where L_x is a bond, C(=O), C(=NH), C(=NH)-NH and SO, SO₂.

[00177] In a farther embodiment, the accelerant is a bifunctional aromatic amine. In a further embodiment, the aromatic amine is selected from the group:

Bifuctional aromatic amines:

<img file="CA2625540C_D0034.tif" />

[00178] In a further embodiment, the accelerant is an oxoamine derivative. In a further embodiment, the oxoamine derivative is selected from the group:

Oxoamine derivatives:

H

Me^N'O^H Me^-

Mett H H il H S nJL

-%^Et Me^xNO-^Bn Me^N\r\ Me to^NH₂ Me Ό N'

[00179] Further, the accelerant include compounds selected from the group consisting of:

»,» '^β γ· ’ NH

H,N’ •NH;

H,N

Η₂Ν-^Νγ^Η

H h,n· wherein 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, Lx-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. Further, in any of the aforementioned aspects or embodiments, the accelerant is selected from the compounds presented in FIG. 5, FIG. 9, or FIG. 10, including by way of example any of compounds 6, 8, 10, 7, and 20 of Figure 5. In any of the aforementioned aspects or embodiments, the accelerant includes an agent that can form a hydrazone upon reaction with a carbonyl-containing group. Further, in any of the aforementioned aspects the accelerant activity depends on the rate of reaction with the ketone moiety and the stability of the resulting intermediate. Further, in any of the aforementioned aspects or embodiments, the pH of the reaction mixture comprising the accelerant, the carbonylcontaining 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.

CA 02625540 2008-04-09

WO 2007/056448

PCT/US2006/043472

<img file="CA2625540C_D0035.tif" />

f ffiadro'iholecular polymers and other molecules can be coupled to the non-natural amino acid polypeptides described herein to modulate biological properties of the non-natural amino acid polypeptide (or the corresponding natural amino acid polypeptide), and/or provide new biological properties to the non-natural amino acid polypeptide (or the corresponding natural amino acid polypeptide). These macromolecular polymers can be coupled to the non-natural amino acid polypeptide via an oxime bond on the non-natural amino acid.

[00181] Water soluble polymers can be coupled to the non-natural amino acid polypeptides described herein. The water soluble polymer may be coupled to the non-natural amino acid by an oxime bond. In some cases, the nonnatural amino acid polypeptides described herein comprise one or more non-natural amino acid(s) linked to water soluble polymers and one or more naturally-occurring amino acids linked to water soluble polymers. Covalent attachment of hydrophilic polymers to a biologically active molecule represents one approach to increasing water solubility (such as in a physiological environment), bioavailability, increasing serum half-life, increasing therapeutic half-life, modulating immunogenicity, modulating biological activity, or extending the circulation time of the biologically active molecule, including proteins, peptides, and particularly hydrophobic molecules. Additional important features of such hydrophilic polymers include biocompatibility, lack of toxicity, and lack of immunogenicity. Preferably, for therapeutic use of the end-product preparation, the polymer will be pharmaceutically acceptable.

[00182] Examples of suitable hydrophilic polymers include: polyalkyl ethers and alkoxy-capped analogs thereof (e.g., polyoxyethylene glycol, polyoxyethylene/propylene glycol, and methoxy or ethoxy-capped analogs thereof, especially polyoxyethylene glycol, the latter is also known as polyethylene glycol or PEG); polyvinylpyrrolidones; polyvinylalkyl ethers; polyoxazolines, polyalkyl oxazolines and polyhydroxyalkyl oxazolines; polyacrylamides, polyalkyl acrylamides, and polyhydroxyalkyl acrylamides (e.g., polyhydroxypropylmethacrylamide and derivatives thereof); polyhydroxyalkyl acrylates; polysialic acids and analogs thereof; hydrophilic peptide sequences; polysaccharides and their derivatives, including dextran and dextran derivatives, e.g., carboxymethyldextran, dextran sulfates, aminodextran; cellulose and its derivatives, e.g., carboxymethyl cellulose, hydroxyalkyl celluloses; chitin and its derivatives, e.g., chitosan, succinyl chitosan, carboxymethylchitin, carboxymethylchitosan; hyaluronic acid and its derivatives; starches; alginates; chondroitin sulfate; albumin; pullulan and carboxymethyl pullulan; polyaminoacids and derivatives thereof, e.g., polyglutamic acids, polylysines, polyaspartic acids, polyaspartamides; maleic anhydride copolymers such as: styrene maleic anhydride copolymer, divinylethyl ether maleic anhydride copolymer; polyvinylalcohols; copolymers thereof; terpolymers thereof; mixtures thereof; and derivatives of the foregoing. The water soluble polymer may be any structural form including but not limited to linear, forked or branched. In some embodiments, polymer backbones that are water-soluble, with from 2 to about 300 termini, are particularly useful. Multifunctional polymer derivatives include, but are not limited to, linear polymers having two termini, each terminus being bonded to a functional group which may be the same or different. In some embodiments, the water polymer comprises a polyethylene glycol) moiety. The molecular weight of the polymer may be of 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 Da and about 100,000 Da, including but not limited to,

<img file="CA2625540C_D0036.tif" />

Da, 500 Da, 400 Da, 300 Da, 200 Da, and 100 Da. In some embodiments, the molecular weight of the polymer is between about 100 Da and about 50,000 Da. In some embodiments, the molecular weight of the polymer is between about 100 Da and about 40,000 Da. In some embodiments, the molecular weight of the polymer is between about

CA 02625540 2008-04-09

WO 2007/056448

PCT/US2006/043472

W'É)a\ Itrsome embodiments, the molecular weight of the polymer is between about 5,000 Da and about 40,000 Da. In some embodiments, the molecular weight of the polymer is between about 10,000 Da and about 40,000 Da. In some embodiments, the polyethylene glycol) molecule is a branched polymer. The molecular weight of the branched chain PEG may be between about 1,000 Da and about 100,000 Da, including but not limited to, 100,000 Da, 95,000 Da, 90,000 Da, 85,000 Da, 80,000 Da, 75,000 Da, 70,000 Da, 65,000 Da, 60,000 Da, 55,000 Da, 50,000 Da, 45,000 Da, 40,000 Da, 35,000 Da, 30,000 Da, 25,000 Da, 20,000 Da, 15,000 Da, 10,000 Da, 9,000 Da, 8,000 Da, 7,000 Da, 6,000 Da, 5,000 Da, 4,000 Da, 3,000 Da, 2,000 Da, and 1,000 Da. In some embodiments, the molecular weight of the branched chain PEG is between about 1,000 Da and about 50,000 Da. In some embodiments, the molecular weight of the branched chain PEG is between about 1,000 Da and about 40,000 Da. In some embodiments, the molecular weight of the branched chain PEG is between about 5,000 Da and about 40,000 Da. In some embodiments, the molecular weight of the branched chain PEG is between about 5,000 Da and about 20,000 Da. Those of ordinary skill in the art will recognize that the foregoing list for substantially water soluble backbones is by no means exhaustive and is merely illustrative, and that all polymeric materials having the qualities described above are contemplated as being suitable for use in methods and compositions described herein. [00183] As described above, one example of a hydrophilic polymer is polyethylene glycol), abbreviated PEG, which has been used extensively in pharmaceuticals, on artificial implants, and in other applications where biocompatibility, lack of toxicity, and lack of immunogenicity are of importance. The polymerpolyeptide embodiments described herein will use PEG as an example hydrophilic polymer with the understanding that other hydrophilic polymers may be similarly utilized in such embodiments.

[00184] PEG is a well-known, water soluble polymer that is commercially available or can be prepared by ringopening polymerization of ethylene glycol according to methods well known in the art (Sandler and Karo, Polymer Synthesis, Academic Press, New York, Vol. 3, pages 138-161). PEG is typically clear, colorless, odorless, soluble in water, stable to heat, inert to many chemical agents, does not hydrolyze or deteriorate, and is generally non-toxic. Poly(ethylene glycol) is considered to he biocompatible, which is to say that PEG is capable of coexistence with living tissues or organisms without causing harm. More specifically, PEG is substantially non-immunogenic, which is to say that PEG does not tend to produce an immune response in the body. When attached to a molecule having some desirable function in the body, such as a biologically active agent, the PEG tends to mask the agent and can reduce or eliminate any immune response so that an organism can tolerate the presence of the agent. PEG conjugates tend not to produce a substantial immune response or cause clotting or other undesirable effects.

[00185] The term “PEG” is used broadly to encompass any polyethylene glycol molecule, without regard to size or to modification at an end of the PEG, and can be represented as linked to a non-natural amino acid polypeptide by the formula:

XO-(CH₂CH₂O)_n-CH₂CH₂-Y where n is 2 to 10,000 and X is H or a terminal modification, including but not limited to, a C_M alkyl, a protecting group, or a terminal functional group. The term PEG includes, but is not limited to, polyethylene glycol) in any of its forms, including bifunctional PEG, multiarmed PEG, derivatized PEG, forked PEG, branched PEG (with each chain having a molecular weight of from about 1 kDa to about 100 kDa, from about 1 kDa to about 50 kDa, or from about 1 kDa to about 20 kDa), pendent PEG (i.e, PEG or related polymers having one or more functional groups pendent to the polymer backbone), or PEG with degradable linkages therein. In one embodiment, PEG in which n is from about 20 to about 2000 is suitable for use in the methods and compositions described herein. In some embodiments, the water polymer comprises a polyethylene glycol) moiety. The molecular weight of the polymer may be of a wide range, including but not limited to, between about 100 Da and about 100,000 Da or more. The

