MX2008007646A - Compositions containing, methods involving, and uses of non-natural amino acids and polypeptides - Google Patents

Abstract

Disclosed herein are non-natural amino acids and polypeptides that include at least one non-natural amino acid, and methods for making such non-natural amino acids and polypeptides. The non-natural amino acids, by themselves or as a part of a polypeptide, can include a wide range of possible functionalities, but typical have at least one aromatic amine group. Also disclosed herein are non-natural amino acid polypeptides that are further modified post-translationally, methods for effecting such modifications, and methods for purifying such polypeptides. Typically, the modified non-natural amino acid polypeptides include at least one alkylated amine group. Further disclosed are methods for using such non-natural amino acid polypeptides and modified non-natural amino acid polypeptides, including therapeutic, diagnostic, and other biotechnology uses.

Description

CONTAINING COMPOSITIONS, METHODS INVOLVING AND USES OF NON-NATURAL AMINO ACIDS AND POLYPEPTIDES FIELD OF THE INVENTION Methods and compositions for making and using non-natural amino acid agents are described herein.

BACKGROUND OF THE INVENTION The ability to incorporate non-genetically encoded amino acids (ie, "non-natural amino acids") into proteins allows the introduction of chemical functional groups that could provide valuable alternatives to the functional groups that are stable in nature. , such as the epsilon -NH2 of lysine, the sulfhydryl -SH of cysteine, the imnino group of histidine, etc. It is known that certain chemical functional groups are inert to the functional groups found in the 20 common genetically encoded amino acids but react cleanly and efficiently to form stable bonds with functional groups that can be incorporated on non-natural amino acids. Methods are now available to selectively introduce chemical functional groups that are not found in proteins, which are chemically inert to all the functional groups found in the 20 common genetically encoded amino acids that can be used for react efficiently and selectively with reagents comprising certain functional groups to form stable covalent bonds.

BRIEF DESCRIPTION OF THE INVENTION Methods, compositions, techniques and strategies for manufacturing, purifying, characterizing and using non-natural amino acids, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides are described herein. In one aspect are methods, compositions, techniques and strategies for deriving a non-natural amino acid and / or a non-natural amino acid polypeptide. In one modality, such methods, compositions, techniques and strategies involve chemical derivation, in other modalities, biological derivation, in other modalities physical derivation, in other modalities a combination of derivations. In other modalities or additional modalities, such referrals are regioselective. In other modalities or additional modalities, such referrals are regiospecific. In other embodiments or additional modalities, such derivations are stoichiometric, almost stoichiometric or stoichiometric-like in both the unnatural amino acid-containing reagent and derivatization reagents. In other modalities or additional modalities, methods are provided that allow the incorporation stoichiometric, almost stoichiometric or similar to stoichiometric of a desired group on a desired non-natural polypeptide. In other embodiments or additional modalities, strategies, reaction mixtures, synthetic conditions are provided that allow the stoichiometric, quasi-stoichiometric or stoichiometric-like of a desired group on an unnatural amino acid polypeptide. In other modalities or additional modalities, such derivations are rapid at room temperature. In other modalities or additional modalities, such derivations occur in aqueous solutions. In other modalities or additional modalities, such derivations occur at a pH between approximately 4 and approximately 10. In other embodiments or additional modalities, such derivations occur at a pH between approximately 4 and approximately 7. In other modalities or additional modalities, such derivations occur at a pH between about 4 and about 5. In other embodiments or additional embodiments, such derivations occur at a pH of about 5. In other embodiments or additional embodiments, such derivations occur at a pH of about 4. In one aspect, amino acids are not found. natural for the chemical derivation of peptides and proteins based on the reactivity of an aromatic amine group. In other embodiments or additional embodiments, at least one of the non-natural amino acids mentioned above is incorporated into a polypeptide that is, such embodiments are polypeptides of non-natural amino acid. In other modalities or additional modalities, the non-natural amino acids are functionalized in their side chains, in such a way that their reaction with a derivatization molecule generates an amine bond. In other embodiments or additional embodiments, the non-natural amino acids are selected from amino acids having aromatic amine side chains. In other embodiments or additional embodiments, the non-natural amino acids comprise a masked side chain, in which a masked aromatic amine group is included. In other embodiments or additional embodiments, the non-natural amino acids comprise aromatic amine side chains, wherein the aromatic amine is selected from an aryl amine or a heteroaryl amine. In a further embodiment or additional embodiment, the non-natural amino acids resemble a natural amino acid in the structure but contain aromatic amine groups. In another embodiment or additional embodiment, the non-natural amino acids resemble phenylalanine or tyrosine (aromatic amino acids). In one embodiment, non-natural amino acids have properties that are different from those of natural amino acids. In one embodiment, such different properties are the chemical reactivity of the side chain; in a further embodiment this different chemical reactivity allows the side chain of the non-natural amino acid to undergo a reaction as long as it is a unit of a polypeptide, although the side chain of the amino acid units that is present in a stable manner in nature in the same polypeptide do not undergo the reaction mentioned above. In a further embodiment, the side chain of the non-natural amino acid has a chemistry orthogonal to that of amino acids that occur stably in nature. In a further embodiment, the side chain of the non-natural amino acid comprises a nucleophil-containing portion; in a further embodiment, the nucleophile containing portion in the side chain of the non-natural amino acid may undergo a reaction to generate a derived amine protein. In a further embodiment, the side chain of the non-natural amino acid comprises an electrophilic-containing portion; in a further embodiment, the electrophilic-containing portion in the side chain of the non-natural amino acid may undergo nucleophilic attack to derive a derived amine protein. In any of the embodiments mentioned above in this paragraph, the non-natural amino acid can exist as a separate molecule or can be incorporated into a polypeptide of any length; if it is the latter, then the polypeptide may naturally incorporate amino acids that occur stably in nature or non-natural amino acids. In another aspect, carbonyl-substituted molecules, such as, for example, aldehydes and ketones, are found for the production of non-natural amino acid polypeptides derived on the basis of an amine linkage. In an additional modality, aldehyde molecules are found substituted ones used to derive non-natural amino acid polypeptides containing aromatic amine via the formation of an amine between the derivatizing molecule and the non-natural amino acid polypeptide containing aromatic amine. Among more embodiments or additional embodiments, the aldehyde-substituted molecules comprise a related group of: a label; a dye; a polymer; a water soluble polymer; a polyethylene glycol derivative; a photo-reticulator; a cytotoxic compound; a drug; an affinity marker; a photoaffinity marker; a reactive compound; a resin; a second polypeptide or polypeptide or polypeptide analogue; an antibody or antibody fragment; a metal queiante; a co-factor; a fatty acid; a carbohydrate; a polynucleotide; a DNA; an RNA; an antisense polynucleotide; a saccharide; a dendrimer soluble in water; a cyclodextrin; a biomaterial; a nanoparticle; a spin marker; a fluorophore; a portion containing metal; a radioactive portion; a new functional group; a group that interacts covalently or non-covalently with other molecules; a photoenaged portion; an excitable portion by actinic radiation; a ligand; a photoisomerizable portion; biotin; a biotin analogue; a portion that incorporates a heavy atom; a chemically cleavable group; a photocleavable group; an elongated side chain; a carbon-bonded sugar; a redox-active agent; an amino thioacid; a toxic portion; an isotopically labeled portion; a biophysical probe; a phosphorescent group; a chemiluminescent group; a dense group of electrons; a magnetic group; an intercalary group; a chromophore; an energy transfer agent; a biologically active agent; a detectable marker; a small molecule; an inhibitory ribonucleic acid; a radionucleotide; a neutron capture agent; a biotin derivative; quantum dot (s); a nanotransmitter; a radio transmitter; an abzyme, an activated complex activator, a virus, an adjuvant, an aglycan, an allergan, an antigioastine; an antihormone, an antioxidant, an aptamer, a guide RNA, a saponin, a shuttle vector, a macromolecule ,. a mimetic, a receptor, a reverse micelle, and any combination thereof. In further embodiments or embodiments, the aldehyde-substituted molecules are aldehyde-substituted polyethylene glycol (PEG) molecules. In a further embodiment, the side chain of the non-natural amino acid has a chemistry orthogonal to that of the amino acids that occur stably in nature allow the non-natural amino acid to react selectively with the aldehyde-substituted molecules. In a further embodiment, the side chain of the non-natural amino acid comprises an aromatic amine-containing portion that selectively reacts with the aldehyde-containing molecule; in a further embodiment, the aromatic amine-containing portion in the side chain of the non-natural amino acid may undergo reaction to generate an alkylated amine-derived protein. In an additional aspect Related to the embodiments described in this paragraph are the modified non-natural amino acid polypeptides resulting from the reaction of the depotting molecule with the non-natural amino acid polypeptides. Additional embodiments include any modifications of the already modified non-natural amino acid polypeptides. In another aspect, substituted aromatic amine molecules, such as, for example, apl amine and heteroaryl amine, are found for the production of non-natural amino acid polypeptides derived on the basis of an amine linkage. In a further embodiment there are substituted aromatic anima molecules used to derive polypeptides from non-natural amino acids containing aldehyde and to the formation of an amine bond between the derivatizing molecule and the non-natural amino acid polypeptide containing aldehyde. In further embodiments or embodiments, substituted aromatic amine comprises a group selected from: a label; a dye; a polymer; a water soluble polymer; a polyethylene glycol derivative; a photo-reticulator; a cytotoxic compound; a drug; an affinity marker; a photo-affinity marker; a reactive compound; a resin; a second protein or polypeptide or polypeptide analogue; an antibody or antibody fragment; a metal chelator; a co-factor; a fatty acid; a carbohydrate; a polynucleotide; a DNA; an RNA; an antisense polynucleotide; a saccharide; a dendrimer soluble in water; a cyclodextrm; a biomaterial; a nanoparticle; a spin marker; a fluorophore; a portion containing metal; a radioactive portion; a new functional group; a group that interacts covalently or non-covalently with other molecules; a photoenaged portion; an excitable portion by actinic radiation; a ligand; a photoisomerizable portion; biotin; a biotin analogue; a portion that incorporates a heavy atom; a group chemically cleaved it; a photocleavable group; an elongated side chain; a carbon-bonded sugar; a redox-active agent; an amino thioacid; a toxic portion; an isotopically labeled portion; a biophysical probe; a phosphorescent group; a chemiluminescent group; a dense group of electrons; a magnetic group; an intercalary group; a chromophore; an energy transfer agent; a biologically active agent; a detectable marker; a small molecule; an inhibitory ribonucleic acid; a radionucleotide; a neutron capture agent; a biotin derivative; quantum dot (s); a nanotransmitter; a radio transmitter; an abzyme, an activated complex activator, a virus, an adjuvant, an aglycan, an allergan, an antigioastine; an antihormone, an antioxidant, an aptamer, a guide RNA, a saponin, a shuttle vector, a macromolecule, a mimotope, a receptor, a reverse micelle, and any combination thereof. In further embodiments or embodiments, the substituted aromatic amine molecules are substituted aromatic amine polyethylene glycol (PEG) molecules. In a In another embodiment, the side chain of the non-natural amino acid has a chemistry orthogonal to that of amino acids that occur stably in nature which allows the non-natural amino acid to react selectively with the substituted aromatic amine molecules. In a further embodiment, the side chain of the non-natural amino acid comprises an aldehyde-containing portion that selectively reacts with the aromatic amine-containing molecule; in a further embodiment, the aldehyde-containing portion of the side chain of the non-natural amino acid may undergo reaction to generate an alkylated amine-derived protein. In a further aspect related to the embodiments described in this paragraph are the modified non-natural amino acid polypeptides resulting from the reaction of the derivatizing molecule with the non-natural amino acid polypeptides. Additional embodiments include any modifications of the already modified non-natural amino acid polypeptides. In another aspect there are mono-, bi-, multi-functional linkers for the derivation of derived non-natural amino acid polypeptides based on an amine linkage. In one embodiment, molecular linkers (bi- and multi-functional) can be used which can be used non-natural amino acid polypeptides containing aromatic amine to other molecules. In another embodiment, the non-natural amino acid polypeptides containing aromatic amine comprise a side chain of apl amine or heteroaryl amine. In one embodiment a non-natural amino acid polypeptide containing aromatic amine is used, the molecular linker contains an aldehyde group in one of its terms. In further embodiments or embodiments, the aldehyde-substituted linker molecules are aldehyde-substituted linker or polyethylene glycol (PEG) molecules. In additional embodiments, the phrase "other molecules" includes, by way of example only, proteins, other polymers and small molecules. In further embodiments or further embodiments, the molecular linkers containing aldehyde comprise the same groups or groups equivalent in all terms, such that after action with an unnatural amino acid polypeptide containing aromatic amine, the resulting product is the homo-multimerization of the unnatural amino acid polypeptide containing aromatic amine. In further embodiments, homo-multimepzation is a homo-dimerization. In other embodiments or additional embodiments, the molecular linkers comprise at least one aldehyde group and a different group in all terms, such that after action with a non-natural amino acid polypeptide containing aromatic amine, the resulting product is hetero-multimerization of the non-natural amino acid polypeptide containing aromatic amine. In additional embodiments, hetero-multimerization is a hetero-dimerization. In a further embodiment, the unnatural amino acid side chain which has a chemistry orthogonal to that of the amino acids that occur stably in nature which allows the non-natural amino acid to react selectively with the substituted aldehyde linker molecules. In a further aspect related to the embodiments described in this paragraph are the unmodified modified or unmodified amino acid polypeptides linked result from the reaction of the linker molecule with the non-natural amino acid polypeptides. Additional embodiments include any additional modifications to the modified or unmodified unnatural amino acid polypeptides already linked. In one aspect there are methods for deriving proteins via the reaction of aromatic amines and aldehyde reagents to an alkylated amine-derived protein. Including such aspects are methods for the derivation of proteins based on the reductive alkylation of aromatic amine and aldehyde containing reagents to generate an amine-derived alkylated protein adduct. In other embodiments or additional embodiments, there are methods for deriving aromatic amine-containing proteins with polyethylene glycol (PEG) aldehyde-functionalized molecules. In still other aspects or additional aspects, the aldehyde-substituted molecule can include proteins, other polymers (unbranched and branched) and small molecules. In another aspect there are methods for synthesis chemistry of aldehyde-substituted molecules for derivation of substituted aromatic amine proteins. In one embodiment, the aldehyde-substituted molecule can comprise peptides, other polymers (unbranched and branched) and small molecules. In one embodiment, there are methods for the preparation of aldehyde-substituted molecules suitable for the derivation of non-natural amino acid-containing polypeptides. In a further embodiment or additional embodiment, non-natural amino acids include, but are not limited to, unnatural amino acids that contain aromatic amine, are specifically incorporated into the site during the in vivo translation of proteins. In other alternative embodiments or embodiments, the non-natural amino acids, which include but are not limited to, non-natural amino acids containing aromatic amine, are specifically incorporated into the site by ribosomal translation. In additional embodiments or alternative embodiments, the non-natural amino acids, which include but are not limited to unnatural amino acids containing aromatic amine, are specifically incorporated into the site during transliteration. In a further embodiment or additional embodiment, aldehyde-substituted molecules allow site-specific derivation of non-natural amine-aromatic-containing amino acids via reductive alkylation of the aromatic amine portion to form an amine-derived polypeptide rented in a specific way from the site. In a further embodiment or additional embodiment, the method for the preparation of aldehyde-substituted molecules provides access to a wide variety of polypeptides specifically derived at the site. In a further embodiment or additional embodiment there are methods for synthesizing polyethylene glycol (PEG) aldehyde-functionalized molecules. In another aspect are methods for the chemical derivation of polypeptides of non-natural amine aromatic substituted amino acids using a bifunctional linker containing aldehyde. In one embodiment methods are found for attaching an aldehyde-substituted linker to a substituted aromatic amine protein via a reductive alkylation reaction to generate an amine linkage. In further embodiments or further embodiments, the non-natural amino acid substituted aromatic amine is an unnatural amino acid aryl amine or substituted heteroaryl amine. In other embodiments or additional modalities, the non-natural amino acid polypeptides are specifically derived at the site and / or with precise control of the three-dimensional structure, using a bifunctional linker containing aldehyde. In one embodiment, such methods are used to attach molecular linkers (mono-, bi- and multi-functional) to unnatural amino acid polypeptides containing aromatic amine, wherein at least one of the linker terms contains a group aldehyde which can be linked to non-natural amino acid polypeptides containing aromatic amine via an amine linkage. In a further embodiment or additional embodiment, these linkers are used to connect the unnatural amino acid polypeptides containing aromatic amine to other molecules, which include, by way of example, proteins, other polymers (branched and unbranched) and molecules little. In some embodiments, the unnatural 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 amino acids linked to a water soluble polymer comprising polyethylene glycol moiety. In some embodiments, the non-natural amino acid polypeptide comprises a substitution, addition or deletion that increases the affinity of the non-natural amino acid polypeptide for a receptor. In some embodiments, the polypeptide of A non-natural amino acid comprises a substitution, addition or cancelation that increases the stability of the non-natural amino acid polypeptide. In some embodiments, the non-natural amino acid polypeptide comprises a substitution, addition or cancelation 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 cancelation 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 cancelation that modulates protease resistance, serum half-life, immunogenicity and / or expression in relation to the amino acid polypeptide are substitution, addition or cancellation. .

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 comprise an unnatural amino acid linked to a water soluble polymer. In some embodiments, the water soluble polymer comprises a polyethylene glycol moiety. In some embodiments, the polypeptide comprising an unnatural amino acid linked to a polymer Water-soluble, for example, can prevent dimerization of the corresponding receptor. In some embodiments, the polypeptide comprising an unnatural amino acid linked to a water soluble polymer modulates the binding of the polypeptide to a binding partner, ligand or receptor. In some embodiments, the polypeptide comprising an unnatural amino acid linked to a water soluble polymer modulates one or more properties or activities of the polypeptide. In some embodiments, the selector codon is selected from the group consisting of an amber codon, ocher codon, opal codon, a single codon, a rare codon, an unnatural codon, a five-base codon, and a four-base codon. In other embodiments or additional embodiments, the unnatural aromatic amine amino acid polypeptides and / or modified non-natural aromatic amine amino acid polypeptides described herein have at least one of the following characteristics: (1) the amine portion of the aromatic amine it is a primary amine; (2) the amine portion of the aromatic amine is a secondary amine; (3) the aromatic portion of the aromatic amine is a heteroaromatic portion; (4) the aromatic portion of the aromatic amine is an aryl portion; (5) the aromatic amine reacts with the functionalized aldehyde groups; (6) is coupled to a water soluble polymer; (7) is coated with PEG; (8) has increased therapeutic half-life relative to the corresponding polypeptide without the unnatural aromatic amino acid amine; (9) has increased serum half-life relative to the corresponding polypeptide without the unnatural aromatic amine amino acid; (10) has an increased circulation time in relation to the corresponding polypeptide without the unnatural aromatic amine amino acid; (11) has increased water solubility relative to the corresponding polypeptide without the non-natural aromatic amine amino acid; (12) has improved bioavailability relative to the corresponding polypeptide without the unnatural amino acid amomatica; (13) has modulated immunogenicity in relation to the corresponding polypeptide without the non-natural aromatic amine amino acid; (14) has a modulated biological affinity of the corresponding polypeptide without the unnatural aromatic amine amino acid; (15) is part of a pharmaceutical composition; (16) is obtained from a cell culture; (17) is chemically synthesized; (18) is used in library selection methods; (19) is used with fixes; (20) is used in protein arrays; (21) is used for gene expression analysis; (22) is coupled to at least one agent; (23) is coupled to a marker; (24) is coupled to a dye; (25) is coupled to a polymer; (26) is coupled to a cytotoxic compound; (27) is coupled to a drug; (28) is coupled to a second protein or polypeptide or polypeptide analogue; (29) is coupled to an antibody or antibody fragment; (30) is coupled to a carbohydrate; (31) is coupled to a polynucleotide; (32) is coupled to an antisense polynucleotide; (33) is coupled to a saccharide; (34) is coupled to a fluorophore; (35) is coupled to a chemically cleaved group; (36) is coupled to a photocleavable group; (37) is coupled to an energy transfer agent; (38) is coupled to a radionucleotide; (39) is modified post-translationally; (40) can be modified post-translationally by reductive alkylation; (41) can be modified post-translationally by reductive alkylation in a pH range between about 4 to about 10; (42) can be specifically derived from the site by post-translation reductive alkylation; (43) can be rapidly modified post-translationally by reductive alkylation at room temperature; (44) can be modified post-translationally by reductive alkylation in aqueous conditions; (45) can be modified post-translationally by reductive alkylation with stoichiometric reaction conditions; (46) can be modified post-translationally by reductive alkylation with almost stoichiometric reaction conditions; (47) can be modified post-translationally by reductive alkylation with stoichiometric-like reaction conditions; (48) is used to treat a mammal suffering from a disease, disorder or condition; (49) is used to treat a human suffering from a disease, disorder or condition; (50) is used to diagnose a mammal suffering from a disease, disorder or condition; (51) is used to diagnose a human suffering from a disease, disorder or condition; (52) is part of sustained release compositions; (53) the amine portion is formed by cross-translation reduction of a masked amine moiety; (54) the amine portion is formed by post-translational reduction of an imma portion; (55) the amine portion is formed by post-translational reduction of an azide moiety; (56) the amine portion by post-reduction reduction of a hydramous portion; (57) the amine portion is formed by post-translation reduction of a nitro portion; (58) is coupled to a prodrug; (59) is obtained from a cell lysate; (60) is translated ribosomally; (61) can be modified post-translationally by reductive alkylation in a pH range between about 4 to about 7; (62) can be modified post-translationally by reductive alkylation in a pH range of from about 4 to about 5; (63) can be modified post-translationally by reductive alkylation at a pH of about 5; (64) can be modified post-translationally by reductive alkylation at a pH of about 4; (65) reacts rapidly under reductive alkylation conditions; (66) reacts in less than about 10 hours under reducing alkylation conditions; (67) reacts in less than about 8 hours under reducing alkylation conditions; (68) reacts in less than about 6 hours under reducing alkylation conditions; (68) reacts in less than about 4 hours under reductive alkylation reaction conditions; (69) reacts in less than about 2 hours in reductive alkylation reactions; (70) reacts in less than about one hour in reductive alkylation reactions or (71) reacts in less than about 30 minutes in reductive alkylation reactions. In other embodiments or alternative embodiments, the amino acid polypeptides of non-natural aromatic amines and / or modified non-natural aromatic amine amino acid polypeptides described herein and having at least two of the above-mentioned characteristics. In other alternative embodiments or embodiments, the non-natural aromatic amine amino acid polypeptides and / or modified non-natural aromatic amine amino acid polypeptides described herein have at least three of the above-mentioned characteristics. In other alternative embodiments or embodiments, the unnatural aromatic amine amino acid polypeptides and / or modified non-natural aromatic amine amino acid polypeptides described herein have at least four of the characteristics mentioned above. In other alternative embodiments or embodiments, the non-natural aromatic amine amino acid polypeptides and / or modified non-natural aromatic amine amino acid polypeptides described herein have at least five of the characteristics mentioned above. In further embodiments or embodiments, the non-natural aldehyde-based amino acid polypeptides and / or modified non-natural aldehyde-based amino acid polypeptides described herein have at least one of the following characteristics: (1) they contain a portion of protected or masked aldehyde; (2) contain a portion of unprotected or unmasked aldehyde; (3) contain a portion of unprotected or unmasked aldehyde that can react with an aromatic amine; (4) contains a portion of unprotected or unmasked aldehyde that can react with a heteroaromatic amine; (5) they contain a portion of unprotected or unmasked aldehyde that can react with an aromatic amine or a heteroaromatic amine by reductive tuning; (6) is coupled to a water soluble polymer; (7) is coated with PEG; (8) have increased therapeutic half-life relative to the corresponding polypeptide without the non-natural aldehyde-based amino acid; (9) has increased serum half-life relative to the corresponding polypeptide without the amino acid a non-natural aldehyde base; (10) has an increased circulation time in relation to the corresponding polypeptide without the non-natural aldehyde-based amino acid; (11) has increased water solubility in relation to the corresponding polypeptide without the non-natural aldehyde-based amino acid; (12) has improved bioavailability relative to the corresponding polypeptide without the non-natural aldehyde-based amino acid; (13) has modulated immunogenicity in relation to the corresponding polypeptide without the amino acid based on the non-natural aldehyde; (14) has a modulated biological affinity of the corresponding polypeptide without the non-natural aldehyde-based amino acid; (15) is part of a pharmaceutical composition; (16) is obtained from cell culture; (17) is chemically synthesized; (18) is used in library selection methods; (19) is used with fixes; (20) is used with protein arrays; (21) is used for gene expression analysis; (22) can be coupled to an agent via reductive tuning; (23) is coupled to a marker; (24) is coupled to a dye; (25) is coupled to a polymer; (26) is coupled to a cytotoxic compound; (27) is coupled to a drug; (28) is coupled to a second protein or polypeptide or polypeptide analogue; (29) is coupled to an antibody or antibody fragment; (30) is coupled to a carbohydrate; (31) is coupled to a polynucleotide; (32) is coupled to a polynucleotide antisense; (33) is coupled to a saccharide; (34) is coupled to a fluorophore; (35) is coupled to a chemically cleavable group; (36) is coupled to a photocleavable group; (37) is coupled to an energy transfer agent; (38) is coupled to a radionucleotide; (39) can be modified post-translationally; (40) can be modified post-translationally by reductive amination; (41) can be modified post-translationally by reductive tuning in a pH range between about 4 to about 10; (42) can be specifically derived from the site by post-translational reductive amination; (43) can be rapidly modified post-translationally by reductive amination at room temperature; (44) can be modified post-translationally by reductive amination under aqueous conditions; (45) can be modified post-translationally by reductive amination with stoichiometric reaction conditions; (46) can be modified post-translationally by reductive amination with almost stoichiometric reaction conditions; (47) can be modified post-translationally by reductive amination with reaction conditions similar to stoichiometric; (48) is used to treat a mammal suffering from a disease, disorder or condition; (49) is used to treat a human suffering from a disease, disorder or condition; (50) is used to diagnose a mammal that suffers from a disease, alteration or condition; (51) is used to diagnose a human suffering from a disease, disorder or condition; (52) is part of sustained release compositions; (53) is coupled to a prodrug; (54) is obtained from cell lysate; (55) is translated ribosomally; (56) can be modified post-translationally by reductive amination in a pH range of from about 4 to about 7; (57) can be modified post-translationally by reductive amination in a pH range of between about 4 to about 5; (58) can be modified post-translationally by reductive amination at a pH of about 5; (59) can be modified post-translationally by reductive amination at a pH of about 4; (60) reacts rapidly under reductive amination conditions; (61) reacts in less than about 10 hours under reductive amination conditions; (62) reacts in less than about 8 hours under reductive amination conditions; (63) reacts in less than about 6 hours under reductive amination conditions; (64) reacts in less than about 2 hours under reductive amination reaction conditions; (65) reacts in less than about one hour in reductive amination reactions or (66) reacts in less than about 30 minutes in reductive amination reactions.

In other alternative embodiments or embodiments, the non-natural aldehyde-based amino acid polypeptides and / or modified non-natural aldehyde-based amino acid polypeptides described herein have at least two of the above-mentioned characteristics. In other embodiments or additional embodiments, the non-natural aldehyde-based amino acid polypeptides and modified non-natural aldehyde-based amino acid polypeptides described herein have at least three of the characteristics mentioned above. Among more alternative modalities or modalities, the amino acid polypeptides based on non-natural aldehydes and / or amino acid polypeptides based on modified non-natural aldehydes described herein have at least four of the characteristics mentioned above. In other alternative embodiments or embodiments, the amino acid polypeptides based on non-natural aldehydes and / or amino acid polypeptides based on modified non-natural aldehydes described herein have at least five of the characteristics mentioned above. Also described herein are methods of making an amino acid polypeptide linked to a water soluble polymer. In some embodiments, the method comprises contacting an isolated polypeptide comprising a non-natural amino acid with a soluble polymer water comprising a portion that reacts with the non-natural amino acid. In some embodiments, the non-natural amino acid incorporated therein is reactive toward a water soluble polymer that is otherwise unreactive toward any of the 20 common amino acids. In some embodiments, the water soluble polymer comprises a polyethylene glycol moiety. The molecular weight of the polymer can be of a wide range, in which is included but not limited to, between about 100 Da and about 100,000 Da or more. The molecular weight of the polymer can be between about 100 Da and about 100,000 Da, which includes but is 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 approximately 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 can be between about 1,000 Da and about 100,000 Da, which include but are 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 Give 4,000 Da, 3,000 Da, 2,000 Da, 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. Also described herein are compositions comprising a polypeptide comprising at least one of the non-natural amino acids described herein and a carrier pharmaceutically acceptable 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 with an unnatural amino acid. In some embodiments, the non-natural amino acid comprises a portion of saccharide. In some embodiments, the water soluble polymer is linked to the polypeptide via a saccharide moiety. Also described herein are cells comprising a polynucleotide encoding the polypeptide comprising a selector codon. In some embodiments, the cells comprise an orthogonal RNA synthetase and / or a tRNA to replace the unnatural amino acid with the polypeptide. In some embodiments, the cells are in a cell culture, while in other embodiments, the cells are part of a multicellular organism, in which amphibians, reptiles, birds and mammals are included. In any of the embodiments of the present cell, additional embodiments include expression of the polynucleotide to produce the non-natural amino acid polypeptide. In some embodiments, the non-natural amino acid polypeptide is produced m Vi tro. In some embodiments, the non-natural amino acid polypeptide is produced in cell lysate. In some embodiments, the amino acid polypeptide Non-natural is produced by ribosomal translation. Methods for the manufacture of a polypeptide comprising an unnatural amino acid are also described herein. In some embodiments the methods comprise culturing cells comprising a polynucleotide or polynucleotides that encode a polypeptide, an orthogonal RNA synthetase and / or an orthogonal tRNA under conditions to allow expression of the polypeptide and purify the polypeptide from the cells and / or culture medium. . Also described herein are libraries of the non-natural amino acids described herein or libraries of the non-natural amino acid polypeptides described herein or libraries of the modified non-natural amino acid polypeptides described herein or combined libraries thereof. Also described herein are those containing at least one non-natural amino acid, at least one non-natural amino acid polypeptide and / or at least one modified non-natural amino acid. Also described herein are those containing at least one polynucleotide encoding a polypeptide comprising a selector codon. The arrangements described herein can be used to select the production of non-natural amino acid polypeptides in an organism (either by detecting the transition of the polynucleotide encoding the polypeptide or by detecting the translation of the polypeptide. polypeptide). Also described herein are methods for selecting libraries described herein for a desired activity or for using the arrangements described herein to select the libraries described herein or for other libraries of compounds and / or polypeptides and / or polmucleotides with a desired activity. Also described herein is the use of such library selection activity data to develop and discover new therapeutic targets, as well as the therapeutic agents themselves. Methods for increasing the therapeutic half-life, average life in the soil or circulation time of a polypeptide are also described herein. In some embodiments, the methods comprise replacing at least one non-natural amino acid with any one or more amino acids in a polypeptide that occurs stably in nature and / or binding the polypeptide to a water soluble polymer. Also described herein are methods for the treatment of a patient in need of such treatment with an effective amount of a pharmaceutical composition comprising a polypeptide comprising an unnatural amino acid and a pharmaceutically acceptable carrier. In some embodiments, the non-natural amino acid is bound to a polymer soluble in water. It will be understood that the methods and compositions described herein are not limited to the methodology, protocol, cell lines, constructs and particular reagents described herein and as such may vary. It will also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the methods and compositions described herein, which will be limited only by the appended claims. As used herein and in the appended claims, the singular forms "a", "" one, and "the" include plural references, unless the context clearly dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood for one of ordinary skill in the art with which the invention described herein is concerned. Although any methods, devices and materials similar or equivalent to those described herein may be used in the practice or test of the invention described herein, the methods, devices and materials referred to are now described. All the publications and patents mentioned in the present are incorporated herein by reference in their entirety for the purpose of describing and disclosing, for example, the constructs and methodologies that are described in the publications, which are used in connection with the presently described invention. The publications discussed herein are provided only as to their disclosure prior to the filing date of the present application. Nothing herein shall be construed as an admission that the inventors described herein have no right to anticipate such disclosure by virtue of prior invention or for any other reason. The term "affinity tag" as used herein, refers to a tag that is reversibly or irreversibly linked to another molecule, either to modify, destroy or form a compound therewith. By way of example, affinity labels include enzymes and their substrates or antibodies and their antigens. The terms "alkoxy", "alkylamino" and "alkylthio" are used in their conventional sense and refer to alkyl groups linked to molecules via an oxygen atom, an amino group, a sulfur atom, respectively. The term "alkyl", by itself or as part of another molecule, means, unless stated otherwise, a straight or branched chain or cyclic hydrocarbon radical or combination thereof, which may be fully saturated, mono- or poly-unsaturated and can include di- and multivalent radicals, having the designated number of atoms (that is, C1-C10 means one to ten carbon atoms). 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 for example, n-pentyl, n-hexyl, n-heptyl, n-octyl and the like. An unsaturated alkyl group is one having one or double bonds or triple bonds. Examples of unsaturated alkyl groups include but are not limited to vinyl, 2-propynyl, crotyl, 2-isopentenyl, 2- (butadienyl), 2,4-pentadienyl, 3- (1,4-pentadienyl), ethynyl 1- and 3 -propynyl, 3-butynyl and homologues and higher isomers. The term "alkyl", unless otherwise indicated, is also intended to include those alkyl derivatives defined in detail hereinbelow, such as "heteroalkyl", "haloalkyl" and "homoalkyl". The term "alkylene" by itself or as part of another molecule means a divalent radical derived from an alkane, as exemplified by (-CH2-) n, wherein n may be from one to about 24. By way of example only, such groups include but are not limited to groups having 10 or fewer carbon atoms such as the structures -CH2-CH2- and -CH2CH2CH2CH2-. A "lower alkyl" or "alkylene" "lower" is a shorter chain alkyl or alkylene group, having generally 8 or less carbon atoms The term "alkylene" unless otherwise indicated, it is also proposed to include those groups described later herein as "heteroalkylene." The term "amino acid" refers to amino acids that occur stably in nature and unnatural amino acids, as well as amino acid analogs and amino acid mimetics that function similarly to amino acids that are presented in a manner stable in nature 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 pyrrolysin and selenocysteine.Amino acid analogs refer to compounds that have the same basic chemical structure as an amino acid that occurs stably in nature, by way of example, an alpha carbon that is linked to a hydrogen, a carboxyl group, an amino group and a R group. Such analogs may have modified R groups (by way of example, norleucine) or they may have modified basic peptide chains, while still retaining the same basic chemical structure as an amino acid that occurs stably in nature. Examples not Limitations of amino acid analogs include homoserin, norleucine, methionine sulfoxide, methionine methylsulfonium. The amino acids can be named herein either by their name, their three-letter symbol commonly known or by the letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Additionally, nucleotides can be named by their commonly accepted single-letter codes. An "amino terminus modification group" refers to any molecule that can be "attached to a terminal amine group." By way of example, such terminal amine groups can be at the end of the polymer molecules, wherein such polymer molecules include but they are not limited to polypeptides, polynucleotides and polysaccharides Term modification groups include but are not limited to various water soluble polymers, peptides or proteins For example only, the term modification groups include polyethylene glycol or serum albumin The term modification groups can be used to modify the therapeutic characteristics of the polymeric molecule, which include but are not limited to increasing the serum half-life of the peptides An "antibody fragment" means any form of an antibody different from the full-length form. Antibody fragments in the presen they include antibodies which are smaller components that exist within the antibodies of full length and antibodies that have been designed. Antibody fragments include but are not limited to Fv, Fe, Fab and (Fab ') 2, single chain Fv (scFv), diabodies, triabodies, tetrabodies, bifunctional hybrid antibodies, CDR1, CDR2, CDR3, CDR combinations, variable regions, regions of structure, constant regions, heavy chains, light chains and variable regions and molecules without alternative scaffold antibodies, bispecific antibodies and the like (Maynard and Georgiou, 2000, Annu Rev. Biomed. Eng. 2: 339- 76; Hudson, 1998, Curr Opin, Biotechnoi, 9: 395-402). Another functional substructure is a single chain Fv (scFv), which consists of the variable regions of the heavy chain and immunoglobulin light chain, covalently linked by a peptide linker (Sz Hu et al., 1996, Cancer Research, 56 , 3055-3061). These small proteins (Mr 25,000) 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 indicated otherwise, the claims and claims of the use of "antibody" or "antibodies" specifically include "antibody fragments" and "antibody fragment". The term "aromatic amine" as used in the present, refers to an aryl moiety that contains an amino moiety. Such amino moieties may include but are not limited to primary amines, secondary amines, tertiary amines, masked amines or protected amines. Such tertiary amines, masked amines or protected amines can be converted to primary amine or secondary amine portions. Additionally, the amine portion may include an amine-like portion that has similar chemical characteristics as the amine moieties, in which but not limited to chemical reactivity is included. The term "aromatic" or "aryl", as used herein, refers to a closed ring structure having at least one ring that has a pi electron system and includes both arylcarbocyclic and arylheterocyclic (or "heteroaryl") groups "or" heteroaromatic "). The carbocyclic or heterocyclic aromatic group may contain from 5 to 20 ring atoms. The term includes covalently linked monocyclic rings or fused ring polycyclic groups (that is, rings that share pairs of adjacent carbon atoms). An aromatic group can be unsubstituted or substituted. Non-limiting examples of "aromatic" or "aryl" groups include phenyl, 1-naphthyl, 2-naphthyl, 4-biphenyl, anthracenyl and phenanthracenyl. The substituents for each of the above indicated aryl and heteroaryl ring systems are selected from the group of acceptable substituents described hereinafter. For brevity, the term "aromatic" or "aryl" when used in combination with other terms (which include but are not limited to aryloxy, arylthioxy, aralkyl) include both aryl and heteroaryl rings as described above. Thus, the term "aralkyl" or "alkaryl" is intended to include those radicals in which an aryl group is attached to an alkyl group (in which but not limited to benzyl, phenethyl, pyridyl ethyl and the like) are included in those which include those groups in which a carbon atom (in which but not limited to a methylene group) has been replaced by a hetero atom, by way of example only, by means of an oxygen atom. Examples of such aryl groups include but are not limited to phenoxymethyl, 2-pyridinoxymethyl, 3- (1-naphthyloxy) propyl and the like. A "bifunctional polymer", also referred to as a "bifunctional linker" which defines a polymer comprising two functional groups that are capable of reacting specifically with other positions to form a covalent or non-covalent bond. Such portions may include but are not limited to the side groups on natural amino acids or non-natural amino acids or peptides containing such natural amino acids or non-natural amino acids. TO As an example only, a bifunctional linker can have a functional group reactive with a group in a first peptide and another functional group that is reactive with a group in a second peptide, thereby forming a conjugate that includes the first peptide, the bifunctional linker and the second peptide. Many methods and linker molecules for the attachment of various compounds to peptides are known. See, for example, European Patent Application No. 188,256; U.S. Patent Nos. 4,671,958, 4,659,839, 4,414,148, 4,699,784: 4,680,338 and 4,569, .789 are incorporated by reference herein in their entirety. A "multifunctional polymer" also referred to as a "multifunctional linker" refers to a polymer comprising two or more functional groups that are capable of reacting with other portions. Such portions may include but are not limited to the side groups of natural or unnatural amino acids or peptides containing such natural or unnatural amino acids (in which they include but are not limited to natural groups of amino acids) to form covalent or non-covalent bonds . A bifunctional polymer or multifunctional polymer may be of any desired length or molecular weight and may be selected to provide a particular desired spacing or conformation between one or more molecules bound to a compound and molecules linked to or the compound.