CA 02625540 2014-01-28 molecular weight of the polymer may be between about 100 Da and about 100,000 Da, including but not limited to, 100,000 Da, 95,000 Da, 90,000 Da, 85,000 Da, 80,000 Da, 75,000 Da, 70,000 Da, 65,000 Da, 60,000 Da, 55,000 Da, 50,000 Da, 45,000 Da, 40,000 Da, 35,000 Da, 30,000 Da, 25,000Da, 20,000 Da, 15,000 Da, 10,000 Da, 9,000 Da, 8,000 Da, 7,000 Da, 6,000Da, 5,000 Da, 4,000 Da, 3,000 Da, 2,000 Da, 1,000 Da, 900 Da, 800 Da, 700 Da, 600 Da, 500 Da, 400 Da, 300 Da, 200 Da, and 100 Da. ht some embodiments, the molecoter weight of the polymer is between about 100 Da and about 50,000 Da. ht some embodiments, file molecular weight of the polymer is between about 100 Da and about 40,000 Da. h some embodiments, the molecular weight of the polymer is between about 1,000 Da and about 40,000 Da. In some embodiments, the molecular weight of the polymer is between about 5,000 Da and about 40,000 Da. In some embodiments, the molecular weight of the polymer is between about 10,000 Da and about 40,000 Da. In some embodiments, the polyethylene glycol) molecule is a branched polymer. The molecular weight of the branched chain PEG may be between about 1,000 Da and about 100,000 Da, including but not limited to, 100,000 Da, 95,000 Da, 90,000 Da, 85,000 Da, 80,000 Da, 75,000 Da, 70,000 Da, 65,000 Da, 60,000 Da, 55,000 Da, 50,000 Da, 45,000 Da, 40,000 Da, 35,000 Da, 30,000 Da, 25,000 Da, 20,000 Da, 15,000Da, 10,000 Da, 9,000 Da, 8,000 Da, 7,000 Da, 6,000 Da, 5,000 Da, 4,000 Da, 3,000 Da, 2,000 Da, and 1,000 Da. In some embodiments, fi» molecular weight of the branched chain PBG is between about 1,000 Da and about 50,000 Da. In some embodiments, the molecular weight of the branched chain PBG is between about 1,000 Da and about 40,000 Da. In some embodiments, the molecular weight of the branched chain PEG is between about 5,000 Da and about 40,000 Da. In. some embodiments, fi» molecular weight of the branched chain PEG 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 Polymws, Inc. catalog, Nektar Therapeutics catalog.

[00186) Specific examples of terminal functional groups in the literature include, but are not limited to, Nsuccmûnidyl carbonate (see e.g., UE. Pat Nos. 5,281,698,5,468,478), amine (see, e.g., Buckmann et al. MakromoL Chem. 182:1379 (1981), Zalipsky et al. Bur. Polym. J. 19:1177 (1983)), hydrazide (See, e.g, Andresz et al. MakromoL Chem. 179:301 (1978)), succininedyl propionate and succiniraidyl butanoate (see, e.g., Olson et al. in Polyethylene glycol) Chemistry &amp; Biological Applications, pp 170-181, Harris &amp; Zalipsky Eds., ACS, Washington, D.C., 1997; sec also U.S. Pat Να 5,672,662), succmimidyl succinate (See, e.g., Abnchowski et al. Cancer Biochem. Biophys, 7:175 (1984) and Joppich et al. Makromol. Chem. 180:1381 (1979), siicciairaidyl ester (see, e.g., U.S. Pat Να 4,670,417), benzotriazole carbonate (see, e.g., U.S. Pat No. 5,650,234), glycidyl ether (see, e.g., Pitfaa et al. Eur. J Biochem. 94:11 (1979), Blling et al., Biotech. AppL Biochem. 13:354 (1991), oxycarbonylimidazole (see, e.g., Beauchamp, et al·, Anal. Biochem. 131:25 (1983), Tondellï et al. J. Controlled Release 1:251 (1985)), pnitrophenyl carbonate (see, e.g,, Veronese, et al., Appl. Biochem. Biotech., 11:141 (1985); and Sartœe et al., Appl. Biochem. Biotech., 27:45 (1991)), aldehyde (see, e.g., Harris et al. J. Polym. Sci. Chem. Ed. 22:341 (1984), U.S. Pat No. 5,824,784, U.S. Pat. No. 5,252,714), maleimide (see, c.g., Goodson et at. Bio/Tediaology 8:343 (1990), Romani ei al. in Chemistry of Peptides and Proteins 2:29 (1984)), and Kogan, Synthetic Comm. 22:2417 (1992)), orthopyridyl-disulfide (see, e.g., Woghiren, et al. Biocoqj. Chem. 4:314(1993)), aczylol (see, e.g., Sawhney ei af Macromolecules, 26:581 (1993)), vtnylsulfone (see, e.g., U.S. Pat No. 5,900,461),

[001871 In some cases, a PEG terminates on one end with hydroxy or methoxy, i.e., X is H or GHj (“methoxy PEG”). Alternatively, the PEG can terminate with a reactive group, thereby forming a bifimctional polymer. Typical reactive groups can include those reactive groups that are commonly used to react with tie functional groups found in the 20 common amino acids (inchiding but not limited to, maleimide groups, activated carbonates (including but not limited to, p-nitropbenyl ester), activated esters (including but not limited to, N-hydroxysuccininiide, p55

CA 02625540 2014-01-28 nitrophenyl ester) and aldehydes) ^W611 as fbnctional groups that an inert to die 20 common amino acids but that react specifically with complementary functional groups present in non-natural amino acids to farm an oxime group in the presence of an accelerant described herein (although such a reaction may be less efficient in fi» absence of an accelerant described herein); examples of file latter include but are not limited to, carbonyl or dicarbonyl and hydroxylamme groups.

[00188] It is noted that file other end of the PEG, which is shown in the above formula by Y, will attach either directly or indirectly to a polypeptide via a non-natural amino acid. When Y is a hydroxylamine group, then the hydroxybrnine-containing PEG reagent can react with a carbonyl- or dicaibonyl-containing non-natural amino acid in a polypeptide to form a PEG grotto coupled to the polypeptide via an oxime bond in the presence of an accelerant described herein (although such a reaction may be less efficient in the absence of an accelerant described herein). When Y is a carbonyl or dicarbonyl group, then the carbonyl- or dicarbonyl-containing PEG reagent can react with a hydroxylamine-containing non-natural amino acid in a polypeptide to form a PEG group coupled to the polypeptide via an oxime bond in the presence of an accelerant described herein (although such a reaction may be less efficient hi the absence of an accelerant described herein).

[00189] Heterobifimctional derivatives are also particularly useful when it is desired to attach different molecules to each terminus of fire polymer. For example, the omega-N-amino-N-azido PEG would allow the attachment of a molecule having an activated electrophilic group, such as an aldehyde, ketone, activated ester, activated carbonate and so forth, to one terminus of the PEG and a molecule having an acetylene group to fire other terminus of the PEG. [00190] In some embodiments, a strong nucleophile (including but not limited to hydroxylamine) cart he reacted with an carbonyl group, including a ketone group present in a non-natural amino acid to form an oxime in fi» presence of an accelerant described herein (although such a reaction may be less efficient in fi» absence of an accélèrent described herein); the subsequent oxime group in some cases can he further reduced by treatment with an appropriate reducing agent. Alternatively, the strong nucleophile can be incorporated into fi» polypeptide via a nonnatural amino acid and used to react preferentially with a carbonyl group, including a ketone group present in the water soluble polymer to form an oxime in the presence of an accelerant described herein (although such a reaction may be less efficient in the absence of an accelerant described herein). Generally, at least one terminus of the PEG molecule is available for reaction with fi» non-natural amino acid,

[00191] TJ» polymer backbone can he linear or branched. Branched polymer backbones are generally known in die art. Typically, a branched polymer has a central branch core moiety and a plurality of linear polymer chains linked to fi» central branch care. PEG is used in branched forms that can be prepared by addition of ethylene oxide to various polyols, such as glycerol, glycerol oligomers, pentaerythritol and sorbitol. The central branch moiety can also be derived from several amino acids, such as lysine. The branched polyethylene glycol) can he represented in general form as Kf-PEG-OHJm in which R is derived from a core moiety, such as glycerol, glycerol oligomers, or pentaerythritol, and m represents the number of arms. Multi-armed PEG molecules, such as those described in U.S. Pat Nos. 5,932,462 5,643,575; 5,229,490; 4,289,872; U.S. Pat Appl. 2003/0143596; WO 96/21469; and WO 93/21259, can also be used as fi» polymer backbone.

[00192] Branched PEG can also be in fi» form of a forked PEG represented by ΡΕΟζ-ΥΟΗΖ^ where Y is a linking group, n is 100-1,000 (i.e., average molecular weight is between about 5 kDa to about 40 kDa), and Z is an activated terminal group linked to CH by a chain of atoms of defined length. Yet another branched ferra, fi» pendant PEG, has reactive groups, such as carboxyl, along the PEG backbone rather than at the end of PEG chains.

CA 02625540 2008-04-09

WO 2007/056448

PCT/US2006/043472

40^!bi^'-’-k&amp;h3r2Jr~W'inakiB^t tffe^i:âesired properties of PEG, the total molecular weight and hydration state of the

PEG polymer or polymers attached to the biologically active molecule must be sufficiently high to impart the advantageous characteristics typically associated with PEG polymer attachment, such as increased water solubility and circulating half life, while not adversely impacting the bioactivity of the parent molecule.

[00194] The methods and compostions described herein may be used to produce substantially homogenous preparations of polymer:protein conjugates. “Substantially homogenous” as used herein means that polymer:protein conjugate molecules are observed to be greater than half of the total protein. The polymer:protein conjugate has biological activity and the present “substantially homogenous” PEGylated polypeptide preparations provided herein are those which are homogenous enough to display the advantages of a homogenous preparation, e.g., ease in clinical application in predictability of lot to lot pharmacokinetics.

[00195] As used herein, and when contemplating hydrophilic polymerrpolypeptide/protein conjugates, the term “therapeutically effective amount” refers to an amount which provides benefit to a patient. The amount will vary from one individual to another and will depend upon 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 present compositions may be readily ascertained by one of ordinary skill in the art using publicly available materials and procedures. By way of example only, a therapeutically effective amount may be an amount which gives an increase in hematocrit for anemia patients; it may be an amount which decreases tumor size in cancer patients; it may be an amount which increases insulin levels in diabetic patients; or it may be an amount which decreases pain in patients suffering form chronic pain.