The term "bioavailability" as used herein refers to the speed and extent to which a substrate or its active portion is provided in a pharmaceutical form and which is available at the site of action or in the general circulation. The term "biologically active molecule", "biologically active portion" or "biologically active agent" when used herein means any substance that can affect any physical or biochemical properties of a biological system, route, molecule or interaction concerned with an organism. , which include but are not limited to viruses, bacteria, bacteriophages, transposon, prion, insects, fungi, plants, animals and humans. In particular, as used herein, biologically active molecules include but are not limited to any substance designed for diagnosis, cure, mitigation, treatment or prevention of disease in humans or other animals or to otherwise improve physical or mental well-being of humans or animals. Examples of biologically active molecules include but are not limited to peptides, proteins, enzymes, small molecule drugs, hard drugs, white drugs, carbohydrates, inorganic atoms or molecules, dyes, lipids, nucleosides, radionuclides, oligonucleotides, toxins, cells, viruses , liposomes, microparticles and miscelas. Classes of biologically active agents that are appropriate for use with methods and compositions described herein include but are not limited to drugs, prodrugs, radionuclides, imaging agents, polymers, antibiotics, fungicides, antiviral agents, anti-inflammatory agents, antitumor agents, cardiovascular agents, anti-anxiety agents, hormones, growth, steroidal agents, microbially derived toxins and the like. The term "biomaterial", as used herein, refers to a biologically derived material, which includes but is not limited to a material obtained from bioreactors and / or from recombinant methods and techniques. The term "biophysical probe" as used herein, refers to probes that can detect or monitor structural changes in molecules. Such molecules include but are not limited to proteins and the "biophysical probe" to detect or monitor the interaction of proteins with other macromolecules. Examples of biophysical probes include but are not limited to spin markers, fluorophores and photoactivatable groups. The term "biotin analog" or also referred to as "biotin mimetic" as used herein, is any molecule, other than biotin, that can be linked with high affinity to aniline and / or streptavidin. The term "term modification group" "carboxy" refers to any molecule that can be analyzed to a terminal carboxy group, By way of example, such carboxy terminal groups can be at the end of polymeric molecules, wherein such polymeric molecules include but are not limited to polypeptides, polynucleotides and polysaccharides The term modification groups include but are not limited to several water-soluble polymers, peptides or proteins.As an example only, the term modification groups include polyethylene glycol or whey albumin.The term modification groups may be used for modifying therapeutic characteristics of the polymeric molecule, which include but are not limited to increasing the serum half-life of the peptides The term "chemically cleavable group", also referred to as "chemically labile" as used herein, is refers to a group that breaks or splits after exposure to acid, base, oxidizing agents, reducing agents, chemical initiators or radical initiators. The term "chemiluminescent group," as used herein, refers to a group that emits light as its state of a chemical reaction and the addition of heat. By way of example only, luminol (5-amino-2,3-dihydro-1,4-phthalazinedione) reacts with oxidants such as hydrogen peroxide (H202) in the presence of a base and a catalyst. metal to produce a product of excited state (3-aminophthalate, 3-APA). The term "chromophore" as used herein, refers to a molecule that absorbs light of visible wavelengths, UV wavelengths or IR wavelengths. The term "cofactor", as used herein, refers to an atom or molecule essential for the action of a large molecule. The cofactors include but are not limited to inorganic ions, coenzymes, proteins or some other necessary factor for the activity of enzymes. Examples include heme in hemoglobin, magnesium in chlorophyll and metal ions for proteins. "Co-folding", as used herein, refers to processes, reactions or re-folding methods that employ at least two molecules that interact with each other and result in the transformation of unfolded or inappropriately folded molecules to properly folded molecules. By way of example only, "co-folding" employs at least two polypeptides that interact with each other and result in the transformation of unfolded polypeptides or inappropriately folded polypeptides to natural, properly folded polypeptides. Such polypeptides may contain natural amino acids and / or at least one non-natural amino acid. A "comparison window", as used herein, refers to a segment of any of the contiguous positions used to compare a sequence with a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned. Such contiguous positions include but are not limited to a group consisting of about 20 to about 600 sequential units, which includes about 50 to about 200 sequential units and about 100 to about 150 sequential units. By way of example only, such sequences include polypeptides and polypeptides that contain non-natural amino acids, the sequential units include but are not limited to natural and non-natural amino acids. In addition, by way of example only, such sequences include polynucleotides with the nucleotides which are the corresponding sequential units. Methods of sequence alignment for comparison are well known in the art. Optimal alignment of sequences by comparison can be carried out, in which they are included but not limited to, by the local homology algorithm of Smith and aterman (1970) Adv. Appl. Ma th. 2: 482c, by the homology alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48: 443, using the similar search method of Pearson and Lipman (1988) Proc. Na t 'l. Acad. Sci, USA 85: 2444, through computerized implementations of these algorithms (GAP, BESTFIT, FASTA and TFASTA in the Element Package of Wisconsin Genetics Programming, Genetics Computer Group, 575 Science Dr. , Madison Wl) or by manual alignment and visual inspection (see, for example, Ausubel et al., Curren t Protocols in Molecular Biology (1995 supplement)). By way of example, an algorithm that can be used to determine percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al (1997) Nuc. Acids Res 25-3389-3402 and Altschul et al (1990) J Mol. Biol. 215: 403-410, respectively. Programming elements to perform BLAST analyzes are publicly available through the National Center for Biotechnology Information. The parameters of the BLAST algorithm W, T and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses a word length (W) of 11 as a default, a hope (E) of 10, M = 5, N = -4 and a comparison of both strands. For amino acid sequences, the BLASTP program uses as a predetermined a word length of 3 and hope (E) of 10 and the scoring matrix BLOSUM62 (see Henikoff and Henikoff (1992) Proc.