[00196] The number of water soluble polymers linked to a (modified) non-natural amino acid polypeptide (i.e., the extent of PEGylation or glycosylation) described herein can be adjusted to provide an altered (including but not limited to, increased or decreased) pharmacologic, pharmacokinetic or pharmacodynamic characteristic such as in vivo half-life. In some embodiments, the half-life of the polypeptide is increased at least about 10,20, 30, 40, 50, 60, 70, 80, 90 percent, two fold, five-fold, 10-fold, 50-fold, or at least about 100-fold over an unmodified polypeptide. [00197] In one embodiment, a polypeptide comprising a carbonyl- or dicarbonyl-containing non-natural amino acid is modified, in the presence of an accelerant, with a PEG derivative that contains a terminal hydroxylamine moiety that is linked directly to the PEG backbone, thus forming an oxime bond (although such a reaction may be less efficient in the absence of an accelerant described herein). In some embodiments, the hydroxylamine-terminal PEG derivative will have the structure:

RO-(CH₂CH₂O)_n-O-(CH₂)_m-O-NH₂ where R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10 and n is 100-1,000 (i.e., average molecular weight is between about 5 kDa to about 40 kDa). The molecular weight of the polymer may be of a wide range, including but not limited to, between abolit 100 Da and about 100,000 Da or more. The molecular weight of the polymer may be between about 100 Da and about 100,000 Da, including but not limited to, 100,000 Da, 95,000 Da, 90,000 Da, 85,000 Da, 80,000 Da, 75,000 Da, 70,000 Da, 65,000 Da, 60,000 Da, 55,000 Da, 50,000 Da, 45,000 Da, 40,000 Da, 35,000 Da, 30,000 Da, 25,000 Da, 20,000 Da, 15,000 Da, 10,000 Da, 9,000 Da, 8,000 Da, 7,000 Da, 6,000 Da, 5,000 Da, 4,000 Da, 3,000 Da, 2,000 Da, 1,000 Da, 900 Da, 800 Da, 700 Da, 600 Da, 500 Da, 400 Da, 300 Da, 200 Da, and 100 Da. In some embodiments, the molecular weight of the polymer is between about 100 Da and about 50,000 Da. In some embodiments, the molecular weight of the polymer is between about 100 Da and about 40,000 Da. In some embodiments, the molecular weight of the polymer is between about 1,000 Da and about 40,000 Da. In some embodiments, the molecular weight of the polymer is between about 5,000 Da and about 40,000 Da. In some embodiments, the molecular weight of the polymer is between about 10,000 Da and about 40,000 Da.

CA 02625540 2008-04-09

WO 2007/056448

PCT/US2006/043472 fffcïcëii :er - éœbotiiment^-â polypeptide comprising a carbonyl- or dicarbonyl-containing amino acid is modified with a PEG derivative that contains a terminal hydroxylamine moiety that is coupled to the PEG backbone by means of an amide bond, thus forming an oxime bond (although such a reaction may be less efficient in the absence of an accelerant described herein) further coupled to the PEG backbone by means of an amide bond. In some embodiments, the hydroxylamine-terminal PEG derivatives have the structure:

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

[00199] In another embodiment, a polypeptide comprising a carbonyl- or dicarbonyl-containing amino acid is modified with a branched PEG derivative that contains a terminal hydroxylamine moiety thus forming an oxime bond (although such a reaction may be less efficient in the absence of an accelerant described herein), with each chain of the branched PEG having an average molecular weight ranging from about 10 kDa to about 40 kDa and, in other embodiments, from about 5 kDa to about 20 kDa. In some embodiments, the PEG derivatives containing a hydoxylamine group will have the structure:

[RO-(CH₂CH₂O)_n-O-(CH₂)₂-C(O)-NH-CH₂-CH₂]₂CH-X-(CH₂)_m-O-NH₂where R is a simple alkyl (methyl, ethyl, propyl, etc.), X is optionally NH, O, S, C(O) or not present, m is 2-10 and n is 100-1,000. The molecular weight of the branched chain PEG may be between about 1,000 Da and about 100,000 Da, including but not limited to, 100,000 Da, 95,000 Da, 90,000 Da, 85,000 Da, 80,000 Da, 75,000 Da, 70,000 Da, 65,000 Da, 60,000 Da, 55,000 Da, 50,000 Da, 45,000 Da, 40,000 Da, 35,000 Da, 30,000 Da, 25,000 Da, 20,000 Da, 15,000 Da, 10,000 Da, 9,000 Da, 8,000 Da, 7,000 Da, 6,000 Da, 5,000 Da, 4,000 Da, 3,000 Da, 2,000 Da, and 1,000 Da. In some embodiments, the molecular weight of the branched chain PEG is between about 1,000 Da and about 50,000 Da. In some embodiments, the molecular weight of the branched chain PEG is between about 1,000 Da and about 40,000 Da. In some embodiments, the molecular weight of the branched chain PEG is between about 5,000 Da and about 40,000 Da. In some embodiments, the molecular weight of the branched chain PEG is between about 5,000 Da and about 20,000 Da.

[00200] Several reviews and monographs on the functionalization and conjugation of PEG are available. See, for example, Harris, Macromol. Chem. Phys. C25: 325-373 (1985); Scouten, Methods in Enzymology 135: 30-65 (1987); Wong et al., Enzyme Microb. Technol. 14: 866-874 (1992); Delgado et al., Critical Reviews in Therapeutic Drug Carrier Systems 9: 249-304 (1992); Zalipsky, Bioconjugate Chem. 6: 150-165 (1995). Methods for activation of polymers can also be found in WO 94/17039, U.S. Pat. No. 5,324,844, WO 94/18247, WO 94/04193, U.S. Pat No. 5,219,564, U.S. Pat. No. 5,122,614, WO 90/13540, U.S. Pat. No. 5,281,698, and more WO 93/15189, and for conjugation between activated polymers and enzymes including but not limited to Coagulation Factor VIII (WO 94/15625), haemoglobin (WO 94/09027), oxygen carrying molecule (U.S. Pat. No. 4,412,989), ribonuclease and superoxide dismutase (Veronese at al., App. Biochem. Biotech. 11: 141-52 (1985)).

[00201] If necessary, the PEGylated non-natural amino acid polypeptides described herein obtained from the hydrophobic chromatography can be purified further by one or more procedures known to those of ordinary skill in the art including, but are not limited to, affinity chromatography; anion- or cation-exchange chromatography (using, including but not limited to, DEAE SEPHAROSE); chromatography on silica; reverse phase HPLC; gel filtration (using, including but not limited to, SEPHADEX G-75); hydrophobic interaction chromatography; size-exclusion chromatography, metal-chelate chromatography; ultrafiltration/diafiltration; ethanol precipitation; ammonium sulfate precipitation; chromatofocusing; displacement chromatography; electrophoretic procedures (including but not limited to preparative isoelectric focusing), differential solubility (including but not limited to ammonium sulfate

CA 02625540 2008-04-09

WO 2007/056448

PCT/US2006/043472

<img file="CA2625540C_D0037.tif" />

protein standards (Preneta AZ, PROTEIN PURIFICATION METHODS, A PRACTICAL APPROACH (Harris &amp; Angal, Eds.) IRL Press 1989, 293-306). The purity of the non-natural amino acid polypeptide:PEG conjugate can he assessed hy proteolytic degradation (including but not limited to, trypsin cleavage) followed by mass spectrometry analysis. PepinskyRB, et.al., J. Pharmcol. &amp;Exp. Ther. 297(3):1059-66 (2001).

D. Use of Linking Groups and Applications, Including Polypeptide Dimers and Multimers

[00202) In addition to adding desired functionality directly to the non-natural amino acid polypeptide, the nonnatural amino acid portion of the polypeptide may first be modified with a multifunctional (e.g., bi-, tri, tetra-) linker molecule that then subsequently is further modified. That is, at least one end of the multifunctional linker molecule reacts with at least one non-natural amino acid in a polypeptide and at least one other end of the multifunctional linker is available for further functionalization. If all ends of the multifunctional linker are identical, then (depending upon the stoichiometric conditions) homomultimers of the non-natural amino acid polypeptide maybe formed. If the ends of the multifunctional linker have distinct chemical reactivities, then at least one end of the multifunctional linker group can react to as to be bound to the non-natural amino acid polypeptide and the other end can subsequently react with a different functionality, including by way of example only, a desired functionality. [00203] The multifunctional linker group has the general structure:

(xix) wherein:

each X is independently NH₂, -C(=O)R₉, -SR’ or -J-R, where R₉ is H or OR’, where

J is

<img file="CA2625540C_D0038.tif" />

R R

<img file="CA2625540C_D0039.tif" />

R , or

R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; each R” is independently H, alkyl, substituted alkyl, or a protecting group, or when more than one R” group is present, two R” optionally form a heterocycloalkyl;

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

each L is independently selected from the group consisting of alkylene, substituted alkylene, alkenylene, substituted alkenylene, -O-, -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(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-, 59

CA 02625540 2008-04-09

WO 2007/056448

PCT/US2006/043472

<img file="CA2625540C_D0040.tif" />

AIL “□&gt; Η, ’.τ, “3, jü-(‘âlKÿierièi^éïjlîstituted 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’)=N-N(R’)-, C(R’)=N-N=,-C(R’)₂-N=N-, and -C(R’)₂-N(R’)-N(R’)-;

Li is optional, and when present, is -C(R’)_p-NR’-C(O)O-(alkylene or substituted alkylene)- where p is 0, 1, or

W is NH₂, -C(=O)R₉, -SR’ or -J-R; and n is 1 to 3 provided that X and L-Lj-W together independently each provide at least one of the following (a) a hydroxylamine group capable of reacting with a carbonyl (including a dicarbonyl) group on a non-natural amino acid or a (modified) non-natural amino acid poylpeptide; (b) a carbonyl group (including a dicarbonyl group) capable of reacting with an hydroxylamine group on a non-natural amino acid or a (modified) nonnatural amino acid poylpeptide; or (c) a carbonyl group (including a dicarbonyl group) capable of undergoing an exchange reaction with an oxime group on a non-natural amino acid or a (modified) non-natural amino acid poylpeptide.

[00204] In a further or alternative illustrative embodiment, the molar ratio of a compound of Formula (I) or Formula (XIV) to the multifunctinal 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. [00205] A bifunctional homolinker in which the linker has two identical ends, i.e., hydroxylamine groups, can be used to form coupled polypeptides via the formation of oxime bonds, wherein the formation of such coupled polypeptides is performed in the presence of at least one accelerant (although the oxime bond may also occur at a slower reaction rate in the absence of the accelerant). The use of an accelerant described herein in the formation of polypeptide dimers and multimers is expected to provide significant benefit because that stoichiometric ratio of the linker to 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 the accelerant than in the absence of the accelerant (or, further, as the molar ratio of accelerant increases, the closer the ratios of the aforementioned reactants will be to stoichiometric). The stoichiometric ratio (or molar ratio) is an important factor in the modification of polypeptides because of the expense of the reagents (including the polypeptide and the molecules for conjugation) and the difficulty in purification. Thus, the use of the accelerants provided herein can be used to reduce the cost and waste resulting from the modification of non-natural amino acid polypeptides, including the formation of polypeptide dimers or multimers, or the linking of any desired group or functionality to a polypeptide.

[00206] Such a linker may be used to form a homodimer of a carbonyl- or dicarbonyl-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 accelerant (although the oxime bond may also occur at a slower reaction rate in the absence of the accelerant). Alternatively, if one end of such a linker is protected, then such a partially protected linker can be used to bind the unprotected hydroxylamine end to a carbonyl- or dicarbonyl-containing non-natural amino acid polypeptide via an oxime bond, leaving the other protected end available for further linking reactions following deprotection. Alternatively, careful manipulation of the stoichiometry of the reagents may provide a similar result (a heterodimer), albeit a result in which the desired heterodimer will likely be contaminated with some homodimer.

[00207] Such a linker may 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 accelerant (although the oxime bond may also occur at a slower reaction rate in the absence of the accelerant). Alternatively, if one end of such a linker is protected, then such a partially protected linker can be used to bind the unprotected carbonyl end to a hydroxylamine-containing non-natural amino acid polypeptide via an oxime bond, leaving the other protected end available for further linking reactions following deprotection. Alternatively, careful

CA 02625540 2008-04-09

WO 2007/056448

PCT/US2006/043472 if ZII if iE£ !pi e. Z if.|f ““’ii 3 manipülâtibn-ôTthe sïôicmbMtetfy· of the reagents may provide a similar result (a heterodimer), albeit a result which the desired heterodimer will likely be contaminated with some homodimer.

[00208] A multifunctional heterolinkers in which each linker has more than one type of terminal reactive group, i.e., hydroxylamine, oxime and thioester groups, can be used to form coupled polypeptides via the formation of at least one oxime bond, wherein the formation of the oxime bond is performed in the presence of at least one accelerant. Such a linker may be used to form a heterodimer of a non-natural amino acid polypeptide using the accelerantpromoted oxime-based chemistry discussed throughout this specification.

[00209] The methods and compositions described herein also provide for polypeptide combinations, such as homodimers, heterodimers, homomultimers, or heteromultimers (i.e., trimers, tetramers, etc.). By way of example only, the following description focuses on the GH supergene family members, however, the methods, techniques and compositions described in this section can be applied to virtually any other polypeptide which can provide 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, C-X-C chemokine, T39765, NAP-2, ENA-78, gro-a, gro-b, gro-c, IP-10, GCP-2, NAP-4, SDF-1, PF4, MIG, calcitonin, c-kit ligand, cytokine, CC chemokine, monocyte chemoattractant protein-1, monocyte chemoattractant protein-2, monocyte chemoattractant protein-3, monocyte inflammatory protein-1 alpha, monocyte inflammatory protein-i beta, RANTES, 1309, R83915, R91733, HCC1, 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, MCP1, 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, four-helical bundle 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 albumin, ICAM-1, ICAM-1 receptor, LFA-1, LFA-1 receptor, insulin, insulin-like growth factor (IGF), IGFI, IGF-II, interferon (IFN), IFN-alpha, IFN-heta, IFN-gamma, any inteferon-like molecule 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, kératinocyte growth factor (KGF), lactoferrin, leukemia inhibitory factor, luciferase, neurturin, neutrophil inhibitory factor (NIF), oncostatin M, osteogenic protein, oncogene product, paracitonin, parathyroid hormone, PD-ECSF, PDGF, peptide hormone, pleiotropin, protein A, protein G, pth, pyrogenic exotoxin A, pyrogenic exotoxin B, pyrogenic exotoxin C, pyy, 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, SEC1, SEC2, SEC3, SED, SEE, steroid hormone receptor, superoxide dismutase, toxic shock syndrome toxin, thymosin alpha 1, tissue plasminogen activator, tumor growth factor (TGF), tumor necrosis factor, tumor necrosis factor alpha, tumor necrosis factor beta, tumor necrosis factor receptor (TNFR), VLA-4 protein, VCAM-1 protein, vascular endothelial growth factor (VEGF), urokinase, mos, ras, raf, met, 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 growth hormone supergene family.

[00210] Thus, encompassed within the methods, techniques and compositions described herein are a GH supergene family member polypeptide containing one or more non-natural amino acids bound to another GH supergene family member or variant thereof or any other polypeptide that is a non-GH supergene family member or variant thereof, either directly to the polypeptide backbone or via a linker. Due to its increased molecular weight compared to

CA 02625540 2008-04-09

WO 2007/056448

PCT/US2006/043472 ihohoMfers; -family member dimer or multimer conjugates may exhibit new or desirable properties, including but not limited to different pharmacological, pharmacokinetic, pharmacodynamic, modulated therapeutic half-life, or modulated plasma half-life relative to the monomeric GH supergene family member. In some embodiments, the GH supergene family member dimers described herein will modulate the dimerization of the GH supergene family member receptor. In other embodiments, the GH supergene family member dimers or multimers described herein will act as a GH supergene family member receptor antagonist, agonist, or modulator. [00211] In some embodiments, the methods and compositions described herein provide multimers comprising one or more GH supergene family member formed by reactions with water soluble activated polymers that have the structure:

R-(CH₂CH₂O)_n-O-(CH₂)_m-X wherein n is from about 5 to 3,000, m is 2-10, X can be a hydroxylamine or carbonyl- or dicarhonyl-containing moiety, and R is a capping group, a functional group, or a leaving group that can be the same or different as X. R can 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, alkenyl, acrylate, methacrylate, acrylamide, active sulfone, amine, aminooxy, protected amine, hydrazide, protected hydrazide, protected thiol, carboxylic acid, protected carboxylic acid, isocyanate, isothiocyanate, maleimide, vinylsulfone, dithiopyridine, vinylpyridine, iodoacetamide, epoxide, glyoxals, diones, mesylates, tosylates, and tresylate, alkene, and ketone.

[00212] Using the chemistry detailed throughout this specification, one of ordinary skill in the art could design a linker in which at least one functional group can form an oxime group, in the presence of the accelerant disclosed herein, with a non-natural amino acid polypeptide; the other functional groups on the linker could utilize other known chemistry, including the nucleophile/electrophile based chemistry well known in the art of organic chemistry.

[00213] The formation of a polypeptide dimer or multimer linked together via at least one oxime group from (a) the reaction of a hydroxylamine-containing non-natural amino acid polypeptide and a carbonyl-containing reagent, or (b) the reaction of a carbonyl-containing non-natural amino acid polypeptide and a hydroxylamine-containing reagent, can be enhanced by addition of an accelerant to the reaction mixture. Such an accelerant is a compound that has at least one of the following properties: (a) increase the rate of reaction between a carbonyl-containing compound and a hydroxylamine-containing compound to form an oxime-containing compound, where the increase in rate is relative to the reaction in the absence of the accelerant; (b) lower the activation energy of the reaction between a carbonyl-containing compound and a hydroxylamine-containing compound to form an oxime-containing compound, where the decrease in activation energy is relative to the reaction in the absence of the accelerant; (c) increase the yield of an oxime-containing compound from the reaction of a carbonyl-containing compound with a hydroxylamine-containing compound, where the increase in yield is relative to the reaction in the absence of the accelerant; (d) lower the temperature at which a carbonyl-containing compound reacts with a hydroxylaminecontaining compound to form an oxime-containing compound, where the decrease in temperature is relative to the reaction in the absence of the accelerant; (e) decrease the time necessary to react a carbonyl-containing compound with a hydroxylamine-containing compound to form an oxime-containing compound, wherein the decrease in time is relative to the reaction in the absence of accelerant; (f) decrease the amount of reagents necessary to form an oxime group on a non-natural amino acid polypeptide, wherein the decrease in amount of reagents is relative to the reaction in the absence of accelerant; (g) decrease the side products resulting from the reaction of a carbonylcontaining compound with a hydroxylamine-containing compound to form an oxime-containing compound, wherein

CA 02625540 2008-04-09

WO 2007/056448 PCT/US2006/043472

-F /ηπ&amp;ηιρ.

the^!'de”creaseinside produbts ïa relative to the reaction in the absence of accelerant; (h) does not irreversibly destroy the tertiary structure of a polypeptide undergoing an oxime-forming reaction in the presence of an accelerant (excepting, of course, where the purpose of the reaction is to destroy such tertiary structure); (i) can he separated from an oxime-containing compound in vacw, and (j) modulate the reaction of a carbonyl-containing compound with a hydroxylamine-containing compound. In further embodiments, the accelerant 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, or all of the aforementioned properties. In a further embodiment, the accelerant has none of the aforementioned properties.

[00214] The use of an accelerant includes the use of a single accelerant or multiple accelerants. In addition, the molar ratio of accelerant 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. Further, the molar ratio of accelerant 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. Further, the accelerant includes compounds that can be substantially removed in vacuo from the resulting oxime-containing compound. Further, the accelerant includes compounds containing a diamine moiety, a semicarhazide moiety, a hydrazine, or a hydrazide moiety. [00215] Further, in any of the aforementioned aspects or embodiments, the accelerant is selected from the group consisting of bifunctional aromatic amines, oxoamine derivatives, and compounds having the following structures:

H

H,N hXV YY ^H2nJY ^Η2Ν'^ν

<img file="CA2625540C_D0041.tif" />

h₂n'Y^H2 h/Y NH O

Η H

Η₂Ν'^Νγ^Ν^

O h₂nY ηΑγ

O 0 wherein R_x, R_y and R_z are selected from the group consisting of: L_x-H, L_x-alkyl, Lx-aryl, L_x-heteroaryl, L_x-alkenyl, Lx-alkynyl, L_:i-alkoxy, and L_x-alkylamine, where L_x is a bond, C(=O), C(=NH), C(=NH)-NH and SO, SO₂.