Nati Acad. Sci. USA 89: 10915) alignments (B) of 50, hope (E) of 10, M = 5, N = -4 and a comparison of both strands. The BLAST algorithm is commonly performed with the "low complexity" filter turned off. The BLAST algorithm also performs an analysis statistical of the similarity between two sequences (see, for example, Karlin and Altschul (1993) Proc. Nati, 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 or match between two nucleotide or amino acid sequences would be presented by probability. 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 with the reference nucleic acid is less than about 0.2, plus or minus about 0.01 or less. approximately 0.001. The term "conservatively modified variants" applies to both natural and non-natural nucleic acid sequences and natural and non-natural amino acids and combinations thereof. With respect to particular nucleic acid sequences, "conservatively modified variants" refer to those natural and non-natural nucleic acids that encode identical or essentially identical natural or non-natural amino acid sequences or where the natural and unnatural nucleic acid does not encode a sequence of natural and unnatural amino acids, to essentially identical sequences. As an example, due to the degeneracy of the genetic code, a large number of Functionally identical nucleic acids encode any given protein. For example, the GCA, GCC, GCG and GCU codons all encode the amino acid alanine. Thus, in each 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 variations of nucleic acid are "silent variations", which are a kind of conservatively modified variants. Thus, by way of example, each naturally occurring or unnatural nucleic acid sequence herein that encodes a natural or unnatural polypeptide also describes every possible silent variation of the natural or unnatural nucleic acid. Those of ordinary skill in the art will recognize that each codon in a natural or unnatural 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 produce a molecule functionally identical. A) Yes, each silent variation of a natural and unnatural nucleic acid encoding a natural or unnatural polypeptide is implicit in each described sequence. With respect to amino acid sequences, substitutions, deletions or individual additions to a nucleic acid, peptide, polypeptide or protein sequence that alters, adds or cancels a single natural or non-natural amino acid or a small percentage of natural amino acids and unnatural in the encoded sequence is a "conservatively modified variant", wherein the alteration results in the cancellation of an amino acid, addition of an amino acid or substitution of a natural and non-natural amino acid with a chemically similar amino acid. Conservative substitution tables that provide functionally similar natural 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. Conservative substitution tables that provide functionally similar amino acids are known to those of ordinary skill in the art. The following eight groups each contain amino acids that are conservative substitutions with each other: 1) Alanma (A), Glycine (G); 2) Aspartic acid (D), glutamic acid (E); 3) Asparagma (N), Glutamine (Q); 4) Argmma (R), Lisma (K); 5) Isoleucm (I), Leucma (L), Metionma (M), Valma (V); 6) Phenylalanine (F), Tyrosma (Y), Tpptophan (W); 7) Sepna (S), Threonine (T); and 8) Cistern (C), Metionma (M) (see, for example, Creighton, Proteins: Structures and Molecular Properties (WH Freeman &Co., 2nd edition (December 1993) The terms "cycloalkyl" and "heterocycloalkyl", by themselves or in combination with other terms, represent, unless stated otherwise, "alkyl" and "heteroalkyl" cyclics, respectively, Thus, a cycloalkyl or heterocycloalkyl include saturated, partially saturated and fully unsaturated ring bonds Additionally, for heterocycloalkyl, a heteroatom may occupy the position in which the heterocycle is attached to the remainder The neteioatome may include, but is not limited to, oxygen, nitrogen or sulfur Examples of cycloalkyl include, but are not limited to, cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl and the like. of heterocycloalkyl include, but are not limited to, 1- (1, 2, 5, 6-tetrahydropyridyl), 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 multi-cyclic structures, which include, but are not limited to, bicyclic and tricyclic ring structures. Similarly, the term "heterocycloalkylene" by itself or as part of another molecule means a divalent radical derived from heterocycloalkyl and the term "cycloalkylene" by itself or as part of another molecule means a divalent radical derived from cycloalkyl. The term "cyclodextrin", as used herein, refers to cyclic carbohydrates consisting of at least six to eight molecules of glucose in a ring formation. The outer part of the ring contains groups soluble in water; In the center of the ring is a relatively non-polar cavity capable of accommodating small molecules. The term "cytotoxic", as used herein, refers to a compound that damages cells. "Denaturing agent" or "denaturing agent", as used herein, refers to any compound or material that will cause a reversible de-folding of a polymer. By way of example only, "denaturing agent" or "denaturing agents", can cause a reversible unfolding of a protein. The strength of a denaturing or denaturing agent will be determined both by the properties and concentration of the particular denaturing or denaturing agent. By way of example, denaturing or denaturing agents include, but are not limited to, chaotropes, detergents, organic, water miscible solvents, phospholipids or a combination thereof. Non-limiting examples of chaotropes include, but are not limited to, urea, guanidine and sodium thiocyanate. Non-limiting examples of detergents may include but are not limited to, strong detergents such as sodium dodecyl sulfate or polyoxyethylene ethers (e.g., Tween or Triton detergents), Sarkosyl, mild nonionic detergents (e.g., digitonma), mild cationic detergents such as N- > 2, 3- (Dioleoxy) -propyl-N, N, N-t-methylammonium, mild ionic detergents (e.g., sodium cholate or sodium deoxycholate) or zwitterionic detergents including, but not limited to, sulfobetaines (Zwitergent) , 3- (3-Clolam? Dopropyl sulfate) d? Met? Lamon? O-1-propane (CHAPS) and 3- (3-chloro? Doprop? L) d? Methalammon? 2-h? Drox? -l-propane (CHAPS?). Non-limiting examples of organic water-miscible solvents include, but are not limited to, acetonitrile, lower alkanols (especially C2-C4 alkanols such as ethanol or isopropanol) or lower alkanols (C2-C4 alcandiols such as ethylene glycol) can be used as denaturing agents . Non-limiting examples of phospholipids include, but are not limited to, phospholipids that occur stably in nature such as phosphatidylethanolamm, phosphatidylchol, phosphatidylserine and phosphatidylsitol or synthetic phospholipid vatives or variants such as dihexanoylphosphatidylcholine or diheptanoylphosphatidylcholine. The term "detectable label", as used herein, refers to a label that may be observable using analytical techniques in which it is included, but not limited to, fluorescence, chemiluminescence, electron spin resonance, ultraviolet / visible absorbance spectroscopy, mass spectrometry, nuclear magnetic resonance, magnetic resonance and electrochemical methods. The term "dicarbonyl", as used herein refers to a group containing at least two portions selected from the group consisting of -C (0) -, -S (0) -, -S (0) 2 -and -C (S) -, which include, but are not limited to, groups 1, 2-d? carbon? lo, groups 1, 3-d? carbon? lo and 1,4-dicarbonyl groups and groups which they contain at least one ketone group and / or at least one aldehyde group and / or at least one ester group, and / or at least one carboxylic acid group, and / or at least one thioester group. Such dicarbonyl groups include diketones, ketoaldehydes, keto acids, ketoesters and ketothioesters. In addition, such groups can be part of linear, branched or cyclic molecules. The term "drug", as used herein, refers to any substance used in the prevention, diagnosis, relief, treatment or cure of a disease or condition. The term "dye", as used herein, refers to a substance of soluble coloration, which contains a chromophore. The term "effective amount", as used in the present, refers to a sufficient amount of an agent or a compound that is administered that will alleviate to some extent one or more of the symptoms of the disease or condition being treated. The result can be reduction and / or relief of the signs, symptoms or causes of a disease or any other desired alteration of a biological system. By way of example, an agent or a compound that is administered includes, but is not limited to, a natural amino acid polypeptide, unnatural amino acid polypeptide, modified natural amino acid polypeptide or modified non-natural amino acid polypeptide. Compositions containing such natural amino acid polypeptides, non-natural amino acid polypeptides, modified natural amino acid polypeptides or modified non-natural amino acid polypeptides can be administered for prophylactic, enhancement and / or therapeutic treatments. An appropriate "effective" amount in any individual case can be determined using techniques, such as a dose escalation study. The term "dense group of electrons," as used herein, refers to a group that disperses electrons when irradiated with an electron beam. Such groups include, but are not limited to, ammonium molybdate, bismuth subnitrate cadmium iodide, 99%, carbohydrazide, ferric chloride hexahydrate, hexamethylene tetramine, 98.5%, anhydrous indium trichloride, iantane nitrate, lead acetate trihydrate, lead citrate trihydrate, lead nitrate, periodic acid, phosphomolybdic acid, phosphotungstic acid, potassium ferrocyanide, potassium ferrocyanide, ruthenium red, silver nitrate, proteinate silver (Ag Analysis: 8.0-8.5%) "Strong", silver tetraphenylporfin (S-TPPS), sodium chloroaurate, sodium tungstate, thallium nitrate, thiosemicarbazide (TSC), uranyl acetate, uranyl nitrate and vanadyl sulphate. The term "energy transfer agent," as used herein, refers to a molecule that can either donate or accept energy from another molecule. By way of example only, the fluorescence resonance energy transfer (FRET) is a dipole-dipole coupling process mdn in which the excited state energy of a fluorescence donor molecule is transferred without irradiation to an acceptor molecule without exciting which then fluorescently emits the donated energy at a longer wavelength. The terms "improve" or "improve" mean increase or prolong either a desired effect or duration. By way of example, "improving" the effect of therapeutic agents refers to the ability to increase or prolong, either in potency or duration, the effect of therapeutic agents in or during the treatment of a disease, alteration or condition. An "effective amount of improvement", as used herein, refers to an amount suitable for improving the effect of a therapeutic agent in the treatment of a disease, disorder or condition. When used in a patient, effective amounts for this use will depend on the severity and course of the disease, alteration or condition, prior therapy, patient's health status and response to the drugs and the judgment of the treating physician. As used herein, the term "eukaryote" refers to organisms belonging to the phylogenetic domain of Eucarya, which include but are not limited to animals (including, but not limited to, mammals, insects, reptiles, birds, etc.), ciliates, plants (including, but not limited to, monocotyledons, dicots and algae), fungi, yeasts, flagellates, microspopies and protists. The term "fatty acid", as used herein, refers to carboxylic acids with about C6 or a longer hydrocarbon side chain. The term "fluorophore", as used herein, refers to a molecule that emits photons in the excitation thereby being fluorescent. The terms "functional group", "active portion", "activating group", "leaving group", "reactive site", "chemically reactive group" and "chemically reactive portion", such as they are used in the present, refer to portions or units of a molecule in which chemical reactions occur. The terms are somewhat synonymous in the chemical arts and are used in the present to indicate the portions of molecules that perform some function or activity and are reactive with other molecules. The term "halogen" includes fluorine, chlorine, iodine and bromine. The term "haloacyl", as used herein, refers to acyl groups containing portions of halogen, wherein, but not limited to, -C (0) CH 3, -C (0) CF, - C (0) CH2OCH3 and the like. The term "haloalkyl", as used herein, refers to alkyl groups containing portions of halogen, wherein, but not limited to, -CF3 and -CH2CF3 and the like. The term "heteroalkyl", as used herein, refers to straight chain or branched or cyclic hydrocarbon radicals or combinations thereof, which consist of an alkyl group and at least one heteroatom selected from the group consisting of O, N, Si and S, wherein the nitrogen and sulfur atoms may optionally be oxidized and the nitrogen heteroatom may optionally be quaternary. The heteroatom (s) 0, N and S and Si can be placed in any position inside the group heteroalkyl or at the position in which the alkyl group is attached to the rest of the molecule. Examples include, but are not limited to, -CH2-CH2-0-CH3, -CH2-CH2-NH-CH3, -CH2-CH2-N (CH3) -CH3, -CH2-S-CH2-CH3, -CH2 -CH2, -S (0) -CH3, -CH2-CH2-S (0) 2-CH3, -CH = CH-0-CH3, -S? (CH3) 3, -CH2-CH = N-OCH3 and -CH = CH-N (CH3) -CH3. In addition, up to two heteroatoms can be consecutive, such as, by way of example, -CH2-NH-OCH3 and -CH2-0-S? (CH3) 3. The term "heteroalkylene", as used herein, refers to a divalent radical derived from heteroalkyl, as exemplified, but not limited to, - H2-CH2-S-CH2-CH2- and -LH2-S-CH2-CH2-NH-CH2-. For heteroalkylene groups, the same or different heteroatoms may also occupy either or both of the chain terms (wherein, but not limited to, alkyleneoxy, alkylenedioxy, alkyleneammo, alkylenediamino, ammooxyalkylene, and the like). Still further, for alkylene and heteroalkylene linker groups, no orientation of the linking group is implied by the direction in which the formula of the linking group is written. By way of example, the formula -C (0) 2R'_ represents both -C (0) 2R'- and -R'C (0) 2-. The term "heteroaplo" or "heteroaromatic", as used herein, refers to aplo groups containing at least one heteroatom selected from N, O and S; wherein the nitrogen and sulfur atoms may optionally be oxidized and the nitrogen atom (s) can optionally be quaternary. Heteroaryl groups may be substituted or unsubstituted. A heteroaryl group can be attached to the rest of the molecule by means of a heteroatom. Non-limiting examples of heteroaryl groups include 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolium, 3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, -oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl , 4-pyridium, 2-pyrimidyl, 4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl, 1-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl and 6-quinolyl . The term "homoalkyl" as used herein refers to alkyl groups that are hydrocarbon groups The term "identical," as used herein, refers to two or more sequences or subsequences that are the same. , the term "substantially identical", as used herein, refers to two or more sequences that have a percentage of sequential units that are the same when compared and are aligned for maximum correspondence in a comparison window or designated region as measured using a comparison algorithm or by manual alignment and visual inspection. example only, two or more sequences may be "substantially identical" if the sequential signals are about 60% identical, about 65% identical, about 70% identical, about 75% identical, about 80% identical, about 85% identical, about 90 % identical or approximately 95% identical in a specified region. Such percentages to describe the "percent identity" of two or more sequences. The identity of a sequence may exist in a region that is at least about 75-100 sequential units in length, in a region that is approximately 50 sec. Units in length or where it is not specified, throughout sequence. This definition also refers to the complement of a test sequence. By way of example only, two or more polypeptide sequences are identical when the amino acid residues are the same, while two or more polypeptide sequences are "substantially identical" if the amino acid residues are approximately 60% identical, approximately 65%. % identical, approximately 70% identical, approximately 75% identical, approximately 80% identical, approximately 85% identical, approximately 90% identical or approximately 95% identical over a specified region. The identity may exist over a region that is at least about 75-100 amino acids in length, in a region that is about 50 amino acids of length or where not specified, through the entire sequence of a polypeptide sequence. In addition, by way of example only, two or more polynucleotide sequences are identical when the nucleic acid residues are the same, while two or more polynucleotide sequences are "substantially identical" if the nucleic acid residues are approximately 60% identical, approximately 65% identical, approximately 70% identical, approximately 75% identical, approximately 80% identical, approximately 85% identical, approximately 90% identical or approximately 95% identical over a specified region. The identity may exist over a region that is at least about 75-100 nucleic acids in length, over a region that is about 50 nucleic acids in length, or where it is not specified, through the entire sequence of a polynucleotide sequence. For sequence comparison, a sequence commonly acts as a reference sequence, to which test sequences are compared. When a sequence comparison algorithm is used, test and reference sequences are introduced to a computer, subsequence coordinates are designed, if necessary and sequence sequence algorithm program parameters are designated. You can use default program parameters or alternatively, the parameters can be designated. Then The sequence comparison algorithm calculates the percentage of sequence identities for the test sequences in relation to the reference sequence, based on the parameters of the program parameters. The term "intercalating agent", also referred to as "intercalating group", as used herein, refers to a chemical compound that can be inserted into the intramolecular space of a molecule or the intermolecular space between molecules. By way of example only one agent or group mtercalador or can be a molecule that is inserted to the stacked bases of the double helix of DNA. The term "isolated", as used herein, refers to the separation and removal of a component component of non-component components. The isolated substances can be either in the dry or semi-dry state or in solution, in which they are included but not limited to an aqueous solution. The isolated component may be in a homogeneous state or the isolated component may be part of a pharmaceutical composition comprising additional pharmaceutically acceptable carriers and / or excipients. Purity and homogeneity can be determined using analytical chemistry techniques which include but are not limited to polyacrylamide gel electrophoresis or high performance liquid chromatography. In addition, when an isolate component is isolated and is the predominant species present in a preparation, the component it is described herein as substantially purified. The term "purified", as used herein, may refer to a component of interest that is at least 85% pure, at least 90% pure, at least 95% pure, at least 99% or greater pure. By way of example only, the nucleic acids or proteins are "isolated" when such nucleic acids or proteins are free of at least some of the cellular components with which they are associated in the natural state or that the nucleic acid has been concentrated to a level greater than the concentration of its production m vivo or vitro. Also, by way of example, a gene is stained when it is separated from open reading frame that flank the gene and encode a different protein from the gene of interest. The term "marker", as used herein, refers to a substance that is incorporated into a compound and is readily detected, whereby its physical distribution can be detected and / or monitored. The term "link", as used herein refers to bonds or chemical portion formed from a chemical reaction between the functional group of a linker and another molecule. Such linkages may include, but are not limited to, covalent bonds and non-covalent bonds, while such chemical moieties may include, but are not limited to, esters, carbonates; Imams, phosphate esters, hydrazones, acetals, orthoesters, peptide bonds and bonds of oligonucleotide. Hydrolytically stable bonds means that the bonds are substantially stable in water and do not react with water at useful pH values, in which it is included but not limited to, under physiological conditions for an extended period of time, perhaps even indefinitely. Hydrolytically unstable or degradable bonds means that the bonds are degradable in water or in aqueous solutions, in which, for example, blood is included. Enzymatically unstable or degradable bonds means that the link can be degraded by one or more enzymes. By way of example only, PEG and related polymers may include degradable linkages in the fundamental chain of the polymer or linker group between the polymer backbone and one or more of the terminal functional groups of the polymer molecule. Such degradable linkages include, but are not limited to, ester linkages formed by the reaction of carboxylic acids PEG or PEG carboxylic acids activated with alcohol groups on a biologically active agent, wherein such ester groups are generally hydrolyzed under physiological conditions to release the biologically active agent. Other hydrolytically degradable linkages include but are not limited to carbonate linkages; imine bonds resulting from the reaction of an amine and an aldehyde; phosphate ester bonds formed by reacting an alcohol with a phosphate group; hydrazone bonds that are the product of reaction of a hydrazide and an aldehyde; acetal bonds which are the reaction product of an aldehyde and an alcohol; orthoester bonds which are the reaction product of a formate and an alcohol; peptide bonds formed by an amine group, wherein, but not limited to, at one end of a polymer such as PEG and a carboxyl group of a peptide; and oligonucleotide linkages formed by a phosphoramidite, wherein but not limited to, at the terminus of a polymer and a 5 'hydroxyl group of an oligonucleotide. The terms "medium" or "means", as used herein, refer to any culture medium used to grow and harvest cells and / or products expressed and / or secreted by such cells. Such "means" or "means" include, but are limited to, solution, solids, semi-solids or rigid supports that can support or contain any host cell, including, by way of example, bacterial host cells, cells yeast host, insect host cells, plant host cells, eukaryotic host cells, mammalian host cells, CHO cells, prokaryotic host cells, E. coli or Pseudomonas host cells and cellular contents. Such "means" or "means" include, but are not limited to, a means or means in which the host cell has been cultured to which a polypeptide has been secreted, in which the medium is included. either before or after a proliferation stage. Such "media" or "media" also include, but are not limited to, pH regulating solutions or reagents containing used host cells, for example an intracellularly produced polypeptide and host cells are lysed or disrupted to release the polypeptide. The term "metabolite", as used herein, refers to a derivative of a natural amino acid polypeptide, an unnatural amino acid polypeptide, a modified natural amino acid polypeptide or a modified non-natural amino acid polypeptide that is formed when the natural amino acid polypeptide, non-natural amino acid polypeptide, modified natural amino acid polypeptide or modified non-natural amino acid polypeptide is metabolized. The term "active metabolite" refers to a biologically active derivative of a natural amino acid polypeptide, an unnatural amino acid polypeptide, a modified natural amino acid polypeptide or a modified non-natural amino acid polypeptide that is formed when the natural amino acid polypeptide is , unnatural amino acid polypeptide, modified natural amino acid polypeptide or modified non-natural amino acid polypeptide is metabolized. The term "metabolized", as used herein, refers to the sum of the processes by means of the which a particular substance is changed by an organism. Such processes include, but are not limited to, hydrolysis reactions and reactions catalyzed by enzymes. Additional information on metabolism can be obtained from The Pharmacological Basis of Therapeutics, 9th Edition, McGraw-Hill (1996). By way of example only, metabolites of natural amino acid polypeptides, unnatural amino acid polypeptides, modified natural amino acid polypeptides or modified non-natural amino acid polypeptides can be identified either by administration of natural amino acid polypeptides, non-natural amino acid polypeptides, modified natural amino acid polypeptides or unnatural amino acid polypeptides modified to a host and analysis of host tissue samples or by incubation of natural amino acid polypeptides, unnatural amino acid polypeptides, modified natural amino acid polypeptides or natural amino acid polypeptides modified with liver cells in vitro and analysis of resulting compounds. The term "metal chelator," as used herein, refers to a molecule that forms a metal complex with metal ions. By way of example, such molecules can form two or more coordination bonds with a central metal ion and can form ring structures. The term "metal-containing portion", as used herein, it refers to a group that contains a metal, atom or particle ion. Such portions include, but are not limited to, cisplatma, chelated metal ions (such as nickel, iron and platinum) and metal nanoparticles (such as nickel, iron and platinum). The term "portion incorporating a heavy atom", as used herein, refers to a group that incorporates an ion of an atom that is usually heavier than the carbon atom. Such ions or atoms include, but are not limited to, silicon, tungsten, gold, lead and uranium. The term "modified", as used herein, refers to the presence of a change to a natural amino acid, an unnatural amino acid, a natural amino acid polypeptide or an unnatural amino acid polypeptide. Such changes or modifications can be obtained by post-synthesis modifications of natural amino acids, non-natural amino acids, natural amino acid polypeptides or unnatural amino acid polypeptides or by post-translational modification of natural amino acids, non-natural amino acids, natural amino acid polypeptides or polypeptides of non-natural amino acids. The "(modified)" form means that the natural amino acid, non-natural amino acid, non-natural amino acid polypeptide or unnatural amino acid polypeptide that is discussed is optionally modified, that is, the natural amino acid, non-natural amino acid, polypeptide of The natural amino acid or non-natural amino acid polypeptide under discussion may be modified or unmodified. As used herein, the term "modulated serum half-life" refers to positive or negative changes in the circulating half-life of a biologically active molecule modified relative to its unmodified form. By way of example, the modified biologically active molecules include but are not limited to natural amino acid, non-natural amino acid, natural amino acid polypeptide or non-natural amino acid polypeptide. By way of example, the average round in the serum is measured by taking blood samples at several points in time after the administration of the biologically active molecule or modified biologically active molecule and determining the concentration of that molecule in each sample. The concentration of the ++++ concentration in the serum over time allows the calculation of the half-life in the serum. By way of example, the half-life in the modulated serum may be an increased serum half-life, which may allow for improved dosage regimens or avoid toxic effects. Such increases in serum may be at least about twice, at least about three times, at least about five times or at least about ten times. The term "modulated therapeutic half-life", as used herein, refers to positive or negative change in the half-life of the therapeutically effective amount of a modified biologically active molecule, relative to its unmodified form. By way of example, the modified biologically active molecules include, but are not limited to, natural amino acid, non-natural amino acid, natural amino acid polypeptide or non-natural amino acid polypeptide. By way of example, the therapeutic half-life is measured by measuring the pharmacokinetic and / or pharmacodynamic properties of the molecule at various points in time after administration. The increased therapeutic half-life may allow a particular beneficial dosage regimen, a particular beneficial total dose or avoid an undesirable effect. By way of example, the increased therapeutic half-life may result from increased potency, increased or decreased bonding of the modified molecule to its target, an increase or decrease in another parameter or mechanism of action of the unmodified molecule or an increased or decreased breakdown of the molecules by enzymes such as for example only, proteases. The term "nanoparticle", as used herein, refers to a particle having a particle size of between about 500 nm to about 1 nm. The term "quasi-stoichiometric", as used herein, refers to the proportion of moles of compounds that participate in a chemical reaction that is from about 0.75 to about 1.5 relative to the number of hydrogen atoms in the aromatic amine side chain of the non-natural amino acid polypeptide. By way of example, a primary aromatic amine has 2 hydrogen atoms, while a secondary amine has a hydrogen. As used herein, the term "non-eukaryotic" refers to non-eukaryotic organisms. By way of example, a non-eucaponeous organism may belong to the phylogenetic domain of Eubacteria, which includes but is not limited to, Escheri chia coli, Thermus thermophi li us or Ba ci ll us tearothermophilus, Pseudomonas fl uorescens, Pseudomonas s aerugmosa , Pseudomonas s putida), or to the phylogenetic domain of Archaea, which includes but is not limited to, Methanococcus jannaschu, Methanobactep um thermoa utotrophi cum, Archaeoglobus fulgidus, Pyrococcus fuposus, Pyrococcus orkoshn, Aeuropyrum pernix or Ha lobacteri um such as Ha loferax vol cann and Haloba cteri um NRC-I or phylogenetic domain. A "non-natural amino acid" refers to an amino acid that is not one of the 20 common amino acids or pyrolisma or selenocysteine. Other terms that can be used synonymously with the term "non-natural amino acid" are "naturally uncoded amino acid", "non-natural amino acid", "amino acids that do not occur stably in the "nature", and several scripted and non-hyphenated versions thereof The term "non-natural amino acid" includes, but is not limited to, amino acids that occur stably in nature by modification of a naturally encoded amino acid (in the which include, but are not limited to, the 20 common amino acids or pyrrolysin and selenocysteine) but which are not themselves incorporated into a growing polypeptide chain by the translation complex Examples of amino acids that occur stably in nature they are not naturally encoded include but are not limited to N-acetylglucosaminyl-L-serine, N-acetylglucosamyl-L-threonine and O-phosphotyrosine Additionally, the term "non-natural amino acid" includes, but is not limited to, amino acids that are not Presented in a stable manner in nature and can be obtained synthetically or can be obtained by non-natural amino acid modification The term "nucleic acid", as used herein, refers to deoxyribonucleotides, deoxypbonucleosides, ribonucleosides or ribonucleotides and polymers thereof either in the form of a single strand or in the form of a double strand. By way of example only, such nucleic acids and nucleic acid polymers include, but are not limited to, (i) natural nucleotide analogs having similar binding properties as a nucleic acid of reference and are metabolized in a manner similar to nucleotides that occur stably in nature; (11) oligonucleotide analogs which include, but are not limited to, PNA (peptide nucleic acid), DNA analogues used in antisense technology (phosphorothioates, phosphoroamidates and the like); (m) conservatively modified variants thereof (in which, but not limited to, degenerate codon substitutions) and complementary sequences and sequences are explicitly indicated. By way of example, degenerate codon substitutions can be obtained by generating sequences in which the third position of one or more selected codons (or all) is substituted with mixed base residues and / or deox osma (Batzer et al., Nucleic Acid Res. 19: 5081 (1991), Ohtsuka et al., J. Biol. Chem. 260 / 2605-2608 (1985), and Rossolmi et al., Mol. Cell. Probes 8: 91-98 (1994)) . The term "oxidizing agent", as used herein, refers to a compound or material that is capable of removing an electron from a compound that is oxidized. By way of example, oxidizing agents include but are not limited to oxidized glutathione, cyst, cistamma, oxidized dithiothreitol, oxidized epititol, and oxygen. A wide variety of oxidizing agents are suitable for use in the methods and compositions described herein. The term "photoafimdad marker", as used in present, refers to a marker with a group which, on exposure to light, forms a bond with a molecule for which the marker has affinity. By way of example only, such a linkage may be covalent or non-covalent. The term "photoenzylated portion", as used herein, refers to a group which, in illumination at certain wavelengths, is covalently or non-covalently bound to other ions or molecules. The term "photocleavable group", as used herein, refers to a group that breaks on exposure to light. The term "photoreticulator", as used herein, refers to a compound comprising two or more functional groups which, on exposure to light, are reactive and form a covalent or non-covalent bond with two or more monomeric molecules or polymeric The term "photoisomerizable portion", as used herein, refers to a group wherein, after illumination with light, it changes from one isomeric form to another. The term "polyalkylene glycol", as used herein, refers to linear or branched polymeric polyether polyols. Such polyalkylene glycols, which include but are not limited to, polyethylene glycol, propylene glycol, polybutylene glycol and derivatives thereof. Other exemplary modalities are listed, for example, in catalogs of commercial suppliers, such as the catalog of Shearwater Corporation's "Polyethylene Glycol and Derivatives for Biomedical Applications" (2001). By way of example only, such polymeric polyether polyols have average molecular weights of between about 0.1 kDa to about 100 kDa. By way of example, such polymeric polyether polyols include, but are not limited to, between about 100 Da and about 100,000 Da or more. The molecular weight of the polymer can be between about 100 Da and about 100,000 Da, which includes but is 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, 0 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 modalities, the weight The molecular weight of the polymer is between approximately 10,000 Da and approximately 40,000 Da. In some embodiments, the poly (ethylene glycol) molecule is a branched polymer. The molecular weight of the branched chain PEG can be between about 1,000 Da and about 100,000 Da, which includes but is 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. The term "polymer", as used herein, refers to a molecule composed of repeated subunits. Such molecules include, but are not limited to polypeptides, polynucleotides or polysaccharides or polyalkylene glycols. The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues, that is, a description directed to a polypeptide is equally applicable to a description of a peptide and a description of a protein and vice versa. The terms apply to amino acid polymers that occur stably in nature as well as amino acid polymers in which one or more amino acid residues is an unnatural amino acid. Additionally, such "polypeptides", "peptides" and "proteins" include amino acid chains of any length, in which full length proteins are included, wherein the amino acid residues are linked by covalent peptide bonds. The term "post-translationally modified" refers to any modification of a natural or non-natural amino acid that occurs after such an amino acid has been translationally incorporated into a polypeptide chain. Such modifications include, but are not limited to, in vivo co-translational modifications, co-translational in vitro modifications (such as in a cell-free translation system), post-translational in vivo modifications, and post-translational in vitro modifications. The term "prodrug", as used herein, refers to an agent that is converted to the original drug in vivo or in vitro. The benefits of such prodrugs include, but are not limited to, (i) ease of administration compared to the original drug; (ii) the prodrug may be bioavailable by oral administration while the original does not; and (iii) the prodrug may also have improved solubility in pharmaceutical compositions compared to the original drug. A prodrug includes a pharmacologically inactive or reduced activity derivative of an active drug. Prodrugs may be designed to modulate the amount of a biologically active drug or molecule that reaches a desired site of action by manipulating the properties of a drug, such as physicochemical, biopharmaceutical or pharmacokinetic properties. The prodrugs are converted to the active drug within the body by means of enzymatic or non-enzymatic actions. Prodrugs may provide improved physicochemical properties such as improved solubility, improved administration characteristics, such as specifically targeting a particular cell, tissue, organ or ligand and improved therapeutic value of the drug. The term "prophylactically effective amount", as used herein, refers to that amount of one of a composition that contains at least one unnatural amino acid polypeptide or at least one polypeptide of modified non-natural amino acid applied prophylactically to a patient that will alleviate to some extent one or more of the symptoms of a disease, condition or disorder being treated. In such prophylactic applications, such amounts may depend on the health status of the patient, weight and the like. It is considered within the skill of the art to determine such prophylactically effective amounts by systematic experimentation, including, but not limited to, a clinical trial of dose escalation. The term "protected", as used herein, refers to the presence of a "protecting group" or moiety that prevents the reaction of the chemically reactive functional group under certain reaction conditions. The protective group will vary depending on the type of chemically reactive group that is protected. By way of example only, (i) if the chemically reactive group is an amine or a hydrazide, the protecting group may be selected from tert-butyloxycarbonyl (t-Boc) and 9-fluorenylmethoxycarbonyl (Fmoc); (ii) if the chemically reactive group is a thiol, the protecting group may be orthopyridyldisulfide; and (iii) if the chemically reactive group is a carboxylic acid, 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 tert-butyl. By way of example only, the blocking / protective groups may also be selected from: H3C ' Et t butyl TBDMS Teoc Additionally, protecting groups include, but are not limited to, photolabile groups such as Nvoc and MeNvoc and other protecting groups known in the art. Other protecting groups are described in Greene and Wuts, Protective Groups in Organic Synthesis, 3rd Ed., John Wiley & Sons, New York, NY, 1999, which is incorporated herein by reference in its entirety. The term "radioactive portion", as used herein, refers to a group whose nuclei spontaneously shed nuclear radiation, such as alpha, beta or gamma particles; where, alpha particles are helium nuclei, beta particles are electrons and gamma particles are high energy photons. The term "reactive compound", as used in the present, refers to a compound that under appropriate conditions is reactive towards another atom, molecule or compound. The term "recombinant host cell", also referred to as "host cell", refers to a cell that includes an exogenous polynucleotide, wherein the methods used to insert the exogenous polynucleotide into a cell include, but are not limited to, direct absorption , transduction, F coupling and other methods known in the art to create recombinant host cells. By way of example only, such an exogenous polynucleotide can be a non-integrated vector, in which it is included but not limited to a plasmid or can be integrated into the host genome. The term "redox-active agent", as used herein, refers to a molecule that oxidizes or reduces another molecule, whereby the active redox agent becomes reduced or oxidized. Examples of redox-active agent include, but are not limited to ferrocene, quinones, Ru2 + / 3 + complexes, Co2 + / 3 + complexes and Os2 + / 3 + complexes. The term "reducing agent", as used herein, refers to a compound or material that is capable of adding an electron to a compound that is reduced. By way of example, reducing agents include, but are not limited to, dithiothreitol (DTT), 2-mercaptoethanol, dithioerythritol, cysteine, cysteamine (2-aminoetiol) and reduced glutathione. Such reducing agents can be used, by way of example only, to maintain the sulfhydryl groups in reduced state and to reduce intra- or inter-molecular disulfide bonds. "Re-folded", as used herein, describes any process, reaction or method that transforms an appropriately folded or unfolded state into a natural folded or properly folded configuration. As an example only, the re-folding transforms polypeptides containing disulfide bond from a misfolded or unfolded state to a natural or properly folded conformation with respect to the iso-sulfide bonds. Such polypeptides containing disulfide linkage can be natural amino acid polypeptides or non-natural amino acid polypeptides. The term "resin", as used herein, refers to high molecular weight insoluble polymer beads. By way of example only, such beads can be used as supports for solid phase peptide synthesis or sites for molecule attachment before purification. The term "saccharide", as used herein, refers to a series of carbohydrates which includes but is not limited to sugars, monosaccharides, oligosaccharides and polysaccharides. The phrase "selectively hybridizes to" or "specifically hybridizes to", as used herein, is linkage, duplexing or hybridization of a molecule to a particular nucleotide sequence under severe hybridization conditions when that sequence is present in a complex mixture in which it is included but not limited to, total cellular or library DNA or RNA. The term "spin marker", as used herein, refers to molecules that contain an atom or group of atoms that exhibit an unpaired electron spin (ie, a stable paramagnetic group) that can be detected by spectroscopy. Electronic spin resonance and can be attached to another molecule. Such spin marker molecules include, but are not limited to, nitrile radicals and nitroxides and may be single-spin markers or double-spin markers. The term "stoichiometric", as used herein, refers to the proportion of the moles of compounds participating in a chemical reaction that is from about 0.9 to about 1.1 relative to the number of hydrogens in the amine side chain aromatic of the non-natural amino acid polypeptide. By way of example, a primary aromatic amine has 2 hydrogens, while the secondary amine has a hydrogen. The term "stoichiometric-like", as used herein, refers to a chemical reaction that becomes stoichiometric or quasi-stoichiometric after changes in reaction conditions or in the presence of additives. Such changes in reaction conditions include, but are not limited to, an increase in temperature or change in pH. Such additives include, but are not limited to, accelerants. The phrase "severe hybridization conditions" refers to hybridization of DNA, RNA, PNA or other nucleic acid mimic sequences or combinations thereof, under conditions of low ionic strength and high temperature. By way of example, under severe conditions a probe will hybridize to its target subsequence in a complex mixture of nucleic acid (in which it is included but not limited to total cell or library DNA or RNA) but which does not hybridize to other sequences in the complex mixture. Severe conditions are sequence dependent and will be different in different circumstances. By way of example, longer sequences hybridize specifically at higher temperature. The severe hybridization conditions include, but are not limited to, (i) about 5-10 ° C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH; (ii) the salt concentration is from about 0.01 M to about 1.0 M at a pH of about 7.0 at a pH of about 8.3 and the temperature is at least about 30 ° C for short probes (which include but are not limited to , from 10 to 50 nucleotides) and at least about 60 ° C for long probes (where, but not limited to, greater than 50 nucleotides); (m) the addition of destabilizing agents including, but not limited to, formamide, (? v) 50% formamide, 5X SSC and 1% SDS, incubation at 42 ° C or 5X SSC, 1 SDS %, incubation at 65 ° C, with washing in 0.2X SSC and 0.1% SDS at 65 ° C for between about 5 minutes to about 120 minutes. By way of example only, the detection of selective or specific hybridization includes, but is not limited to, a positive signal at least twice the background. An extensive guide to the hybridization of nucleic acids is found in T ± jssen, Labora tory Techniques m Biochemi s try and Molecular Biology - Hybridization of Nuclei c Probes, "Overview of principles of hybridization and the strategy of nucleic acid assays" (1993). The term "subject" as used herein, refers to an animal that is the object of treatment, observation or experiment. By way of example only, a subject may be, but is not limited to, a mammal in which it is included, but not limited to a human. The term "substantially purified", as used herein, refers to an ingredient component that may be substantially or substantially free of other components that normally accompany or interact with the component of the prior art before purification. As an example only, a component of interest can be "substantially purified", when the preparation of the component of interest contains less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10% , less than about 5%, less than about 4%, less than about 3%, less than about 2% or less than about 1% (dry weight) of contaminating components. Thus, a "substantially purified" component of interest may have a purity level of about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, approximately 99% or greater. By way of example only, a natural amino acid polypeptide or unnatural amino acid polypeptide can be purified from a natural cell or host cell in the case of naturally occurring recombinantly produced amino acid polypeptides or non-natural amino acid polypeptides. By way of example a preparation of a natural amino acid polypeptide or an unnatural amino acid polypeptide can be "substantially purified" when the preparation contains less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less about 2% or less than about 1% (dry weight) of contaminating material. By way of example when a natural amino acid polypeptide or an unnatural amino acid polypeptide is recombinantly produced by host cells, the natural amino acid polypeptide or unnatural amino acid polypeptide 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. By way of example when a natural amino acid polypeptide or non-natural amino acid polypeptide is recombinantly produced by host cells, the natural amino acid polypeptide or non-natural amino acid polypeptide may be present in the culture medium at about 5 g / 1, approximately 4 g / 1, approximately 3 g / 1, approximately 2 g / 1, approximately 1 g / 1, approximately 750 mg / l, approximately 500 mg / l, approximately 250 mg / l, approximately 100 mg / l, approximately 50 mg / l, approximately 10 mg / l or approximately 1 mg / l or less of the dry weight of the cells. By way of example, natural amino acid polypeptides or "substantially purified" unnatural amino acid polypeptides can have a purity level of about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 99% or greater as determined by appropriate methods, in the which is included but not limited to analysis of SDS / PAGE analysis, RP-HPLC, SEC and capillary electrophoresis. The term "substituents" also referred to as "non-interfering substituents" refers to groups that can be used to replace another group in a molecule. Such groups include, but are not limited to, halo, Ci-Cio alkyl, C2-C? Or alkenniio, C2-C? Or aikinyl, Ci-Cio alkoxy, C5-C12 aralkyl, C3-C12 cycloalkyl, C4-C] 2-cycloalkenyl, phenyl, substituted phenyl, toluyl, xylenyl, biphenyl, C2-C? 2 alkoxyalkyo, C5-C12 alkoxyaryl, C5-C12 aryloxyalkyl, C7-C12 oxyaryl, C? -C6 alkylsulfinyl, C1-C10 alkylsulfonyl, - (CH2 ) m-0- (C1-C10 alkyl) wherein m is from 1 to 8, aryl, substituted aryl, substituted alkoxy, fluoroalkyl, heterocyclic radical, substituted heterocyclic radical, nitroalkyl, -N02, -CN, -NRC (O) - (d-C10 alkyl), -C (O) - (C1-C10 alkyl), C2-C ? 0 alkythioalkyl, -C (O) O- (C1-C10 alkyl), -OH, -S02, = S, -COOH, -NR2, carbonyl, -C (O) - (C1-C10 alkyl) -CF3, -C (0) -CF3, -C (0) NR2, - (C1-C10 aryl) -S- (C6-C? 0 aryl), -C (0) - (C6-C10 aryl), - (CH2 ) m-0- (CH2) m-0- (C1-C10 alkyl) wherein each m is from 1 to 8, -C (0) NR2, -C (S) NR2, -S02NR2, -NRC (0) NR2, -NRC (S) NR2, salts thereof and the like. Each R group in the list The foregoing includes, but is not limited to, H, alkyl or substituted alkyl, aryl or substituted aryl or alkaryl. When the substituent groups are specified by their conventional chemical formulas, written from left to right, they also include the chemically identical substituents that would result from writing the structure from right to left, for example, -CH20- is equivalent to -OCH2-. By way of example only, substituents for alkyl and heteroalkyl radicals (wherein those groups referred to as alkylene, alkenyl, heteroalkylene, heteroalkylene, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl and heterocycloalkenyl) include but are not limited to: -OR , = 0, = NR, = N-0R, -NR2, -SR, -halogen, -SiR3, -0C (0) R, -C (0) R, -C02R, -C0NR2, 0C (0) NR2, -NRC (0) R, -NR-C (0) NR2, -NR (0) 2R, -NR-C (NR2) = NR, S (0) R, -S (0) 2R, -S (0) 2NR2, -NRS02R, -CN and -N02. Each group R in the foregoing list includes, but is not limited to, hydrogen, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, in which but not limited to, aryl substituted with 1-3 halogens, substituted alkyl or unsubstituted, alkoxy or thioalkoxy groups or aralkyl groups. When two R groups are attached to the same nitrogen atom, it can be combined with the nitrogen atom to form the 5-, 6- or 7-membered ring. For example, -NR2 is proposed to include, but not be limited to, 1-pyrrolidinyl and 4- morpholinyl. By way of example, substituents for aryl and heteroaryl groups include, but are not limited to, -OR, = 0, = NR, = N-0, -NR2, -SR, -halogen, -SiR3, -OC (0) R, -C (0) R, -C02R, -C0NR2, -0C (0) NR2, -NRC (0) R, -NR-C (0) NR2, -NR (0) 2R, -NR-C ( NR2) = NR, -S (0) R, -S (0) 2R, -S (0) 2NR2, -NRS02R, -CN, -N02, -R, -N3, -CH (Ph) 2, fluoro ( C? -C4) alkoxy and fluoro (C1-C4) alkyl, in a number ranging from zero to the total number of open valencies in the aromatic ring system; and wherein each group R in the preceding list includes, but is not limited to, hydrogen, alkyl, heteroalkyl, aryl and heteroaryl. The term "therapeutically effective amount", as used herein, refers to the amount of a composition that contains at least one unnatural amino acid polypeptide and / or at least one modified non-natural amino acid polypeptide administered to a patient who already suffers from a disease, condition or alteration, sufficient to cure or at least partially stop or alleviate to some extent one or more of the symptoms of the disease, disorder or condition being treated. The effectiveness of such compositions depends on conditions in which it is included, but not limited to, the severity and course of the disease, alteration or condition, prior therapy, the patient's health status and response to the drugs and the physician's judgment of treatment. As an example only, quantities Therapeutically effective can be determined by systematic experimentation, which includes but is not limited to a clinical study of dose escalation. The term "thioalkoxy," as used herein, refers to alkyl groups containing sulfur linked to molecules via an oxygen atom. The term "thermal melting point" or Tm, is the temperature (under ionic strength, pH and defined n-acid concentration) at which 50% of the probes complementary to a target are hybridized to the target sequence in equilibrium. The term "toxic portion", as used herein, refers to a compound that can cause injury or death. The terms "treat", "treating" or "treatment", as used herein, include alleviating, reducing or ameliorating a disease or condition symptoms, preventing further symptoms, improving or preventing the fundamental metabolic causes of the symptoms, inhibit the disease or condition, for example, stop the development of the disease or condition, alleviate the disease or condition, bring about regression of the disease or condition, alleviate a condition caused by the disease or condition or stop the symptoms of the disease or condition. The terms "treat", "treating" or "treatment" include, but are not limited to, prophylactic and / or therapeutic treatments.