[00216] In a further embodiment, the accelerant is a bifunctional aromatic amine. In a further embodiment, the aromatic amine is selected from the group:

Bifuctional aromatic amines:

<img file="CA2625540C_D0042.tif" />

[00217] In a further embodiment, the accelerant is an oxoamine derivative. In a further embodiment, the oxoamine derivative is selected from the group:

CA 02625540 2014-01-28

PCT/DS2006/043472

Oxoamino derivatives?’

N1»'^'O'^H Μ··'^\Τ^β# Μί^Ό^

[00218] Further, the accelerant include compounds selected from the group consisting of:

Μ·γ«’ „,3y ,,Α, _H,A.

&gt;

wherein R„ R, andRc are selected from the group consisting où L*-H, I^-alkyl, L»-aryl, L,-heteroaryl, U-alkenyl, Lx-alkynyl, [^-alkoxy, and I^-alkylamine, where L, is a hand, C(“O), C(“NH), and C(NH)-NH. Further, in any of the aforementioned aspects or embodiments, the accelerant is selected from the compounds presented in FIG. 5, FIG. 9, or FIG. 10, including by way of example any of compounds 6, 8,10,7, and 20 of Figure 5. In any of the aforementioned aspects or embodiments, the accelerant includes an agent that can form a hydrazone upon reaction with a carbonyl-containing group. Further, in any of the aforementioned aspects the accelerant activity depends on the rate of reaction with (he ketone moiety and die stability of the resulting intermediate. Further, in any of the aforementioned aspects or embodiments, the pH of the reaction mixture comprising the accelerant; 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 3D 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 45; about 5.0; about 5.5; about 6.0; about 6.5; and about 7.0.

Expression in Alternate Systems

[00219] Several strategies have been employed to introduce non-natural amino acids into proteins in nonrecombinant host cells, mutagenized host cells, or in cell-free systems. These systems are also suitable for use in malting the non-natural amino acid polypeptides described herein. Derivatization of amino acids with reactive side* chains such as Lys, Cys and Tyr resulted in the conversion of lysine to N’-acetyl-Iysine. Chemical synthesis also provides a straightforward method to incorporate nan-natural amino adds. With foe recent development of enzymatic ligation and native chemical ligation of peptide fragments; it is possible to make larger proteins. See, e.g„ P. £. Dawson and S. B. H, Kent, Annu, Rev, Biochem, 69:923 (2000). Chemical peptide ligation and native chemical ligation are described in U.S. Patent No. 6,184,344, U.S. Patent Publication No. 2004/0138412, U.S. Patent Publication No. 2003/0208046, WO 02/098902, and WO 03/042235

A general in vitro biosynthetic method in which a suppressor tRNA chemically acylated with the desired non-natural amino acid is added to an in vitro extract capable of supporting protein biosynthesis, has been used to site-specifically incorporate over 100 non-natural amino acids into a variety of proteins of virtually any size. See, eg., V. W. Cornish, D. Mendel and P. G. Schultz, Aneaw. Chem. lot Bd. Bnel.· 1995,34:621 (1995); GJ. Noren, S.J. Anthony-CahiD, M.G Griffith, P.G. Schultz, A general method for site-specific incorporation of non-natural amino acids into proteins, Science 244:182-188 (1989); and, JD. Bain, GG. Giabe, T.A. Dix, A.R. Chamberlin, E.S. Diala, Biosynthetic site-specific incorporation tf a non-natural amino acid Into a polypeptide, J, Am. Cheat, Soc. 111:8013-8014 (1989). A broad range of functional groups has been introduced into proteins for studies of protein stability, protein folding, enzyme mechanism, and signal transduction.

[00220] An in vivo method, termed selective pressure incorporation, was developed to exploit foe promiscuity of wild-type synthetases. See, e.g., N. Budisa, C. Minks, S. Alefelder, W. Wenger, F. M. Dona L. Moroder and R.

Huber, FASEB 1.13:41 (1999). An auxotrophic strain, in which foe relevant metabolic pathway supplying foe cell 64

CA 02625540 2014-01-28 with a particular natural amino acid is switched off, is grown in minimal media cantaining.limitcd Concentrations of the natural amino acid, while transcription of the target gene is repressed. At the onset of a stationary growth phase, the nature! amino acid is depleted and replaced with the non-natural ammo acid analog. Induction of expression of the recombinant protein results in the accumulation of a protein containing the rum-natural analog. For example, using this strategy, o, m and p-fluoropbenylalanines have been incorporated into proteins, and exhibit two characteristic shoulders in the UV spectrum which can be easily identified, see, eg, C. Minks, R. Huber, V Moroder and N. Budisa, Anal. Biochem.· 284:29 (2000); trifluoromethionine has been used to replace methionine in bacteriophage T4 lysozyme to study its interaction with chitooligosaccharide ligands by ”F NMR, see, eg., H. Duewel, B. Daub, V. Robinson and J. F. Honek, Biochemistry, 36:3404 (1997); and trifluoioleucine has been incorporated in place of leucine, resulting in increased thermal and chemical stability of a leudne-zipper protein. See, eg., Y. Tang, G. Ghirlanda, W. A. Petin, T. Nakajima, W. F. DeGrado and D. A Tinell, Antew. Chem, Int Ed, Broti- 40:1494 (2001). Moreover, selenomethionine and teUuromethionine are incorporated into various recombinant proteins to facilitate the solution of phases inX-ray crystallography. See, eg., W. A Hendrickson, J. R. Horton andD. M. lemaster, EMBOJ.. 9:1665 (1990); J. O. Boles, K. Lewinski, M. Kunkle, 3. D. Odom, B. Dunlap, L. Lebioda and M. Hatada, Nat Struct, Biot. 1:283 (1994); N. Budisa, B. Stripe, P. Demange, C. Eckerakom, I Kellennann and R. Huber, Bur. J. Biochem.. 230:788 (1995); and, N- Budisa, W. Kanibrock, S. Steinbacher, A Hunun, L. Prude, T. Neuefeind, L. Moroder and R. Huber, I. Mol. Biol. 270:616 (1997). Methionine analogs with alkene or alkyne functionalities have also been incorporated efficiently, allowing firr additional modification of proteins by chemical means. See, e.g, J. C. van Hest and D. A Titrait, PEBSLett, 428:68 (1998); 3. C. van Host, K. L. Kiick and D. A Tirrell, J. Am. Chem, Soc., 122:1282 (2000); and, K. L. Kiick and D. A. Tinell, Tetrahedron, 56.-9487 (2000); U.S. Patent No. 6,586,207; U.S. Patent Publication 2002/0042097.

[00221] The success of this method depends on the recognition of the non-natural amino acid analogs by armnoacjd-fRNA synthetases, which, in general, require high selectivity to insure tire fidelity of protein translation. One way to expand the scope of this method is to relax the substrate specificity of aminoaoyl-tRNA synthetases, which has been achieved in a limited number of cases. For example, replacement of Ala²⁵* by Gly in Escherichia colt phenylaianyl-tRNA synthetase (PheRS) increases the size of substrate binding pocket, and results in the acylation of tRNAPhe by p-Cl-phenyialanine (p-Cl-Phe). See, M. Ibba, P. Kast and H. Hennecke, Biochemistry. 33:7107 (1994). An Escherichia coli strain harboring this mutant PheRS allows the mcorparatinn of p-Clphenylalanine or p-Br-phenylahmme in place of phenylalanine. See, eg., M. Ibba and H. Hennecke, FBBS Lett.. 364:272 (1995); and, N. Sharma, R. Furter, P. Kast and D. A Tiirell, FBBS Lett., 467:37 (2000). SMarty, a point mutation Phel30Ser near the amino acid binding site of Escherichia coli tyrosyl-tRNA synthetase was shown to allow azatyrosine to be incorporated more efficiently than tyrosine. See F. Hamsno-Takaku, T. Iwatna, S. SaitoYano, K. Takaku, Y. Monden, M. Kitabatake, D. Soil and S. Nishimura, J, Biol· Chan., 275:40324 (2000).

[00222] Another strategy to incorporate non-naturai amino acids into proteins in vivo is to modify synthetases that .have proofreading mechanisms. These synthetases cannot discriminate and therefore activate amino acids that are structurally similar to the cognate natural amino acids. This error is corrected at a separate site, which deacyletes the misebarged amino acid ftom the tRNA to maintain the fidelity of protein translation. If the proofreading activity of tire synthetase is disabled, structural analogs that are misactivated may escape the editing function and be incorporated. Ibis approach has been demonstrated recently with the valyl-tRNA synthetase (ValRS). See, V. Doling, H. D. Mootz, L. A Nangle, T. L. Hendrickson, V. de Ctecy-Ugard, P. Schimmel and P. Mariiere, Science, 292:501 (2001). ValRS can misaminoacylate fRNAVal with Cys, Thr, or aminobutyrate (Abu); these noncognate

CA 02625540 2008-04-09

WO 2007/056448 PCT/US2006/043472 a’mno'ULiSyaM-SubsêqüënÛy'fy'dralyzed by the editing domain. After random mutagenesis of the Escherichia coli chromosome, a mutant Escherichia coli strain was selected that has a mutation in the editing site of ValRS. This edit-defective ValRS incorrectly charges tRNAVal with Cys. Because Abu sterically resembles Cys (—SH group of Cys is replaced with -CH3 in Abu), the mutant ValRS also incorporates Abu into proteins when this mutant Escherichia coli strain is grown in the presence of Abu. Mass spectrometric analysis shows that about 24% of valines are replaced by Abu at each valine position in the native protein.

[00223] Solid-phase synthesis and semisynthetic methods have also allowed for the synthesis of a number of proteins containing non-natural amino acids. For example, see the following publications and references cited within, which are as follows: Crick, F.H.C., 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 activating potency of an S-peptide fragment, J. Am Chem, 88(24):5914-5919 (1966); Kaiser, E.T. Synthetic approaches to biologically active peptides and proteins including enyzmes, Acc Chem Res, 22:47-54 (1989); Nakatsuka, T., Sasaki, T., Kaiser, E.T. Peptide segment coupling catalyzed by the semisynthetic enzyme thiosubtilisin, J Am Chem Soc. 109:3808-3810 (1987); Schnolzer, M., Kent, S B H. Constructing proteins by dovetailing unprotected synthetic peptides: backbone-engineered HIV protease, Science. 256(5054):221-225 (1992); Chaiken, I.M. Semisynthetic peptides and proteins, CRC Crit Rev Biochem, 11(3):255-301 (1981); Offord, R.E. Protein engineering by chemical means? Protein Eng., 1(3):151-157 (1987); and, Jackson, D.Y., Burnier, J., Quan, C., Stanley, M., Tom, J., Wells, J.A. A Designed Peptide ligase for Total Synthesis of Ribonuclease A with Non-natural Catalytic Residues, Science, 266(5183):243 (1994).

[00224] Chemical modification has been used to introduce a variety of non-natural side chains, including cofactors, spin labels and oligonucleotides into proteins in vitro. See, e.g., Corey, D.R., Schultz, P.G. Generation of a hybrid sequence-specific single-stranded deoxyribonuclease, Science, 238(4832):1401-1403 (1987); Kaiser, E.T., Lawrence D.S., Rokita, S.E. The chemical modification of enzymatic specificity, Annu Rev Biochem, 54:565-595 (1985); Kaiser, E.T., Lawrence, D.S. Chemical mutation of enyzme active sites, Science, 226(4674):505-511 (1984); Neet, K.E., Nanci A, Koshland, D.E. Properties of thiol-subtilisin, J Biol. Chem, 243(24):6392-6401 (1968); Polgar,

L. et M.L. Bender. A new enzyme containing a synthetically formed active site. Thiol-subtilisin. J. Am Chem Soc, 88:3153-3154 (1966); and, Pollack, S.J., Nakayama, G. Schultz, P.G. Introduction of nucleophiles and spectroscopic probes into antibody combining sites, Science, 242(4881):1038-1040 (1988).

[00225] Alternatively, biosynthetic methods that employ chemically modified aminoacyl-tRNAs have been used to incorporate several biophysical probes into proteins synthesized in vitro. See the following publications and references cited within: Brunner, J. New Photolabeling and crosslinking methods, Annu. Rev Biochem, 62:483-514 (1993); and, Krieg, U.C., Walter, P., Hohnson, A.E. Photocrosslinking of the signal sequence of nascent preprolactin of the 54-kilodalton polypeptide of the signal recognition particle, Proc. Natl. Acad. Sci, 83(22):86048608 (1986).

[00226] Previously, it has been shown that non-natural amino acids can be site-specifically incorporated into proteins in vitro by the addition of chemically aminoacylated suppressor tRNAs to protein synthesis reactions programmed with a gene containing a desired amber nonsense mutation. Using these approaches, one can substitute a number of the common twenty amino acids with close structural homologues, e.g., fluorophenylalanine for phenylalanine, using strains auxotropic for a particular amino acid. See, e.g., Noren, C.J., Anthony-Cahill, Griffith,

M. C., Schultz, P.G. A general method for site-specific incorporation of non-natural amino acids into proteins, Science, 244: 182-188 (1989); M.W. Nowak, et al., Science 268:439-42 (1995); Bain, J.D., Glabe, C.G, Dix, T.A., Chamberlin, A.R., Diala, E.S. Biosynthetic site-specific Incorporation of a non-natural amino acid into a

CA 02625540 2014-10-16 polypeptide J. Am Chem. Soe, III ^013-8014 («89); N. Budisa et al., FASSEA 13:41-51 (1999); Ellman, J.A., Mendel, D, Anthony-Cahilf S., Noren, C.Î., Schultz, P.G. diosynthetic method for introducing non-natural amino acids site-specifically into proteins. Methods in Eoz.. vol. 202,301-336 (1992); and, Mendel, D., Cornish, V.W. &amp; Schultz, P.G. Site-Directed Mutagenesis with an Expanded Genetic Code, Aimu Rev Bioohva, Blomol Struct. 24, 435-62 (1995).

(00227] For in vivo methods for incorporating non-natural amino aoids into proteins and other polypeptides, and for methods for producing the appropriate synthetases/tRNAs see e.g. US. Patent Nos. 7,045,337 and 7,083,970.

(00228] For example, a suppressor tRNA was prepared that recognized the stop codon UAG and was chemically aminoaeylated with an non-natural amino acid. Conventional site-directed mutagenesis was used to introduce the atop codon TAG, at the site of interest in tho protein gene. See, e.g., Sayers, I.R., Schmidt, W, Eckstein, F. 5'-3’ Exomideases in phosphoroihioate-based oligtioucleoflde-directed mitagensis, Nucleic Acids Res. 16(3):791-802 (1988). When the acylated suppressor tRNA and the mutant gene were combined in an in vitro transcriptfon/translation system, the non-natural amino acid was incorporated in response to the UAG codon which gave a protein containing that amino acid at the specified position. Experiments using [’Hj-Phe and experiments with α-hydroxy acids demonstrated that only the desired amino acid is incorporated at the position specified by die UAG codon and that this amino acid is not incorporated at any other site in the protein. See, e.g., Noren, et al, supra; Kobayashi el al, (2003) Nature Structural Biology 10(6):425-432; and, Ellman, J A., Mendel, D., Schultz, P.G, Sitespecific incorporation of novel backbone structures into pivtelns, Science, 255(5041):197-200 (1992).

(00229] A tRNA may be aminoaeylated with a desired amino acid by any method or technique, including but not limited to, chemical or enzymatic aminoacylation.

(00230) Aminoacylation may be accomplished by aminoacyl tRNA synthetases or by other enzymatic molecules, including but not limited to, ribozymes. The term “ribozyme” is interchangeable with “catalytic RNA. Cech end coworken (Cech, 1987, Science, 236:1532-1539; McCorkle et al., 1987, Concepts Biodicm. 64:221-226) demonstrated the presence of naturally occurring 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 splicing, the recent development of artificial evolution of ribozymes has expanded potential catalysis to various chemical reactions. Studies have identified SNA tnolecnles that can catalyze aoiinoacyl-RNA bonds on their own (293'tennjni (Illangakekate et al., 1995 Science 267:643-647), and an RNA molecule which can transfer an amino acid from one RNA molecule to another (Lohse etal., 1996, Nature 381:442-444).

[00231] U.S. Patent Application Publication 2003/0228593, describes methods to construct ribozymes and their use in aminoacylation of (RNAs with naturally encoded and non-natural amino acids. Substrate-immobilized forma of enzymatic molecules that can aminoacylate tRNAs, including but not limited to, ribozymes, may enable officient affinity purification of the aminoaeylated products. Example of suitable substrates include agarose, sepharose, and magnetic beads. The production and use of a substrate-immobilized form of ribozyme for aminoacylation is described in Chemistry and Biology 2003, 10:1077-1084 and U.S. Patent Application Publication 200370228593.

)00232] Chemical aminoacylation methods include, but ate not limited to, those introduced by Hecht and coworkers (Hecht, S. M. Acc. Chem. Res. 1992,25,545; Heckler, T. G.; Roesser, J. R·; Xu, C.; Chang, P.; Hecht, S. M. Biochemistry 1988, 27, 7254; Hecht, S. M.; Alford, B. L·; Kuroda, Y.; Kitano, S. J. BioL Chem. 1978, 253, 4517) and by Schultz, Chamberlin, Dougherty and others (Cornish, V. W.; Mendel, D.; Schulte, P. G. Angew. Chem. Int Ed. Engl 1995, 34, 621; Robertson, S. A.; Ellman, J. A.; Schultz, P. G. J. Am. Chem. Soe. 1991,113,

CA 02625540 2014-01-28

2722; Norcn, C. J. Anthony Cahill; S. J. ^{: M}- ^C-1 Schulte, P. G. Science 1989,244,182; Bain, J. D.; Glabe,

C. G,; Dix, T. A.; Chamberlin, A. R. J. Am. Chem. See. 1989, 111, 8013; Bain, J. D. et al Nature 1992,356,537; Gallivan, J. P.; Lester, H. A.; Dougherty, D. A. Chem. Biot 1997,4,740; TnrcaW, et al. J. Biol. Chem. 1996,271, 19991; Nowak, M. W. et ai Science, 1995,268,439; Saks, M. B. et at J. Biol. Cham. 1996,271,23169; Hohsaka, T. et al. I Am. Chem. Soc. 1999,121,34), to avoid the use of synthetases in aminoacylation. Such methods or other chemical sminoacylation methods may be used to aminoacylate iRNA molecules described herein.

100233] Methods for generating catalytic RNA may involve generating separate pools of randomized ribozyme sequences, performing directed evolution on the pools, screening the pools for desirable sminoacylation activity, and selecting sequences of those ribozymes exhibiting desired aminoaejdation activity.

(00234] Ribozymes can conquise motifs and/or regions that facilitate acylation activity, such as a GGU motif and a U-rich region. For example, it has been reported that U-rich regions can facilitate recognition of an amino acid substrate, and a GGU-motif can form base pairs with the 3* termini of a tRNA, In combination, the GGU and motif and Urich region facilitate simultaneous recognition of both file amino acid and tRNA simultaneously, and thereby facilitate aminoacylation of the 3' terminus of die tRNA (00235] Ribozymes can be generated by in vitro selection using a partially randomized i24ntim conjugated with «NA*®;». followed by systematic engineering of a consensus sequence found in the active clones. An exemplary ribozyme obtained by this method is termed *Tx3 ribozyme and is described in U.S. Pub. App. Να. 2003/0228593, acts as a versatile catalyst for foe synthesis of various aminoacyi-tRNAs charged with cognate non-natural amino acids.

[00236] Aminoacylate tRNAs ribozymes can be immobilized on a substrate so as to ratable efficient affinity purification of the anrinoacylated tRNAs. Examples of suitable substrates include, but are not limited to, agarose, sepharose, and magnetic beads. Ribozymes can be immobilized on resins by taking advantage of the chemical structure of RNA, such as foe 3-ras-diol on the ribose of RNA can be oxidized with periodate to yield flic corresponding dialdehyde to facilitate immobilization of foe RNA on the resin. Various types of résina can be used including inexpensive hydrazide resins wherein reductive amination makes foe interaction between the resin and foe ribozyme an irreversible linkage. Synthesis of aminoacyl-fRNAs can be significantly facilitated by this on-column aminoacylation technique. Kourouklis et at Methods 2005; 36:239-4 describe a column-based aminoacylation system.