As used herein, the term "water-soluble polymer" refers to any polymer that is soluble in aqueous solvents. Such water-soluble polymers include, but are not limited to, polyethylene glycol, polyethylene glycol propionaldehyde, C1-C10 alkoxy or aryloxy mono derivatives thereof (described in U.S. Patent No. 5,252,714 which is incorporated by reference herein), monomethoxy polyethylene glycol, polyvinyl pyrrolidone, polyvinyl alcohol, polyamino acids, divinyl ether maleic anhydride, N- (2-hydroxypropyl) -methacrylamide, dextran, dextran derivatives including dextran sulphate, propylene glycol, polypropylene oxide copolymer / ethylene oxide, polyoxyethylated polyol, heparin, heparin fragments, polysaccharides, oligosaccharides, glycans, cellulose and cellulose derivatives, in which but not limited to methylcellulose and carboxymethyl cellulose, serum albumin, 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) -DL-aspartamide and the like or mixtures thereof. By way of example, the binding of such water-soluble polymers to a natural amino acid polypeptide or non-natural polypeptide can result in changes in which include, but are not limited to, serum half-life. increased or modulated, increased or modulated therapeutic half-life in relation to the unmodified form, modulated immunogenicity, modulated physical association characteristics in which it is included, but not limited to, aggregation and multimer formation, altered receptor binding, altered linkage to one or more link partners and dimerization or multimerization of altered receiver. In addition, such water soluble polymers may or may not have their own biological activity. Unless indicated otherwise, conventional methods of mass spectroscopy, NMR, HPLC, protein chemistry, biochemistry, recombinant DNA techniques and pharmacology are used within the skill of the art. The compounds (wherein, but not limited to, non-natural amino acids, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides and reactive to produce the aforementioned compounds) presented herein include isotopically-labeled compounds, which are identical those mentioned in the various formulas and structures presented herein, but by 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, 180, 170, 35S, 18F, 36C1, respectively. Certain isotopically-labeled compounds described herein, for example those in which radioactive isotopes such as 3 H and 14 C are incorporated, are useful in tissue and / or substrate tissue distribution analyzes. In addition, substitution with isotopes such as deutepo, that is, H, can provide certain therapeutic advantages resulting from increased metabolic stability, for example increased live half-life or reduced dosage requirements. Some of the compounds herein (which include, but are not limited to, non-natural amino acids, non-natural amino acid polypeptides, and unnatural amino acid polypeptides modified and reactive to produce the compounds mentioned above) have asymmetric carbon atoms and can therefore exist as enantiomers or diastereomers. The diastereomeric mixtures can be separated into their individual diastereomers based on their physicochemical differences by known methods, for example by chromatography and / or fractional crystallization. The enantiomers can be separated by converting the enantiomeric mixture to a diastereomeric mixture by reaction with an appropriate optically active compound (e.g., alcohol), separating the diastereomers and converting (for example, by hydrolyzing) the individual diastereomers to the corresponding pure enantiomers. All such isomers, in which diastereomers, enantiomers, and mixtures thereof are included are considered part of the compositions described herein. In other embodiments or additional embodiments, the compounds described herein (including, but not limited to, non-natural amino acids, unnatural amino acid polypeptides and unnatural amino acid polypeptides modified and reactive to produce the compounds mentioned above) they are used in the form of pro-drugs. In other embodiments or additional embodiments, the compounds described herein (wherein, but not limited to, non-natural amino acids, unnatural amino acid polypeptides and unnatural amino acid polypeptides modified and reactive to produce the compounds mentioned above) are metabolized upon administration to an organism in need of producing a metabolite which is then used to produce a desired effect, in which a desired therapeutic effect is included. In addition or additional embodiments, active metabolites of non-natural amino acids and polypeptides of modified or unmodified non-natural amino acids are found. The methods and formulations described herein include the use of N-oxides, crystalline forms (also known as polymorphs) or pharmaceutically acceptable salts of non-natural amino acids, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides. In certain embodiments, non-natural amino acids, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides can exist as tautomers. All tautomers are included within the scope of the non-natural amino acids, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides presented herein. In addition, non-natural amino acids, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides described herein may exist in unsolvated forms 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, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides presented herein are also considered disclosed herein. Some of the compounds herein (which include, but are not limited to, non-natural amino acids, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides and reagents to produce the compounds mentioned above) may exist in various tautomeric forms. All of such tautomeric forms are considered as part of the compositions described herein. Also, for example all the enol-keto forms of any compounds (including, but not limited to, non-natural amino acids, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides and reactants for producing the aforementioned compounds) of the present are considered as part of the compositions described herein. Some of the compounds of the present invention (which include, but are not limited to, non-natural amino acids, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides and reactants to produce either one or the other of the compounds mentioned above) are acids and can form a salt with a pharmaceutically acceptable cation. Some of the compounds of the present invention (in which they include, but are not limited to non-natural amino acids, non-natural amino acid polypeptides and unnatural amino acid polypeptides modified and reactive to produce the compounds mentioned above) can be basic and thus, can forming a salt with a pharmaceutically acceptable anion. All such salts, in which di-salts are included, are within the scope of the compositions described herein and can be prepared by conventional methods. For example, salts can be prepared by contacting the acidic and basic entities, either in an aqueous, non-aqueous or partially aqueous medium. The salts are recovered by using at least one 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. The salts, for example, include: (1) acid addition salts, formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like; or formed with organic acids such as acetic acid, propionic acid, hexanoic acid, cyclopentanpropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, acid benzoic acid, 3- (4-hydroxybenzoyl) benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethanedisulfonic acid, 2-hydroxy-ethanesulfonic acid, bezenesulfonic acid, 2-naphthalenesulfonic acid, 4-met? lb? c? clo- [2.2.2] oct-2-en-l-carboxylic acid, glucoheptonic acid, 4,4 '-methylenebis- (3-h? drox? -2-en-l- carboxyl? co), 3-phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid, lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxamftoic acid, salicylic acid, stearic acid, muconic acid and the like; (2) salts formed when a proton The acid present in the original compound is either replaced by a metal ion, for example, an alkali metal ion, an alkaline earth metal ion or an aluminum ion; or coordinated with an organic base. Acceptable organic bases include ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine and the like. Acceptable inorganic bases include aluminum hydroxide, calcium hydroxide, potassium hydroxide, sodium carbonate, sodium hydroxide and the like. It should be understood that a reference to a salt includes solvent addition forms or crystalline forms thereof, particularly solvates or polymorphs. The solvates contain either stoichiometric or non-stoichiometric amounts of a solvent and are frequently formed during the crystallization process. Hydrates are formed when the solvent is water or alcoholates are formed when the solvent is alcohol. Polymorphs include different crystal packing arrangements of the same elemental composition of a compound. Polymorphs usually have different patterns of x-ray diffraction, infrared spectra, melting points, density, hardness, crystalline form, optical and electrical properties, stability and solubility. Various factors such as recrystallization solvent, crystallization rate and storage temperature can cause a single crystalline form dominate BRIEF DESCRIPTION OF THE FIGURES A better understanding of the elements and advantages of the present methods and compositions can be obtained by reference to the following detailed description which summarizes illustrative modalities, in which the principles of the methods, compositions, devices and apparatuses are used. and the accompanying figures of which: Figure 1 illustrates a non-limiting aspect of methods for selecting and designing a polypeptide to be modified using the methods, compositions and techniques described herein. Figure 2 presents illustrative non-limiting examples of the types of non-natural amino acids described herein. Such non-natural amino acids can be used in or incorporated into any of the methods, compositions, techniques and strategies for making, purifying, characterizing and using non-natural amino acids, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides described herein. Figure 3 presents illustrative non-limiting examples of the types of non-natural amino acids described herein. Such non-natural amino acids can be used in or incorporated into any of the methods, compositions, techniques and strategies for making, purifying, characterizing and using non-natural amino acids, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides described herein. For Figure 3 only, X represents a halogen. Figure 4 presents illustrative non-limiting examples of the types of non-natural amino acids described herein. Such non-natural amino acids can be used in or incorporated into any of the methods, compositions, techniques and strategies for making, purifying, characterizing and using non-natural amino acids, non-natural amino acid poly-peptides and modified non-natural amino acid polypeptides described herein. Figure 5 presents illustrative non-limiting examples of the types of non-natural amino acids described herein. Such non-natural amino acids can be used in or incorporated into any of the methods, compositions, techniques and strategies for making, purifying, characterizing and using non-natural amino acids, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides described herein. Figure 6 presents illustrative non-limiting examples of the types of non-natural amino acids described herein. Such non-natural amino acids can be used in or incorporated into any of the methods, compositions, techniques and strategies for making, purifying, characterizing and using non-natural amino acids, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides described herein. For Figure 6 only, X represents a halogen. Figure 7 presents illustrative non-limiting examples of the types of non-natural amino acids described herein. Such non-natural amino acids can be used in or incorporated into any of the methods, compositions, techniques and strategies for making, purifying, characterizing and using non-natural amino acids, non-natural amino acid polypepdates and modified non-natural amino acid polypeptides described herein . Figure 8 presents illustrative non-limiting examples of the types of non-natural amino acids described herein. Such non-natural amino acids can be used in or incorporated into any of the methods, compositions, techniques and strategies for making, purifying, characterizing and using non-natural amino acids, unnatural amino acid polypeptides and modified non-natural amino acid polypeptides described herein. Figure 9 presents illustrative non-limiting examples of the types of non-natural amino acids described herein. Such non-natural amino acids can be used in or incorporated into any of the methods, compositions, techniques and strategies for making, purifying, characterizing and using non-natural amino acids, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides described herein. For Figure 9 only, X represents a halogen. Figure 10 presents illustrative non-limiting examples of the types of non-natural amino acids described herein. Such non-natural amino acids can be used in or incorporated into any of the methods, compositions, techniques and strategies for making, purifying, characterizing and using non-natural amino acids, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides described herein. Figure 11 presents a non-limiting example illustrating the synthesis methodology used to manufacture the natural amino acids described herein. Such non-natural amino acids can be used in or incorporated into any of the methods, compositions, techniques and strategies for making, purifying, characterizing and using non-natural amino acids, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides described herein. Figure 12 presents a non-limiting example illustrating the synthesis methodology used to make the non-natural amino acids described herein. Such non-natural amino acids can be used in or incorporated into any of the methods, compositions, techniques and strategies for making, purifying, characterizing and using non-natural amino acids, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides described herein. Figure 13 presents a non-limiting example illustrating the synthesis methodology used to make the non-natural amino acids described herein. Such non-natural amino acids can be used in or incorporated into any of the methods, compositions, techniques and strategies for making, purifying, characterizing and using non-natural amino acids, unnatural amino acid polypeptides and modified non-natural amino acid polypeptides described herein. Figure 14 presents non-limiting illustrative examples of non-natural amino acids described herein that contain masked and / or protected amine moieties that can be converted to unmasked and / or unprotected amine moieties. Such non-natural amino acids can be used in or incorporated into any of the methods, compositions, techniques and strategies for making, purifying, characterizing and using non-natural amino acids, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides described herein.

Figure 15 shows illustrative non-limiting examples of non-natural amino acids described herein that contain amine side chains masked and / or protected for reductive amination with an aldehyde-containing reagent. Such non-natural amino acids can be used in or incorporated into any of the methods, compositions, techniques and strategies for making, purifying, characterizing and using non-natural amino acids, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides described herein. Figure 16 presents illustrative non-limiting examples for the formation of polypeptides with non-natural amino acids containing aromatic amine by reducing alkylations with aldehyde-containing reagents. Figure 17 presents illustrative non-limiting examples of non-natural aromatic protected aldehyde-containing amino acids described herein. Such non-natural amino acids can be used in or incorporated into any of the methods, compositions, techniques and strategies for making, purifying, characterizing and using non-natural amino acids, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides described herein. Figure 18 presents illustrative non-limiting examples of polypeptide formation with non-amino acid aldehyde-containing natural products by deprotection / unmasking post-translation of a protected / masked precursor followed by reducing amino acids with aromatic amine-containing reagents. Figure 19 presents illustrative non-limiting examples of selective reductive site killing and reductive amination of non-natural amino acid polypeptides. Figure 20 presents illustrative non-limiting examples of reduced urotension-II reducing alkylations (UT-II-SH) with propionaldehyde (I) and benzaldenido (II) and the corresponding HPLC chromatograms. Figure 21 presents illustrative non-limiting examples of urotension-reducing alkylations II (UT-II) with propionaldehyde (I) and benzaldehyde (II) and the corresponding HPLC chromatograms. Figure 22 shows illustrative non-limiting examples of reductive alkylations of the peptide XT-8 with propionaldehyde (I) and benzaldehyde (II), isobutaldehyde (III) and pivalaldehyde (IV) and the corresponding HPLC chromatograms. Figure 23 presents illustrative non-limiting examples of reductive alkylations of the SXT-9 peptide with propionaldehyde (I), benzaldehyde (II), isobutaldehyde (III) and pivalaldehyde (IV) and HPLC chromatograms corresponding. Figure 24 shows illustrative non-limiting examples of reductive alkylations of the HXT-9 peptide with propionaldehyde (I), benzaldehyde (II), isobutaldehyde (III) and pivalaldehyde (IV) and the corresponding HPLC chromatograms. Figure 25 presents illustrative non-limiting examples of reductive alkylations of the WXT-9 peptide with propionaldehyde (I), benzaldehyde (II) and isobutaldehyde (III) and the corresponding HPLC chromatograms. Figure 26 presents illustrative non-limiting examples of reductive alkylations of the peptide NXT-9 with propionaldehyde (I) and benzaldehyde (II) and the corresponding HPLC chromatograms. Figure 27 presents illustrative non-limiting examples of reductive alkylations of the peptide RXT-10 with propionaldehyde (I) and benzaldehyde (II) and the corresponding HPLC chromatograms. Figure 28 presents illustrative non-limiting examples of reductive alkylations of the peptide AXT-11 with propionaldehyde (I) and benzaldehyde (II) and the corresponding HPLC chromatograms. Figure 29 presents illustrative non-limiting examples of reductive alkylations of the peptide AXT-11 with different aldehydes (I-III) and HPLC chromatograms corresponding. Figure 30 presents illustrative non-limiting examples of reductive alkylations of the AXT-11 peptide with different aldehydes (IV-VII) and the corresponding HPLC chromatograms. Figure 31 presents non-limiting examples illustrative of competitive reactions of peptide AXT-11 with an aldehyde and a 1,3-diketone (I-III) and the corresponding HPLC chromatograms. Figure 32 presents illustrative non-limiting examples of competitive reactions of peptide NXT-9 with an aldehyde and a 1,2-diketone (I-III) and the corresponding HPLC chromatograms. Figure 33 presents non-limiting examples illustrating competitive reactions of the peptide MXT-9 with different aldehydes and ketones (I-V) and the corresponding HPLC chromatograms. Figure 34 presents illustrative non-limiting examples of the reduction of the peptide MXT-9-N3 to MXT-9NH2 (I), followed by reductive alkylations with propionaldehyde (II) and benzaldehyde (III) and the corresponding HPLC chromatograms. Figure 35 presents illustrative non-limiting examples and the PEG coating of peptide comparison by coating with N-terminal PEG and PEG coating of unnatural amino acids containing aromatic amine described herein. Figure 36 presents illustrative non-limiting examples of PEG aldehyde reagents used to coat PEG with non-natural amino acids containing aromatic amine incorporated into peptides. Figure 37 presents illustrative non-limiting examples of PEG coating of MT-9 by N-thermal reduction alkylation. Figure 38 presents illustrative non-limiting examples of the PEG coating of MXT-9 by reductive alkylation of unnatural amino acids containing aromatic amine incorporated into MXT-9. Figure 39 presents illustrative non-limiting examples of the PEG coating of hGH and IFNa by reductive alkylation of non-natural amino acids containing aromatic amine incorporated into hGH and IFNa. Figure 40 presents a non-limiting illustrative image of an electrophoresis gel of various coatings with hGH PEG.

DETAILED DESCRIPTION OF THE INVENTION I. Introduction Recently, a completely new technology in the science of proteins has been reported that promises overcome many of the limitations associated with site-specific protein modifications. Specifically, new components have been added to the biosthetic protein machinery of the procaponte Eschep chia coli (E. coli) (eg, L. Wang, et al., (2001), Science 292: 498-500) and the eucapone Sa ccharomyces cerevi siae (S. cerevi siae) (for example, J. Chin et al., Science 301: 964-7 (2003)), which has enabled the incorporation of non-natural amino acids into proteins in vivo. A number of new amino acids with new chemical, physical or biological properties, in which they include photoaffinity markers and photoisomerizable amino acids, crosslinking amino acids (See, for example, Chin, J. W., et al. (2002) Proc. Nati.

Acad. Sci. U.S.A. 99: 11020-11024; and, Chin, JW, et al., (2002) J. Am. Chem. Soc. 124: 9026-9027), amino acids containing heavy atoms, ketoammo acids and glycosylated amino acids have been incorporated efficiently and with high fidelity to proteins in E . coli and in yeast in response to the amber codon, TAG, using this methodology. See, for example, J. W. Chin et al., (2002), Journal of the American Chemical Society 124: 9026-9027 (incorporated by reference in its entirety); J. W. Chin, & P. G. Schultz, (2002), ChemBioChem 3 (11): 1135-1137 (incorporated by reference in its entirety); J. W. Chin, et al., (2002), PNAS United States of America 99: 11020-1 1024 (incorporated by reference in its entirety); Y L. Wang & P. G. Schultz, (2002), Chem. Comm. , 1: 1-11 (incorporated by reference in its entirety). These studies have shown that it is possible to selectively and systematically introduce chemical functional groups that are not found in proteins, which are chemically inert to all the functional groups found in the 20 genetically encoded common amino acids and that can be used to react efficiently and selectively to form stable covalent bonds.

II. General View Figure 1 presents an overview of the compositions, methods and techniques that are described herein. At one level, the tools (methods, compositions, techniques) for creating and using a polypeptide comprising at least one non-natural amino acid or non-natural amino acid modified with an aromatic amine or a heteroaromatic amine are described herein. The aromatic group includes, but is not limited to, furans, pyrrole, thiophenes, pyridines, quinolines, isoquinolines, imidazoles, thiazoles, pyrimidines, pyridazines, pyrazines, benzothiazoles, thiazolopyridines, oxazoles, benzoxazoles, oxazolopyridines, thiazolopyrimidines, oxazolopyrimidines, benzoxazines and benzothiazines. . Such non-natural amino acids may contain additional functionality, in which is included but not limited to, a marker; a dye; a polymer; a water soluble polymer; a polyethylene glycol derivative; a reticulator photo; a cytotoxic compound; a drug; an affinity marker; a photoaffinity marker; a reactive compound; a resin; a second protein or polypeptide or polypeptide analogue; an antibody or antibody fragment; a metal chelator; a co-factor; a fatty acid; a carbohydrate; a polynucleotide; a DNA; an RNA; an antisense polynucleotide; a saccharide, a water-soluble dendrimer, a cyclodextrin, a biomaterial; a nanoparticle; a spin marker; a fluorophore, a portion containing metal; a radioactive portion; a new functional group; a group that interacts covalently or non-covalently with other molecules; a photoenaged portion; an excitable portion by actinic radiation; a ligand; a photoisomerizable portion; biotin; a biotin analogue; a portion that incorporates a heavy atom; a group chemically cleaved it; a photo-scissors group; an elongated side chain; a carbon-bonded sugar; a redox-active agent; an amino thioacid; a toxic portion; an isotopically labeled portion; a biophysical probe; a phosphorescent group; a chemiluminescent group; a dense group of electrons; a magnetic group; an intercalary group; a chromophore; an energy transfer agent; a biologically active agent; a detectable marker; a small molecule; a ribonucleic acid inhibitor; a radionucleotide; a neutron capture agent; a biotin derivative; quantum dot (s); a nanotransmitter; a radio transmitter; an abzyme, an activated complex activator, a virus, an adjuvant, an aglycan, an allergan, an angiostatin, an antihormone, an antioxidant, an aptamer, a guide RNA, a saponin, a shuttle vector, a macromolecule, a mimotope, a receptor, a reverse micelle and any combinations thereof. As shown in Figure 1, in one aspect there are methods for selecting and designing a polypeptide to be modified using the methods, compositions and techniques described herein. The new polypeptide can be designed de novo, in which it is included by way of example only, as part of high-throughput screening processes (in which case, numerous polypeptides can be designed, synthesized, characterized and / or tested) based on to the interests of the researcher. The new polypeptide can also be designed based on the structure of a known or partially characterized polypeptide. By way of example only, the superfamily of the growth hormone gene (see below) has been the subject of intense study by the scientific community; A new polypeptide can be designed based on the structure of a member or members of this genetic superfamily. The principles for selecting which amino acid (s) to substitute and / or modify are described separately in the present. The choice of which modification to employ is also described herein and can be used to satisfy the need of the experimenter or end user. Such needs may include but are not limited to, manipulating the therapeutic effectiveness of the polypeptide, improving the safety profile of the polypeptide, adjusting the pharmacokinetics, pharmacology and / or pharmacodynamics of the polypeptide, such as, by way of example only, increasing the solubility in the polypeptide. water, bioavailability, increase the half-life in the serum, increase the therapeutic half-life, modulate the immunogenicity, modulate the biological activity or prolong the circulation time. In addition, such modifications include, by way of example only, providing additional functionality to the polypeptide, incorporating a detectable tag, label or signal into the polypeptide, facilitating the isolation properties of the polypeptide and any combination of the modifications mentioned above. Also described herein are non-natural amino acids that have or can be modified to contain an aromatic amine or a heteroaromatic amine. The heteroaromatic group includes, but is not limited to, furans, pyrrole, thiophenes, pyridines, quinolines, isoquinolines, imidazole, thiazoles, pyrimidines, pyridazines, pyrazines, benzothiazoles, thiazolopyridines, oxazoles, bepzoxazoles, oxazolopyridmas, thiazolopyrimidines, oxazolopyrimidines, benzoxazmas and benzotiazmas. Included with this aspect are methods for producing, purifying, characterizing and utilizing such non-natural amino acids. In another aspect described herein are methods, strategies and techniques for incorporating at least one such non-natural amino acid to a polypeptide. Also included with this aspect are methods for producing, purifying, characterizing and using such polypeptides that contain at least one such non-natural amino acid. Also included with this aspect are compositions of and methods for producing, purifying, characterizing and using oligonucleotides (in which DNA and RNA are included) that can be used to produce, at least in part, a polypeptide that contains at least a non-natural amino acid. Also included with this aspect are compositions of and methods for producing, purifying, characterizing and using cells that can express such oligonucleotides that can be used to produce, at least in part, a polypeptide that contains at least one unnatural amino acid. Thus, polypeptides comprising at least one non-natural amino acid or non-natural amino acid modified with an aromatic amine or a heteroaromatic amine are provided and described herein. The heteroaromatic group includes, but is not limited to, furans, pirróles, thiophenes, pyridines, qumolmas, isoquinolinas, ímidazol, tlazoles, pirimidmas, piridazmas, pyrazines, benzotiazoles, thiazolopiridmas, oxazoles, benzoxazoles, oxazolopiridmas, thiazolopirimidmas, oxazolopirimidmas, benzoxazmas and benzotiazmas. In certain embodiments, polypeptides with at least one non-natural amino acid or non-natural amino acid modified with an aromatic amine or a heteroaromatic amine include at least one post-translation modification at some position in the polypeptide. In such embodiments, the non-natural amino acids modified by heterocycle may include, but are not limited to, furans, pyrroles, thiophenes, pipdmas, quamo, isomers, imidazole, tlazoles, pyrimidines, pipdazines, pyrazmas, benzothiazoles, thiazolopyridines, oxazoles, benzoxazoles , oxazolopiridmas, thiazolopyrimidmas, oxazolopipmidmas, benzoxazmas and benzotiazmas. In some modalities the post-translation modification occurs via the cellular machinery (for example, glycosylation, acetylation, acylation, lipid modification, palmitoylation, addition of palmitate, phosphorylation, glycolipid bond modification and the like), in many instances, such post-translational modifications based on cellular machinery occur at the amino acid sites that they are stably present in nature in the polypeptide, however, in certain modalities, post-translational modifications based on Cellular machinery occurs in the site (s) of non-natural amino acids in the polypeptide. In other embodiments, the post-translation modification does not use cellular machinery, but the functionality is instead provided by attaching a molecule (in which, but not limited to, a marker, a dye, a polymer, a polymer water soluble, a polyethylene glycol derivative, a photoreticulator, a cytotoxic compound, a drug, an affinity tag, a photoaffinity tag, a reactive compound, a resin, a second polypeptide or polypeptide protein or analogue, an antibody or fragment of antibody, a metal chelator, a co-factor, a fatty acid, a carbohydrate, a polynucleotide, a DNA, an RNA, an antisense polynucleotide, a saccharide, a water-soluble dendrimer, a cyclodextrin, a biomaterial, a nanoparticle a spin marker, a fluorophore, a metal-containing portion, a radioactive portion, a new functional group, a group that interacts covalently or non-covalently, n other molecules, a photoenaged portion; an excitable portion by actinic radiation; a ligand; a photoisomerizable portion; biotin; a biotin analogue; a portion that incorporates a heavy atom; a chemically cleavable group; a photo-scissors group; an elongated side chain; a carbon-bonded sugar; a redox-active agent; an amino thioacid; a toxic portion; a marked portion isotopically; a biophysical probe; a phosphorescent group; a chemiluminescent group; a dense group of electrons; a magnetic group; an intercalary group; a chromophore; an energy transfer agent; a biologically active agent; a detectable marker; a small molecule; an inhibitory ribonucleic acid; a radionucleotide; a neutron capture agent; a biotin derivative; quantum point (s); a nanotransmitter; a radio transmitter; an abzyme, an activated complex activator, a virus, an adjuvant, an aglycan, an allergana, an angiostatma, an antihormone, an anti-oxidant, a ptamero, a guide RNA, a saponin, a shuttle vector, a macromolecule, a mimotope, a receptor, a reverse micelle and any combinations thereof) comprising a second group reactive to at least one non-natural amino acid comprising a first reactive group (in which, but not limited to, an amino acid is not natural containing an aromatic amine or a heteroaromatic amine functional group) using reductive alkylation methodology. In certain embodiments, such secondary reactive groups can be carbonyl-containing groups, in which is included, but not limited to, aldehydes and ketones. In certain embodiments, the post-translational modification is made live in a eucaponic cell or in a non-eukaryotic cell. In certain modalities, the post-translation modification is done in vitro. Also included with this aspect there are found methods for producing, purifying, characterizing and using such polypeptides that contain at least one such non-natural modified amino acid post-translationally. Also included within the scope of the methods, compositions, strategies and techniques described herein are reagents capable of reacting with a non-natural amino acid (which contains either an aromatic amine group, a heteroaromatic amine group or protected forms thereof) which is part of a polypeptide to produce any of the post-translational modifications mentioned above. In general, the resulting post-translationally modified non-natural amino acid will contain at least one reductively aldehyde or ketone, which may undergo further modification reactions. Also included with this aspect are methods for producing, purifying, characterizing and using such reagents which are capable of any such post-translational modifications of such non-natural amino acid (s). In certain embodiments, the protein includes at least one post-translational modification that is made in vivo by a host cell, wherein post-translational modification is not normally done by another type of host cell. In certain embodiments, the protein includes at least one post-translational modification that is made in vivo by a cell eukaryotic, where post-translational modification is not normally done by a non-eukaryotic cell. Examples of such post-translation modifications include, but are not limited to, glycosylation, acetylation, acylation, lipid modification, palmitoylation, palmitate addition, phosphorylation, glycolipid linkage modification and the like. In one embodiment, post-translational modification comprises attachment of an oligosaccharide to an asparagine by a GlcNAc-asparagine binding (in which but not limited to, wherein the oligosaccharide comprises (GlcNAc-Man) 2 ~ Man-GlcNAc -GlcNAc and the like). In another embodiment, the post-translational modification comprises) the annexation of an oligosaccharide (in which, but not limited to, Gal-GalNAc, Gal-GlcNAc, etc.) to a serine or threonine via a GalNAc-serine linkage , a GalNAc-threonine linkage, a GlcNAc-serine linkage or a GlcNAc-threonine linkage. Examples of secretion signal sequences include, but are not limited to, a prokaryotic secretion signal sequence, a eukaryotic secretion signal sequence, a 5 'eukaryotic secretion signal sequence-optimized for bacterial expression, a new sequence of secretion signal, a pectate lyase secretion signal sequence, Omp A secretion signal sequence and a phage secretion signal sequence. Examples of secretion signal sequences include, but are not limited to, STII (procapone), Fd Gilí and M13 (phage), Bgl2 (yeast) and the signal sequence bla derived from a transposon. In certain embodiments, a protein or polypeptide may comprise a secretion sequence or localization sequence, an epitope tag, a FLAG tag, a polyhistidy tag, a GST fusion and / or the like. Also included with this aspect are methods for producing, purifying, characterizing and using such polypeptides that contain at least one such post-translation modification. In other embodiments, the non-natural glycosylated amino acid polypeptide is produced in an unglycosylated form. Such a non-glycosylated form of a non-natural glycosylated amino acid can be produced by methods that include chemical or enzymatic removal of oligosaccharide groups from a glycosylated unnatural amino acid polypeptide isolated or substantially purified or unpurified; production of the non-natural amino acid in a host that does not glycosylate such an unnatural amino acid polypeptide (such a host includes procaryotes or eucapone designed or mutated to not glycosylate such a polypeptide), the introduction of a glycosylation inhibitor into the cell culture medium in which such a non-natural amino acid polypeptide is produced by a eucapone which would normally glycosylate such a polypeptide, or a combination of any such methods. Also disclosed herein are such non-glycosylated forms of polypeptides of non-naturally occurring, normally glycosylated amino acids (usually glycosylated means a polypeptide that would be glycosylated when produced under conditions in which polypeptides that occur stably in nature are glycosylated). Of course, such non-glycosylated forms of non-naturally occurring glycosylated amino acid polypeptides can be in an unpurified form, a substantially purified form or in an isolated form. In certain embodiments, the protein includes at least one post-translational modification that is done in vitro, wherein the post-translational modification is stoichiometric, stoichiometric-like, or quasi-stoichiometric. Examples of such post-translation modifications include, but are not limited to, reductive alkylations of aromatic amine groups or heteroaromatic amine groups with carbonyl-containing reagents using a reducing agent. In certain embodiments, such post-translational modifications include, but are not limited to, reductive alkylations of aromatic amine groups or heteroaromatic amine groups with reagents containing aldehyde using a reducing agent. In certain embodiments, such post-translational modifications include, but are not limited to, reductive alkylations of aromatic amine groups or heteroaromatic amine groups with aldehyde-containing reagents using reductive reducing agent. sodium cyanoborohydride. The polypeptide containing unnatural amino acid may contain at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine or ten or more non-natural amino acids containing an aromatic amine, heteroaromatic amine or protected forms thereof. The non-natural amino acids may be the same or different, for example, there may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more different sites in the piotema comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18 , 19, 20 or more different non-natural amino acids. In certain embodiments, at least one, but less than all, of a particular amino acid present in a version that occurs stably in the nature of the protein is substituted with a non-natural amino acid. The non-natural amino acid polypeptide may also contain one or more non-natural amino acids that are non-natural amino acids containing an aromatic amine, heteroaromatic amine or protected forms thereof. The methods and compositions provided and described herein include polypeptides comprising at least one non-natural amino acid containing an aromatic amine, heteroaromatic amine or protected forms thereof. The introduction of at least one such Non-natural amino acids to a polypeptide may allow the application of conjugation chemistries that involve specific chemical reactions, in which they include, but are not limited to, one or more non-natural amino acids while not reacting with the 20 common amino acids that are Presented in a stable manner in nature. Such specific chemical reactions involve such incorporation of amino acid side chains include but are not limited to, reductive alkylation chemistry methodology appropriate for the particular functional groups or substituents present. Once incorporated, the side chains of amino acids can be modified by using known chemistry methodologies for those of ordinary skill in the art as appropriate for the particular functional groups or substituents present in the naturally encoded amino acid. The methods and compositions of non-natural amino acids described herein provide conjugates of substances having a wide variety of functional groups, substituents or moieties, with other substances in which it is included but not limited to a label; a dye; a polymer; a water soluble polymer; a polyethylene glycol derivative; a photo-reticulator; a cytotoxic compound; a drug; an affinity marker; a photoaffinity marker; a reactive compound; a resin; a second protein or polypeptide or polypeptide analogue; an antibody or antibody fragment; a metal chelator; a co-factor; a fatty acid; a carbohydrate; a polynucleotide; a DNA; an RNA; an antisense polynucleotide; a saccharin, a water-soluble dendimer, a cyclodextrm, a biomaterial; a nanoparticle; a spin marker; a fluorophore, a portion containing metal; a radioactive portion; a new functional group; a group that interacts covalently or non-covalently with other molecules; a photoenaged portion; a portion excitable by actinic radiation; a ligand; a photoisomerizable portion; biotma; a biotome analogue; a portion that incorporates a heavy scale; a chemically cleavable group; a photo-scissors group; an elongated side chain; a carbon-bonded sugar; a redox-active agent; an amino thioacid; a toxic portion; an isotopically labeled portion; a biophysical probe; a phosphorescent group; a chemiluminescent group; a dense group of electrons; a magnetic group; a michellating group; a chromophore; an energy transfer agent; a biologically active agent; a detectable marker; a small molecule; an inhibitory ribonucleic acid; a radionucleotide; a neutron capture agent; a biotome derivative; quantum point (s); a nanotransmitter; a radio transmitter; an abzyme, an activated complex activator, a virus, an adjuvant, an aglycan, an allergan, an angiostatma, an antihormone, an antioxidant, an aptamer, a guide RNA, a saponin, an shuttle vector, a macromolecule, a mimotope, a receptor, a reverse micelle and any combination thereof. In another aspect of the compositions, methods, techniques and strategies described herein are methods for studying or using any of the modified or unmodified non-natural amino acid polypeptides mentioned above. Included in this aspect, by way of example only, there are therapeutic, diagnostic, analytical, industrial, cosmetic, plant biology, environmental energy production and / or military uses that would benefit from a polypeptide that it comprises a modified or unmodified non-natural amino acid polypeptide or protein.