[00237] Isolation of the anrinoacylated tRNAs can be accomplished in a variety of ways. One suitable method la to elute foe anrinoacylated tRNAs from a column with a buffer such as a sodium acetate solution with 10 mM BDTA a buffer containing 50 mM N-(24iydroxyethyl)piperazine-N'-(3-propanesulfonic acid), 12.5 mM KCi, pH 7.0,10 mM EDl’A, or simply an EDTA buffered water (pH 7.0).

[00238] ’ The anrinoacylated tRNAs can be added to translation reactions in order to incorporate the amino acid with which the tRNA was anrinoacylated in a position of choice in a polypeptide made by the translation reaction. Examples of translation systems in which foe anrinoacylated tRNAs described herein may be used include, but are not limited to cell lysates. CeU lysates provide reaction components necessary for in vitro translation of a polypeptide from an input mRNA. Examples of such reaction components include but are not limited to ribosomal proteins, iRNA, amino acids, tRNAs, GTP, ATP, translation initiation and elongation factors and additional factors associated wifo translation. Additionally, translation systems may be batch translations or compartmentalized translation. Batch translation systems combine reaction components in a single compartment while compartmentalized translation systems separate the translation reaction components from reaction products that can inhibit the translation efficiency. Such translation systems are available commercially,

CA 02625540 2008-04-09

WO 2007/056448 PCT/US2006/043472

[00239]’ a^v‘cSip3i“iansiriptioii/translation system may be used. Coupled transcription/translation systems allow for both transcription of an input DNA into a corresponding mRNA, which is in turn translated by the reaction components. An example of a commercially available coupled transcription/translation is the Rapid Translation System (RTS, Roche Inc.). The system includes a mixture containing E, coli lysate for providing translational components such as ribosomes and translation factors. Additionally, an RNA polymerase is included for the transcription of the input DNA into an mRNA template for use in translation. RTS can use compartmentalization of the reaction components by way of a membrane interposed between reaction compartments, including a supply/waste compartment and a transcription/translation compartment.

[00240] Aminoacylation of tRNA may be performed by other agents, including but not limited to, transferases, polymerases, catalytic antibodies, multi-functional proteins, and the like.

[00241] Stephan in Scientist 2005 Oct 10; pages 30-33 describes additional methods to incorporate non-natural amino acids into proteins. Lu et al. in Mol Cell. 2001 Oct;8(4):759-69 describe a method in which a protein is chemically ligated to a synthetic peptide containing non-natural amino acids (expressed protein ligation).

[00242] Microinjection techniques have also been use incorporate non-natural amino acids into proteins. See, e.g., M. W. Nowak, P. C. Kearney, J. R. Sampson, M. E. Saks, C. G. Labarca, S. K. Silverman, W. G. Zhong, J. Thorson, J. N. Abelson, N. Davidson, P. G. Schultz, D. A. Dougherty and H. A. Lester, Science, 268:439 (1995); and, D. A. Dougherty, Curr. Opin. Chem. Biol.. 4:645 (2000). A Xenopus oocyte was coinjected with two RNA species made in vitro: an mRNA encoding the target protein with a UAG stop codon at the amino acid position of interest and an amber suppressor tRNA aminoacylated with the desired non-natural amino acid. The translational machinery of the oocyte then inserts the non-natural amino acid at the position specified by UAG. This method has allowed in vivo structure-function studies of integral membrane proteins, which are generally not amenable to in vitro expression systems. Examples include the incorporation of a fluorescent amino acid into tachykinin neurokinin-2 receptor to measure distances by fluorescence resonance energy transfer, see, e.g., G. Turcatti, K. Nemeth, M. D. Edgerton, U. Meseth, F. Talabot, M. Peitsch, J. Knowles, H. Vogel and A. Chollet, J. Biol. Chem., 271:19991 (1996); the incorporation of biotinylated amino acids to identify surface-exposed residues in ion channels, see, e.g., J. P. Gallivan, H. A. Lester and D. A. Dougherty, Chem. Biol., 4:739 (1997); the use of caged tyrosine analogs to monitor conformational changes in an ion channel in real time, see, e.g., J. C. Miller, S. K. Silverman, P. M. England, D. A. Dougherty and H. A. Lester, Neuron, 20:619 (1998); and, the use of alpha hydroxy amino acids to change ion channel backbones for probing their gating mechanisms. See, e.g., P. M. England, Y. Zhang, D. A. Dougherty and H. A. Lester, Cell, 96:89 (1999); and, T. Lu, A. Y. Ting, J. Mainland, L. Y. Jan, P. G. Schultz and J. Yang, Nat. Neurosci.. 4:239 (2001).

[00243] The ability to incorporate non-natural amino acids directly into proteins in vivo offers a wide variety of advantages, including by way of example only, high yields of mutant proteins, technical ease, the potential to study the mutant proteins in cells or possibly in living organisms and the use of these mutant proteins in therapeutic treatments and diagnostic uses. The ability to include non-natural amino acids with various sizes, acidities, nucleophilicities, hydrophobicities, and other properties into proteins can greatly expand our ability to rationally and systematically manipulate the structures of proteins, both to probe protein function and create new proteins or organisms with novel properties.

[00244] In one attempt to site-specifically incorporate para-F-Phe, a yeast amber suppressor tRNAPheCUA /phenylalanyl-tRNA synthetase pair was used in a p-F-Phe resistant, Phe auxotrophic Escherichia coli strain. See, e.g., R. Furter, Protein Sci., 7:419 (1998).

CA 02625540 2014-01-28

[00245] It may also be possible to obtain expression ofa hob-mîumI amino add polypeptide described herein using a coll-ftee (in-vino) translational system. Translation systems may be cellular or cell-free, end may be prokaryotic or eukaryotic. Cellular translation systems include, but are not limited to, t«We cell preparations such as penseabtiized cells or cell cultures wherein a desired nudoic acid sequence can be tatnscrflied to niRNA and tin mRNA translated. Cell-See translalloaa systems are commercially available and many different types and systems are well-known. Bxamples of cell-free systems inohide, but arc 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, phosphorylsted or otherwise modified because many such modifications are only possible in eukaryotic systems. Some of these extracts and lysates are available commercially (Fromoga; Madison, Wis.; Stratagene; La Jolla, Calif; Amersham; Arlington Heights, III.; OIBCO/BRL; Grand Island, N,Y.&gt; Membranous extracts, such as the canino pancreatic extracts containing microsomal membranes, are also available which are uae&amp;l for translating secretory proteins. In these systems, which can include either niRNA as a template (in-vitro translation) or DNA aa a template (combined in-vitro transcription and translation), the in vitro synthesis is directed by the ribosomes. Considerable «Sut has been applied to the development of ceD-fiee protein expression systems. See, e.g., Kim, DM and J.R. Swartz, Biotechnology and Bioengineering, 74 309-316 (2001); Kim, DM and J.R, Swartz, Biotechnology Letters, 22, 1537-1542, (2000); Kim, DM., and JR. Swartz, Biotechnology Progress, 16, 385-390, (2000); Kim, D.M., and J.R. Swartz, Biotechnology and Bioengineering, 66, 180-188, (1999); andPatoaik, R. and JR. Swartz, Bloiechaiqna 24,862-8«, (1998); U.S. Patent No. 6337,191; UR. Patent Publication No. 2002/0081660; WO 00/55353; WO 90/05785. Another approach that may be applied to the expression of non-natural amino acid polypeptides includes the niRNA-peptide fbaiou technique. See, e.g., R. Roberts and L Szostak, Proc. Natl Acad. SA (USA) 94:12297-12302 (1997); A. Frankel, el al., Chemistry &amp; Biology 10:1043-1050 (2003). In this approach, an niRNA template linked to puromycin is translated into peptide on the ribosome. If one or more tRNA molecules has been modified, nonnatural amino acids can be incorporated into the peptide as well. After tire last mRNA codon has been read, puromycin captures the C-terminus of the peptide. If the resulting niRNA-peptide conjugate is found to bave interesting properties in an in vitro assay, its identity can be easily revealed from flic niRNA sequence. In tin's way, one may screen libraries of non-natural amino acid polypeptides comprising one or more non-natural amino adds to identify polypeptides having desired properties. More recently, in vitro ribosome translations with purified components have been reported that permit the synthesis of peptides substituted with non-natural amino acids. See, e.g,, A. Forster et A, Proc. Natl Acad. SA (USA) 100:6353 (2003).

[00246] Reconstituted translation systems may also be used. Mixtures of purified translation factors have also been used successfully to translate mRNA into protein as well ts combinations of lysates or lysates supplemented with purified translation factors such as initiation factor-1 (IF-1), IF-2, IF-3 (a or B), elongation fbetor T (EF-Το), or termination factors. Cell-free systems may also be coupled transcription/translation systems wherein DNA is introduced to the system, transcribed into mRNA and the mRNA translated as described in Current Protocols In Molecular Biology (F. M. Ausubel et al. editors, Wiley Interscience, 1993).

RNA transcribed in eukaryotic transcription system may be in tire form of heteronucleac

RNA (hnRNA) or 5'-end caps (7-mcthy) guanosine) and 3'-end poly A tailed mature niRNA, which can he an advantage in certain translation systems. For example, capped roRNAs are translated with high efficiency in the reticulocyte lysate system.

CA 02625540 2008-04-09

WO 2007/056448

PCT/US2006/043472

EXAMPLES

Example 1 : Improved oxime formation in the presence of accelerant

[00247] A carbonyl-containing compound (non-natural amino acid polypeptide) is shown in Scheme 1A. The keto group of the amino acid para-acetylphenylalanine incorporated into the protein reacts with a hydroxylaminecontaining compound to form a relatively stable oxime. This reaction is highly specific. A faster reaction was observed in the presence of an accelerant (Scheme IB).