II. Location of non-natural amino acids in polypeptides The methods and compositions described herein include the incorporation of one or more non-natural amino acids into a polypeptide. One or more non-natural amino acids can be incorporated in one or more particular positions that do not disrupt the activity of the polypeptide. This can be obtained by making "conservative" substitutions, which include but are not limited to substituting hydrophobic amino acids with natural or unnatural hydrophobic amino acids, amino acids bulky by non-natural or natural voluminous amino acids, hydrophilic amino acids by non-natural or natural hydrophilic amino acids and / or inserting the unnatural amino acid to a site that is not required by activity. A variety of biochemical and structural processing may be employed to select the desired sites for substitution by an unnatural amino acid within the polypeptide. Any position of the polypeptide chain is suitable for selection to incorporate an unnatural amino acid and the selection may be based on rational design or by random selection for any or no particular desired purpose. The selection of desired sites may be to produce an unnatural amino acid polypeptide (which may be further modified or remain unmodified) having any desired property or activity, including, but not limited to agonists, super-agonists, agonists inverses, antagonists, receptor binding modulators, receptor activity modulators, link modulators to link partners, link partner activity modulators, link partner conformation modulators, dimer or multimer formation, no change to the activity or property compared to the natural molecule or manipulate any physical or chemical property of the polypeptide such as solubility, aggregation or stability. For example, sites in the polypeptide required for biological activity of a polypeptide can be identified using point mutation analysis, alanine scanning or homology scanning methods known in the art. Residues other than those identified as critical for biological activity by alanine scanning mutagenesis or homolog may be good candidates for substitution with unnatural in amino acid depending on the desired activity sought for the polypeptide. Alternatively, sites identified as critical to biological activity may also be good candidates for substitution with an unnatural amino acid, again depending on the desired activity sought for the polypeptide. Another alternative would be to simply make serial substitutions at each position on the polypeptide chain with an unnatural amino acid and observe the effect on polypeptide activities. Any means, technique or method for selecting a position for substitution with an unnatural amino acid to any polypeptide is suitable for use in the methods, techniques and compositions described herein. The structure and activity of mutants that occur stably in the nature of a polypeptide containing cancellations can also be determined to determine regions of the proteins that are likely to be tolerant for substitution with a non-natural amino acid. A Once the residues that are likely to be intolerant of substitution with non-natural amino acids have been eliminated, the impact of substitutions proposed in each of the remaining functions can be examined from the three-dimensional structure of the relevant polypeptide and any ligands or proteins associated links. X-ray crystallographic structures and NMR structures of many polypeptides are available in the Protein Data Bank (PDB, www.rcsb.org), an updated database containing three-dimensional structural data of large protein molecules and nucleic acids. In addition, models can be made that investigate the secondary and tertiary structure of the polypeptide, if three-dimensional structural data are not available. Thus, the identity of amino acid positions that can be substituted with non-natural amino acids can be easily obtained. Exemplary incorporation sites of a non-natural amino acid include, but are not limited to, those that are excluded from potential receptor binding regions or regions for binding to binding proteins or ligands, may be fully or partially exposed to solvent, have minimal or no hydrogen bonding interaction with a nearby residue, they may be minimally exposed to nearby reactive residues and may be in regions that are highly flexible as predicted by the crystal structure of a particular polypeptide with its associated receptor, ligand or binding protein. A wide variety of non-natural amino acids can be substituted for or incorporated into a given function in a polypeptide. In general, a particular non-natural amino acid is selected for incorporation based on an examination of the three-dimensional crystal structure of a polypeptide with its associated ligand, receptor and / or binding protein, a preference for conservative substitutions (ie, non-amino acid natural, aryl-based, such as p-acetylphenylalanine or O-propargyltyrosine replaced by Phe, Tyr or Trp) and the specific conjugation chemistry that one wishes to introduce to the polypeptide protein. In one embodiment, the method further includes incorporating the non-natural amino acid into the protein, wherein the non-natural amino acid comprises a first reactive group and contacting the protein with a molecule (in which, but not limited to, a marker is included; a dye, a polymer, a water soluble polymer, a polyethylene glycol derivative, a photocrosslinker, a cytotoxic compound, a drug, an affinity tag, a photoaffinity tag, a reactive compound, a resin, a second protein or polypeptide or polypeptide analogue, an antibody or antibody fragment, a metal burner, a co-factor, a fatty acid, a carbohydrate, a polynucleotide, a DNA, an RNA, a antisense polynucleotide; a saccharide, a water-soluble dendrimer, a cyclodextrin, a biomaterial; a nanoparticle; a spin marker; a fluorophore, a portion containing metal; a radioactive portion; a new functional group; a group that interacts covalently or non-covalently with other molecules; a photo-charged portion; an excitable portion by actinic radiation; a ligand; a photoisomerizable portion; biorin; a biotin analogue; a portion that incorporates a heavy atom; a chemically cleavable group; a photosecisible group, an elongated side chain: a carbon-bonded sugar; a redox-active agent; an amino thioacid; a toxic portion; an isotopically labeled portion; a biophysical probe; a phosphorescent group; a chemiluminescent group; a dense group of electrons; a magnetic group; an intercalary group; a chromophore; an energy transfer agent; a biologically active agent; a detectable marker; a small molecule; an inhibitory ribonucleic acid; a radionucleotide; a neutron capture agent; a biotin derivative; quantum dot (s); a nanotransmitter; a radio transmitter; an abyss, an activated complex activator, a virus, an adjuvant, an aglycan, an allergan, an angiostatin, an antihormone, an antioxidant, an aptamer, a guide RNA, a saponin, a shuttle vector, a macromolecule, an mimotope, a receptor, a reverse miscella and any combination thereof) that it comprises a second reactive group. In certain embodiments, the first reactive group is an amine moiety on an aromatic amine and the second reactive group is an aldehyde, wherein the amine group is reductively alkylated on contact with the aldehyde in the presence of a reducing agent, such as sodium cyanoborohydride. . In other embodiments, the first reactive group is an amine moiety on a heteroaromatic amine and the second reactive group is an aldehyde, wherein the amine group is reductively alkylated on contact with -X-aldehyde in the presence of a reducing agent, such as cyanoborohydride. sodium. In some cases, the substitution (s) or incorporation (s) of non-natural amino acid will be (are) combined with other additions, substitutions or cancellations within the polypeptide to affect other biological traits. In some cases, other additions, substitutions or deletions may increase the stability (in which they include but not limited to, resistance to proteolytic degradation) of the polypeptide or increase the agility of the polypeptide by its appropriate receptor, ligand and / or protein. link. In some cases, the other additions, substitutions or cancellations may increase the solubility (in which, but not limited to, when expressed in E. coli or other host cells) of the polypeptide. In some modalities, sites are selected for substitution with an amino acid coded naturally or unnaturally in addition to other sites for incorporation of a non-natural amino acid for the purpose of increasing the amount of polypeptide following the expression of E. coli or other recombinant host cells. In some embodiments, the polypeptides comprise another addition, substitution or cancelation that modulates activity by the associated ligand, binding protein and / or receptor, modulates (in which the dimerization of the receptor is included but not limited to, increases or decreases) , stabilizes receptor dimers, modulates the circulating half-life, modulates the release to bioavailability, facilitates purification or improves or alters a particular route of administration. Simultaneously, the polypeptide may comprise chemical or enzymatic cleavage sequences, protease cleavage sequences, reactive groups, antibody binding domains (in which they include but not limited to FLAG or poly-His) or other affinity-based sequences (including, but not limited to, FLAG, poly-His, GST, etc.) or linked molecules (including but not limited to biotin) that improve detection (including, but not limited to) GFP), transport of purification through tissues or cell membranes, release or activation of prodrugs, reduction in size or other features of the polypeptide.

IV. Family of growth hormone supergen as an example The methods, compositions, strategies and techniques described herein are not limited to a particular type, class or family of the polypeptide or protein. Of course, virtually any polypeptides can be designed or modified to include at least one modified or unmodified non-natural amino acid described herein. By way of example only, the polypeptide can be homologous to a therapeutic protein selected from the group consisting of: alpha-1 hostin, angiostatin, antihemolytic factor, antibody, antibody fragment, apolipoprotein, apoprotein, atrial natriuretic factor, atrial natriuretic polypeptide , atrial peptide, chemokine CXC, T39765, NAP-2, ENA-78, calcitonin, c-kit ligand, cytokine, chemokine C, monocyte chemoattractant protein-1, monocyte chemoattractant protein-2, monocyte chemoattractant protein-3 , monocyte inflammatory protein-1 alpha, monocyte inflammatory beta-1 protein, 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, peptide-78 epithelial neutrophil activator, MIP-16, MCP-1, epidermal growth factor (EGF), peptide gone epithelial neutrophil activator, erythropoietin (EPO), exfoliating toxin, Factor IX, Factor VII, Factor VIII, Factor X, fibroblast growth factor (FGF), fibrinogen, fibronectin, four-helix bundle protein, G-CSF, glp-1, GM-CSF, glucocerebrosidase, gonadotropin, growth factor, receptor of growth factor, 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), IFN-alpha, IFN-beta, IFM -gamma, any interferon-like molecule or a member of the IFN family, interleukin (IL), IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, keratinocyte growth factor (KGF), lactoferrin, leukemia inhibitory factor, luciferase, neuroturin, neutrophilic inhibiting factor (NIF), oncostatin M , osteogenic protein, oncogene product, paracytoline, parathyroid hormone, PD-ECSF, PDGF, peptide hormone, pleiotropin, protein A, protein G, prh, pyrogenic exotoxin A, pyrogenic exotoxin B, pyrogenic exotoxin C, pyy, relaxin, renin, SCF, small biospecific protein, soluble complement receptor I, soluble I-CAM 1, soluble interleukin receptor, soluble TNF receptor, somatomedin, somatostatin, somatotropin, streptokinase, superantigens, Staphylococcal enterotoxin, SEA, DEB, SEC1, SEC2, SEC3, DES, SEE, steroid hormone receptor, superoxide dismutase, toxic shock syndrome toxin, alpha 1 limosis, tissue plasminogen activator, tumor growth factor (TGF), tumor necrosis factor, tumor necrosis factor alpha, tumor necrosis beta factor, receptor of tumor necrosis factor (TNFR), VLA-4 protein, VCAM-1 protein, vascular endothelial growth factor (VEGF), urokinase, mos, ras, raf, met, p53, tat, fos, myc, un, myb , laughing, estrogen receptor, progesterone receptor, testosterone receptor, aldosterone receptor, LDL receptor and corticosterone. Thus, the following description of the growth hormone (GH) supergene family is provided for illustrative purposes and by way of example only and not as a limit on the scope of the methods, compositions, strategies and techniques described herein. In addition, the reference to GH polypeptides in this application is intended to use the generic term as an example of any member of the GH supergene family. Thus, it will be understood that modifications and chemistries described herein with reference to GH or protein polypeptides can equally be applied to any member of the GH supergene family, in which those specifically listed herein are included. The following proteins include those encoded by genes of the hormone supergene family of Growth (GH) (Bazan, F., Immunology Today 11: 350-354 (1990), Bazan, JF Science 257: 410-413 (1992), Mott, HR and Campbell, ID, Curren t Opinion m Structural Biology 5: 114-121 (1995); Silvenno in, O. and Ihle, JN, SIGNALING BY THE HEMATOPOIETIC CYTOKINE RECEPTORS (1996)): growth hormone, prolactma, placental lactogen, eptropoieme (EPO), thrombopoietic a (TPO), heterologous ? na-2 (IL-2), IL-3, OL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, (p35 subunit), IL-13, IL-15, oncostat a M, ciliary neurotropic factor, leukemia inhibitor, alpha interferon, internal beta, epsteron interferon, inferred gamma, inferred omega, inferred tau, iactor granulocyte colony stimulator (G-CSF), macro-granulocyte colony stimulator (GM-CDF), macrophage colony stimulator (M-CSF) and card? otropma-1 (CT-1), (" the supergen family GH ") It is anticipated that additional members of this genetic family will be identified in the future through cloning and genetic sequencing. Members of the GH supergene family have similar secondary and tertiary structures despite the fact that they have in general a limited amino acid or DNA sequence identity. The shared structural elements allow new members of the genetic family to be easily identified and the methods and compositions of non-natural amino acids described herein similarly applied.

Structures of a diversity of cytokines, in which G-CSF is included (Zink et al., FEBS Lett 314: 435 (1992), Zink et al., Biochemistry 33: 8453 (1994), Hill et al., Proc. Nati, Acad. Sci., USA 90: 5167 (1993)), GM-CSF (Diederichs, K., et al., Science 154: 1779-1782 (1991), Walter et al., J. Mol. Biol. 224: 1075-1085 (1992)), IL-2 (Bazan, JF and McKay, DB Science 257: 410-413 (1992)), IL-4 (Redfield et al., Biochemis try 30: 11029-11035 ( 1991), Powers et al., Science 256: 1673-1677 (1992)) and IL-5 (Milburn et al., Na ture 363: 172-1 6 (1993) 1 have been determined by X-ray diffraction and NMR studies and show a surprising conservation with the GH structure, despite a lack of significant primary sequence homology.It is considered that IFN is a member of this family based on modeling and other studies (Lee et al., J Inferred Cytokine Res. 15: 341 (1995); Murgolo et al., Proteins 17: 62 (1993); Radhakrishnan et al., Structure 4: 1453 (1996); Klaus et al., J. Mol. Biol. 274: 661 (1997)). A large number of additional cytokines and growth factors including ciliary neurotrophic factor (CNTF), leukemia inhibitory factor (LIF), thrombopoietin (TPO), oncostatin M, macrophage colony stimulating factor (M-CSF) , IL-3, IL-6, IL-7, IL-9, IL-12, IL-13, IL-15, and granulocyte colony factor (G-CSF), as well as IFNs such as alpha, beta , omega, tau, epsilon and interferon gamma belong to this family (reviewed in Mott and Caml, Curren t Opinion in Structural Biology 5: 114-121 (1995); Silvennoinen and Ihle (1996) SIGNALING BY THE HEMATOPOIETIC CYTOKINE RECEPTORS). All previous cytokines and growth factors are considered to comprise a large genetic family. In addition to sharing similar secondary and tertiary structures, members of this line share the property that they must oligomerize cell surface receptors to activate intracellular signaling pathways. Some members of the GH family, which include but are not limited to: GH and EPO, bind to a single type of receptor and cause it to form homodimers. Other members of the family, including but not limited to IL-2, IL-4 and IL-6, bind to more than one type of receptor and cause the receptors to form heterodimers or higher order aggregates (Davis et al., (1993) Science 260: 1805-1808; Paonessa et al., 1995) EMBO J. 14: 1942-1951; Mott and Caml, Curren t Opinion in Structure Biology 5: 114-121 (1995)). Mutagenesis studies have shown that, as GH, these other cytokines and growth factors contain multiple receptor binding sites, commonly two and bind to their cognate receptors sequentially (Mott and Caml, Curren t Opinion in Struct ura l Biology 5 : 114-121 (1995); Matthews et al., (1996) Proc. Na ti, Acad. Sci., USA 93: 9471-9476). As GH, the primary receptor binding sites for these and other members of the family they occur mainly in the four alpha helices and the A-B loop. The specific amino acids in the helical bundles that participate in the receptor binding differ among the members of the family. The majority of cell surface receptors that interact with members of the GH supergene family are structurally related and comprise a second large multigenetic family. See, for example, U.S. Patent No. 6,608,183, which is incorporated herein by reference in its entirety. A general conclusion reached from mutational studies of several members of the GH supergene family is that the loops that bind alpha helices tend not to be involved in the receptor binding in general. In general, the short B-C loop seems to be non-essential for the receptor binding in most, if not all, members of the family. For this reason, the B-C loop can be constituted with non-natural amino acids as described herein in members of the GH supergene family. Buvle A-B, the C-D loop (and D-E loop of interferon / IL-10 like members of the GH superfamily) can also be substituted with a non-natural amino acid. Amino acids close to helix A and distal to the final helix also tend not to be involved in the linkage of the reductant and may also be sites for introducing non-natural amino acids. In some embodiments, an unnatural amino acid is substituted in any function within a loop structure in which are included but not limited to the first 1, 2, 3, 4, 5, 6, 7 or more amino acids of loop AB, BC, CD or DE. In some embodiments, an unnatural amino acid is substituted within the last 1, 2, 3, 4, 5, 6, 7 or more amino acids of loop A-B, B-C, C-D or D-E. Certain members of the GH family including but not limited to EPO, IL-2, IL-3, IL-4, IL-6, GM-CSF, TPO, IL-10, 11-12, p35, IL-13, IL-15 and terferon beta contain N-linked and / or O-linked sugars. The glycosylation sites in the proteins occur almost exclusively in the loop regions and not in the alpha helical bundles. Because the loop regions are generally not involved in the receptor binding and because they are sites for the covalent attachment of sugar groups, they can be useful sites for introducing non-natural amino acid substitutions to the proteins. The amino acids comprising the N- and O-linked glycosylation sites in the proteins may be sites for non-natural amino acid substitutions because these amino acids are exposed on the surface. Accordingly, the natural protein can tolerate bulky sugar groups attached to the proteins at these sites and at the glycosylation sites they have to be located away from the receptor binding sites.