<img file="CA2625540C_D0043.tif" />

<img file="CA2625540C_D0044.tif" />

<img file="CA2625540C_D0045.tif" />

Scheme IB

Example 2: Screening of potential accelerants using 30 K PEG hGH-pAcF conjugation

[00248] Human growth hormone (hGH) with para-acetylphenylalanine substituted for tyrosine at position 35 was used to screen a panel of 20 compounds (FIG. 5). hGH was buffer exchanged into 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 a Centrocon (10 K MWCO) concentrator. hGH (10 μϊ) was mixed with 3.6 μϊ mono hydroxylamine 30 K PEG (2.5 mM), 3 μΐ of potential accelerant solution (200 mM in hGH reaction buffer) and buffer to give a final volume of 30 μϊ. The molar ratio of hGH:PEG was 1:2. The reaction mixtures were incubated at 28°C for 16 horns and 36 hours and analyzed by SDS-PAGE (FIG. 6). Each lane of the gels shown in Figure 6 is labeled with the compound tested as shown in Figure 5. The last lane in each gel was a control reaction (no accelerant). Compounds 6, 7, 8, 10, and 20 were accelerants. Two compounds from the panel, compounds 7 and 20 (acetic hydrazide) (pictured in Figure 5), were accelerants and were further evaluated under similar reaction conditions with a higher hGH protein concentration (8 mg/ml). The reaction mixtures were incubated at 28°C for 16 hours, and the results from SDS-PAGE analysis are shown in FIG. 7. Lane 1 was a reaction mixture with a hGH:PEG molar ratio of 1:2 and 50 mM of accelerant compound 7. Lane 2 was a reaction mixture with a hGH:PEG molar ratio of 1:2 and 50 mM of accelerant compound 20. Lane 3 was a reaction mixture with a hGH:PEG molar ratio of 1:2 with no accelerant. Lane 4 was a reaction mixture with a hGH:PEG molar ratio of 1:5 with no accelerant. After 16 hours, bolh compounds were shown to catalyze the reaction.

Example 3: LCMS analysis of hGH after incubation with accelerant

[00249] In hGH reaction buffer, wild type hGH (5.8 mg/ml) was incubated with various concentrations of the accelerant acetic hydrazide (200 mM, 100 mM, 50 mM, 25 mM, 12.5 mM, 6.25 mM and 0 mM) at 28 °C for 48 hours. The accelerant was removed through dialysis (10 k MWCO). The resulting protein solutions were analyzed by LCMS (FIG. 8).] Figure 10A shows the total LCMS trace. Figure 8B shows the mass spectrum of hGH without accelerant. Figure 8C shows the mass spectrum of hGH with 200 mM accelerant acetic hydrazide.

[00250] An accelerant for protein conjugation preferably does not exert any deteriorative effects on protein, such as fragmentation, precipitation, and undesired covaler&gt;+ modification. No fragmentation was observed when using 71

CA 02625540 2008-04-09

WO 2007/056448

PCT/US2006/043472 ,- ? il ii C: iT’fi c .-·· u.,» « it. ::¾.

accélérants:¹ 7^!drid-^:‘2O ^!!is&amp;Wh'Trf-Pi (s&amp;^h^,!‘iii'-Pigure 5) based on SDS-PAGE analysis and protein precipitation in all the conditions used with both scFv and hGH. For example, no covalent modification could be detected with LCMS after 48 hour of incubation of wild type hGH with up to 200 mM accelerant 20 (acetic hydrazide). The measured molecular weights of hGH in all the conditions are same and match theoretical value 22256.

Example 4: One step dimerization of scFv-pAcF

[00251] Single chain Fv (scFv) 108 protein with para-acetylphenylalanine substituted at position 259 (scFv 108 259-pAcF) was used the following conjugation experiment. scFv 108 259-pAcF in storage buffer was buffer exchanged into 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 hydroxylamine homobifunctional 2 K PEG linker 2.5 mM stock solution and supplemented with acetic hydrazide as accelerant. The final reaction mixture consisted of 147 fiM homobifunctional 2 K PEG linker, the corresponding concentration of pAcF-substituted scFv and 47 mM acetic hydrazide. The reaction mixtures were incubated at 28 °C and analyzed at different time points (6 hours, 20 hours, 44 hours) by SDS-PAGE (FIG. 2).

[00252] To make the one-step reaction more efficient and more practical with the homobifunctional 2 K PEG linker, accelerants were used to facilitate the dimerization process. Without accelerant, very little dimer product could be detected after 20 hours by SDS-PAGE. Lane 4 of the 6 hour, 20 hour, and 44 hour gels show reaction mixtures with a scFv:PEG linker molar ratio of 2.0:1 without the accelerant acetic hydrazide. On the other hand, in the presence of 47 mM acetic hydrazide, the dimer product appeared after 6 hours. As shown in Figure 2, different protein and linker molar ratios, 1.6:1,2.0:1 and 2.4:1, were tested to scan for the best conjugation conditions. Lane 1 of the 6 hour, 20 hour, and 44 hour gels show reaction mixtures with a scFv:PEG linker molar ratio of 1.6:1 with acetic hydrazide. Lane 2 of the 6 hour, 20 hour, and 44 hour gels show reaction mixtures with a scFvdinker molar ratio of 2.0:1 with acetic hydrazide. Lane 3 of the 6 hour, 20 hour, and 44 hour gels show reaction mixtures with a scFvdinker molar ratio of 2.4:1 with acetic hydrazide. As a control, scFv 108 was incubated with 47 mM acetic hydrazide in the absence of the homobifunctional PEG linker for 44 hours. No dimer formation was observed. Lane 5 of the 6 hour, 20 hour, and 44 hours gels show reaction mixtures of scFv and accelerant acetic hydrazide without the PEG linker. This result indicates that the accelerant, acetic hydrazide, does not facilitate the formation of intermolecular disulfide bonds among scFv. In the presence of homobifunctional PEG linker, the dimer is produced through the oxime formation between the PEG linker and protein.

Example 5: Mono hydroxylamine 30 K PEG and scFv-pAcF conjugation

[00253] scFv 108 259-pAcF 0.5 mM stock solution in reaction buffer mentioned above (10 μΐ ) is mixed with various amounts of mono hydroxylamine 30 K PEG 2.5 mM stock solution, 2.2 μΐ of 200 mM acetic hydrazide stock solution and reaction buffer. The reaction mixtures with a final reaction volume of 22 μΐ have different molar ratios of scFv:PEG (1:3 or 1:5). The accelerating effect of acetic hydrazide on conjugation of mono hydroxylamine 30 K PEG and scFv was evaluated at protein and PEG molar ratio of 1:3 and 1:5 with and without accelerant. The reaction mixtures were incubated at 28 °C and were analyzed at different time points (20 hours, 44 hours) by SDSPAGE (FIG. 3). Lanes 1, 2, and 3 show 100%, 20%, and 10% of the starting scFv-pAcF, respectively. Lane 4 of the 20 hour and 44 hour gels show reaction mixtures with a scFv:PEG molar ratio of 1:3 with 20 mM acetic hydrazide. Lane 5 of file 20 hour and 44 hour gels show reaction mixtures with a scFv:PEG molar ratio of 1:3 without accelerant. Lane 6 of the 20 hour and 44 hour gels show reaction mixtures with a scFv:PEG molar ratio of 1:5 with 20 mM acetic hydrazide. Lane 7 of the 20 hour and 44 hour gels show reaction mixtures with a scFv:PEG molar ratio of 1:5 without accelerant. The conjugation results demonstrated that acetic hydrazide accelerates the

CA 02625540 2014-01-28 conjugation reaction. The reaction at a &amp; I'·³ molar ratio of protsinFEG with accelerant proceeded faster than the reaction at a 1:5 ratio ofprotein:PEG without accelerant

[00254] Similarly, the relative conjugation efficiency of different concentrations of the accelerant acetic hydrazide (5 mM, 20 mM, 80 mM) with a εοΡν.30 K PEG mono hydroxyiamine molar ratio of 1:2 were evaluated (FIG. 4). The reaction mixtures were incubated at 28°C. Lane 1 shows the reaction mixture with 5 mM acetic hydrazide (scFv:30 K PEG mono hydraxylamino molar ratio of 1:2). Lane 2 shows the reaction mixture with 20 mM acetic hydrazide (scFv;30 K PEG mono hydroxyiamine molar ratio of 1:2). Lane 3 shows the reaction mixture with 80 mM acetic hydrazide (scFv:30 K PEG mono hydroxyiamine molar ratio of 1:2). Lane 4 shows the reaction mixture with no acetic hydrazide (scFwJO KPEG mono hydroxyiamine molar ratio of 1:5). Lanes 5, 6, and 7 show 10%, 20%, and 100% of the starting scFv-pAcF, respectively. SDS-PÀGE analysis showed that the higher concentration of accelerant, the faster the conjugation. The conjugation at a scFv:PEG molar ratio of 1:2 h * presence of 80 mM accelerant is fisfer than the conjugation at a scFw.PBG molar ratio of 1:5 without the aceeleram.

Example 6: Small Molecule Studies of Accelerated Oxime formation

[00235) Acetophenone (0J mM) was reacted with ethylhydroxyhmine (1 mM) in aqueous solution buffered to a pH of about 4.0; a series of accelerants (20 mM) was added to thia reaction mixture to determine the effect of accelerant identity on the rate and yield of oxime formation (see FIG. 10(a)). The accelerants that were tested in this model reaction are presented in FIG. 10(b). Aliquots were taken fiom the reaction mixture and analyzed by highperformance liquid chromatography after 2 h, 5 h, 9 h, and 24 h. In addition, for each of these samples, the ketone peak absorbance was compared to the oxime peak absofoaace at 260 nm using TJV/Via spectoscopy. FIG. 11· presents the results after 2 h and 9 h of reaction. As can be seen, all accelerants increase the rate of oxime formation; however, accelerants 1 and 7 (shown in Figure 10(b)) provide the highest yield, with accelerant 1 providing the highest yield following longer reaction times. Without being bound to a particular theory, foe activity of the accelerant appears to depend on both the rate of reaction with foe ketone and on the stability of the hydrazone intermediate.

[00256] It is understood that the examples and embodiments described herein are for illustrative purposes only and that the scope of the claims is not to be limited by any example or embodiment but are to be given the broadest interpretation consistent with the description as a whole,