Additional members of the genetic family of GH are likely to be discovered in the future. New members of the GH supergene family can be identified by computer-aided secondary and tertiary structure analysis of predicted protein sequences and by screening techniques designed to identify mixtures that bind to a particular target. Members of the GH supergene family commonly possess four or five antipathetic helices linked by non-helical amino acids (the loop regions). The proteins may contain a hydrophobic signal sequence and its N terminus to promote cell secretion. Such members discovered later in the GH supergene family are also included within the methods and compositions described herein.

V. Unnatural Amino Acids A very wide variety of non-natural amino acids are suitable for use in the methods and compositions described herein, as long as they have at least one of the following four properties: (1) at least one functional group on the side chain of the non-natural amino acid having at least one characteristic and / or activity and / or reactivity orthogonal to the chemical reactivity of the 20 common genetically purified amino acids 8this is, alamine, argmam, asparagma, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine) or at least orthogonal to the chemical reactivity of the amino acids that occur stably in the nature present in the polypeptide and includes the non-natural amino acid; (2) the non-natural amino acids produced are substantially chemically inert toward the 20 common genetically encoded amino acids; (3) the non-natural amino acid can be stably incorporated into a polypeptide, preferably with the stability conditioned by the amino acids that occur stably in nature or under typical physiological conditions and further preferably such incorporation can occur via a system in vi and (4) the non-natural amino acid includes an aromatic amine or heteroaromatic amine or a functional group that can be transformed to an aromatic amine or heteroaromatic amine, by reacting with a reagent, preferably under conditions that do not destroy the biological properties of the polypeptide which includes the non-natural amino acid (unless, of course, such destruction of biological properties is the purpose of the modification / transformation) or wherein the transformation may occur under aqueous conditions at a pH of between about 4 and about 10 or where the reactive site in the non-natural amino acid is a s nucleophilic nucleus.

Illustrative non-limiting examples of amino acids that satisfy those four properties for non-natural amino acids that can be used with the compositions and methods described herein are presented in all Figures and the text herein. Any number of non-natural amino acids can be introduced into the polypeptide. The non-natural amino acids can also include an aromatic amine or protected or masked heteroaromatic amine, whereby, after deprotection or unmasking, the aromatic amine or protected or masked heteroaromatic amine is transformed to an aromatic amine or heteroaromatic amine. Unnatural amino acids of interest that may be suitable for use in the methods and compositions described herein include but are not limited to amino acids comprising a photoactivatable regulator, spin-labeled amino acids, fluorescent amino acids, metal-binding amino acids, amino acids containing metal, radioactive amino acids, amino acids with new functional groups, amino acids that interact covalently or non-covalently with other molecules, photoengineered and / or photoisomerizable amino acids, amino acids comprising biotin or a biotin analog, glycosylated amino acids such as a serine substituted with sugar, other Modified carbohydrate amino acids, amino acids that contain keto, amino acids that they comprise polyethylene glycol or polyether, amino acids substituted by heavy atoms, chemically cleavable and / or photocleavable amino acids, amino acids with elongated side chains in comparison to natural amino acids, in which but not limited to polyethers or long chain hydrocarbons are included, but are included but not limited to, greater than about 5 or greater than about 10 carbon atoms, amino acids containing carbon-linked sugar, redox-active amino acids, amino acid-containing amino acid and amino acids comprising one or more toxic moieties. In some embodiments, the non-natural amino acids comprise a saccharide moiety. Examples of such amino acids include-acetyl-L-glucosaminyl-L-serine, N-acetyl-L-galactosaminyl-L-serine,? F-acetyl-L-glucosaminyl-L-threonine,? J-acetyl-L-glucosamin- L-asparagine and O-manosaminyl-L-serine. Examples of such amino acids also include examples wherein the N- or 0- linkage that occurs stably in nature between the amino acid and the saccharide is replaced by a covalent bond not commonly found in nature - in which they are included but not limited to, an amine bond and the like. Examples of such amino acids also include saccharides that are not commonly found in proteins that occur stably in nature, such as 2-deoxy-glucose, 2-deoxygalactose and the like. The chemical portions via non-natural amino acids that can be incorporated into proteins offer a variety of advantages and manipulations of the protein. For example, an unnatural amino acid of heavy atom may be useful for X-ray structure databases. The site-specific introduction of heavy atoms using non-natural amino acids also provides that selectivity and flexibility to choose positions for heavy atoms. Non-natural photoreactive amino acids (including but not limited to amino acids with benzophenone and arylazides (in which they are included but limited to phenylazide) side chains), for example, allow in vitro and efficient in vitro photorecovery of protein. Examples of non-natural photoreactive amino acids include but are not limited to p-azido-phenylalanine and p-benzoyl-phenylalanine. The protein with non-natural photoreactive amino acids can then be crosslinked at will by excitation of the photoreactive group that provides temporal control. In one example, the methyl group of an unnatural amino acid can be substituted with an isotopically-labeled methyl group, which includes but is not limited to a methyl group, such as a probe of local structure and dynamics, in which are included but not limited to, with the use of nuclear magnetic resonance and vibrational spectroscopy.

A. Structure and synthesis of non-natural amino acids: Alkylated aromatic amine groups Non-natural amino acids with nucleophilic reactive groups, such as by way of example only, an aromatic amine group (in which secondary and tertiary amino groups are included), a Masked aromatic amine group (which can be easily converted to an aromatic amine group) or a protected aromatic amine group (which has similar reactivity to an aromatic amine group after deprotection) allow a variety of reasons for linking molecules via various reactions, in those which include but are not limited to reductive alkylation reactions with aldehyde-containing reagents. Such non-natural amino acids containing aromatic amine include amino acids having the structure of formula (A): • K,,. \ ?? Xs -B 'and 8"« l where . "- V ... X XX is selected from the group consisting of a monocyclic aryl ring, a bicyclic aryl ring, a multicyclic aryl ring, a heteroaryl ring monocyclic, a bicyclic heteroaryl ring and a multicyclic heteroaryl ring; A is independently CRa or N; B is independently CRa, N, 0 or S; each Ra is independently selected from the group consisting of H, halogen, alkyl, - (NR ') 2, -C (0) R', C (0) N (R ') 2, -OR and -S (0) 2R ', where k is 1, 2 or 3 and n is 0, 1, 2, 3, 4, 5, or 6; Ri is H, an amino protecting group, resin, amino acid, polypeptide or poly nuci eófido and R2 is OH, a protective group ester, resin, amino acid, polypeptide or polynucleotide; each R3 and R4 is independently H, halogen, lower alkyl or substituted lower alkyl or R and R4 or two R3 groups optionally form a cycloalkyl or heterocycloalkyl; M is H or -CH2R5; or the portion M-N-C (R5) can form a ring structure of 4 to 7 members; R5 is alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, substituted alkoxy, alkylalkoxy, substituted alkylalkoxy, polyalkylene oxide, substituted polyalkylene oxide, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocycle , substituted heterocycle, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl, , -C (0) R ", -C (0) OR", -C (0) N (R) 2, -C (O) NHCH (R ") 2, - (alkylene or alkylene) -N (R ") 2" - (alkenylene or substituted alkenylene) -N (R'J 2, - (alkylene or substituted alkylene) - (aryl or substituted aryl), - (alkenylene or substituted alkenylene) - (aryl or substituted aryl), - (alkylene or substituted alkylene) -ON (R ") 2, - (alkylene or substituted alkylene) -C (0) SR", - (alkylene or substituted alkylene) -SS- (aryl or substituted aryl), wherein each R "is independently hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocycle, substituted heterocycle, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl or -C (0) 0R '; or R5 is LX, wherein X is selected from the group consisting of a marker, a dye, a' polymer ', a water soluble polymer, a polyethylene glycol derivative, a photocrosslinker, a cytotoxic compound, a drug; a m affinity frame; a photoaffinity marker; a reactive compound; a resin; a second protein or polypeptide or polypeptide analogue; an antibody or antibody fragment; a metal chelator; a co-factor; a fatty acid; a carbohydrate; a polynucleotide; a DNA; an RNA; an antisense polynucleotide; a saccharide, a water-soluble dendrimer, a cyclodextrin, a biomaterial; a nanoparticle; a spin marker; a fluorophore, a portion that contains metal; a radioactive portion; a new functional group; a group that interacts covalently or non-covalently with other molecules; a photoenaged portion; an excitable portion by actinic radiation; a ligand; a photoisomerizable portion; biotin; a biotin analogue; a portion that incorporates a heavy atom; a group chemically cleaved it; a photocleavable group; an elongated side chain; a carbon-bonded sugar; a redox-active agent; an amino thioacid; a toxic portion; an isotopically labeled portion; a biophysical probe; a phosphorescent group; a chemiluminescent group; a dense group of electrons; a magnetic group; an intercalary group; a chromophore; an energy transfer agent; a biologically active agent; a detectable marker; a small molecule; an inhibitory ribonucleic acid; a radionucleotide; an electron capture agent; a biotin derivative; quantum dot (s); a nanotransmitter; a radio transmitter; an abzyme, an activated complex activator, a virus, an adjuvant, an aglycan, an allergen, an angiostatin, an antihormone, an antioxidant, an aptamer, a guide RNA, a saponin, a shuttle vector, a macromolecule, an mimotope, a receptor, a reverse micelle and any combination thereof and L is optional and when present is a linker selected from the group consisting of alkylene, substituted alkylene, alkenylene, substituted alkenylene, -0-, -0- (alkylene) or substituted alkylene), - (alkylene or substituted alkylene) -0-, -C (0) -, -C (0) - (alkylene or substituted alkylene), (alkylene or substituted alkylene) -C (0) -, -C (0) N (R ') -, -C (0) N (R') - (alkylene or substituted alkylene) -, - (alkylene or substituted alkylene) -0C (0) N (R ') -, -N (R ') C (0) -, -N (R') C (0) - (alkylene or substituted alkylene) -, (alkylene or substituted alkylene) -NR 'C (0) -, -S-, -S - (alkylene or substituted alkylene) -, -S (0) k- wherein k is 1, 2 or 3, -S (0) k ~ (alkylene or substituted alkylene) -, -C (S) -, -C (S) - (alkylene or substituted alkylene) -, CSN (R ') -, CSN (R') - (alkylene or substituted alkylene) -, -N (R ') CO- (alkylene or substituted alkylene) -, - N (R ') C (0) 0-, - (alkylene or substituted alkylene) -0-N = CR' -, - (alkylene or substituted alkylene) -C (0) NR '- (alkylene or substituted alkylene) - , (alkylene or substituted alkylene) -S (0) - (alkylene or substituted alkylene) -S-, - (alkylene or substituted alkylene) -SS-, S (0) kN (R ') -, N (R ') C (O) N (R') -, - N (R ') C (S) N (R') -, - N (R ') S (0) kN (R') -, -N (R ') -N =, -C (R') = N-, -C (R ') = NN (R') -, -C (R ') = NN =, -C (R') 2-N = N and -C (R ') 2-N (R') -N (R ') -; or two R5 groups optionally form a cycloalkyl or heterocycloalkyl; or R5 and any Ra optionally form a cycloalkyl or a heterocycloalkyl and each R 'is independently H, alkyl or substituted alkyl. Such non-natural amino acids may also be in the form of a salt or can be incorporated into a non-naturally occurring amino acid polypeptide, polymer, polysaccharide or reductively alkylated polynucleotide and optionally. ? > .- The structure as it is presented in all the examples in the present one does not present the relative orientations of "A", "B", "NH-M" and "Ra"; rather, these four aspects of this structure can be oriented in any chemically firm manner (along with other aspects of this structure) as illustrated by the examples herein. Unnatural amino acids containing a portion of aromatic amine having the structure of Formula (A) include the non-natural amino acids having the structure of Formula (I), Formula (II), Formula (III), Formula (IV) and Formula (V): wherein each A1 is independently selected from t R ,, N \ 1 or C-Nlj and more than two A 'can be < - H with the condition that A1 is selected from < '< , or \ Such non-natural amino acids may be in the form of a salt, or they may be incorporated into a non-natural amino acid polypeptide, polymer, polysaccharide or a polynucleotide and optionally reductively alkylated. Non-limiting examples of non-natural amino acids containing an aromatic amine moiety having the structure of Formula (A) include the non-natural amino acids having the structure of Formula (VI) and Formula (VII), K. "7 1, f :? "• '" "H¿ 1 i, wherein G is an amine protecting group, in which are included but not limited to.

Such non-natural amino acids may be in the form of a salt or may be incorporated into a polypeptide of an unnatural amino acid, polymer, polysaccharide or a polynucleotide and optionally reductively alkylated. Nonnatural amino acids that contain a portion of aromatic amine have the following structures: wherein each Ra is independently selected from the group consisting of H, halogen, alkyl, -N02, -CN, substituted alkyl, -N (R ') 2, -C (0) kR', -C (0) N ( R ') 2, -OR' and -S (0) kR ', wherein k is 1, 2 or 3; M is H or -CH2R5; or the portion M-N-C (R5) can form a ring structure of 4 to 7 members; Ri is H, an amino protecting group, resin, amino acid, polypeptide or polynucleotide; R2 is OH, a protecting group or ester, resin, amino acid, polypeptide or polynucleotide; R5 is alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, substituted alkoxy, alkylalkoxy, substituted alkylalkoxy, polyalkylene oxide, substituted polyalkylene oxide, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocycle , substituted heterocycle, alkapyl, substituted alkaryl, aralkyl, substituted aralkyl,, -C (0) R ", -C (0) OR", -C (0) N (R ") 2, -C (0) NHCH (R'J 2 , - (alkylene or alkylene) -N (R'J 2 »- (alkenylene or substituted alkenelene) -N (R") 2, - (alkylene or substituted alkylene) - (aryl or substituted aryl), - (alkenylene or alkenylene substituted) - (aplo or substituted aplo), - (alkylene or substituted alkylene) -ON (R ") 2, - (alkylene or substituted alkylene) -C (0) SR", - (alkylene or substituted alkylene) -SS- (substituted aryl or aryl), wherein each R "is independently hydrogen, alkyl, substituted alkyl, alkenylc, substituted alkenyl, alkoxy, substituted alkoxy, aplo, substituted aryl, heteroaryl, substituted heteroaryl, heterocycle, substituted heterocycle, alkaryl, alkaryl substituted, aralkyl, substituted aralkyl or -C (0) 0R '; or R5 is LX, wherein X is selected from the group consisting of a label; a dye; a polymer; "a water-soluble polymer; a polyethylene glycol derivative; a fo torreticulator; a cytotoxic compound; a drug; an affinity marker; a photo-quality marker; a reactive compound; a resin; a second protein or polypeptide or polypeptide analogue; an antibody or antibody fragment; a metal chelator; a co-factor; a fatty acid; a carbohydrate; a polynucleotide; a DNA; an RNA; an antisense polynucleotide; a saccharin, a water-soluble dendrimer, a cyclodextrm, a biomaterial; a nanoparticle; a spin marker; a fluorophore, a portion containing metal; a radioactive portion; a new functional group; a group that interacts covalently or non-covalently with other molecules; a photoenaged portion; a portion excitable by actinic radiation; a ligand; a photoisomerizable portion; biotin; a biotome analogue; a portion that incorporates a heavy atom; a group chemically cleaved it; a photo-compact group; an elongated side chain; a carbon-bonded sugar; a redox-active agent; an amino thioacid; a toxic pomón; an isotopically labeled portion; a biophysical probe; a phosphorescent group; a chemiluminescent group; a dense group of electrons; a magnetic group; an intercalary group; a chromophore; an energy transfer agent; a biologically active agent; a detectable marker; a small molecule; an inhibitory ribonucleic acid; a radionucleotide; an electron capture agent; a biotin derivative; quantum dot (s); a nanotransmitter; a radio transmitter; an abzyme, an activated complex activator, a virus, an adjuvant, an aglycan, an allergen, an angiostatma, an antihormone, an antioxidant, an aptamer, a guide RNA, a saponin, a shuttle vector, a macromolecule, an mimotope, a receptor, a reverse micelle and any combination thereof and L is optional and when present is a linker selected from the group consisting of alkylene, substituted alkylene, alkenylene, substituted alkenylene, -0-, -0- (alkylene or substituted alkylene), - (alkylene or substituted alkylene) -0-, -C (0) -, -C (0) - (alkylene or substituted alkylene), (alkylene or substituted alkylene) -C (0) -, -C (0) N (R ') -, -C (0) N (R') - (alkylene or substituted alkylene) -, - (alkylene or substituted alkylene) ) -0C (0) N (R ') -, -N (R') C (0) -, -N (R ') C (0) - (alkylene or substituted alkylene) -, (alkylene or substituted alkylene) -NR 'C (O) -, -S-, -S- (alkylene or substituted alkylene) -, -S (0) k- where k is 1, 2 or 3, -S (0) k- (alkylene) or substituted alkylene) -, -C (S) -, -C (S - (alkyl ene or alkylene suctitide) -, CSN (R ') -, CSN (R') - (alkylene or substituted alkylene) -, -N (R ') CO- (alkylene or substituted alkylene) -, -N (R') C (0) 0-, - (alkylene or substituted alkylene) -0-N = CR '-, - (alkylene or alkylene substituted) -C (0) NR '- (alkylene or substituted alkylene) -, (alkylene or substituted alkylene) -S (0) k- (alkylene or substituted alkylene) -S-, - (alkylene or alkylene) ituido) -SS-, S (0) kN (R ') -, -N (R') C (0) N (R ') -, -N (R') C (S) N (R ') - , -N (R ') S (0) kN (R') -, -N (R ') - N =, -C (R') = N-, -C (R ') = NN (R') -, -C (R ') = NN =, -C (R') 2-N = N and -C (R ') 2-N (R') -N (R ') -; or R5 and any Ra optionally form a cycloalkyl or heterocycloalkyl and each R 'is independently H, alkyl or substituted alkyl. Such non-natural amino acids can also be in the form of a salt or can be incorporated into a non-natural amino acid polypeptide, polymer, polysaccharide or a polynucleotide. Other non-limiting examples of non-natural amino acids containing an aromatic amine moiety having the structure of Formula (A) are shown in Figures 2-10. Such non-natural amino acids can also be in the form of a salt or can be incorporated into a non-natural amino acid polypeptide, polymer, polysaccharide or a polynucleotide. Non-limiting examples of synthesis of compounds of Formula (A) are shown in Figures 11-13. Such examples show the synthesis of non-natural amino acids containing aromatic amine portions, wherein the amine moiety created is a primary or secondary amine. Such non-natural amino acids may be in the form of a salt or they may be incorporated into a non-naturally occurring amino acid polypeptide, polymer, polysaccharide or a polynucleotide and optionally reductively alkylated. A non-limiting formation of an unnatural amino acid containing aromatic amine having the structure of Formula (A) is shown in Figure 14 wherein by way of example only, non-natural amino acids of Formula (A) can be formed by reducing amine portions protected or masked on the aromatic portion of a non-natural amino acid. Such protected or masked amine portions include but are not limited to substituents imine, hydrazines, nitro or azide. Agents used to reduce such protected or masked portions of amine include but are not limited to TCEP, Na2S, Na2S204, LiAlH4, NaBH4 or NabCNH3. Such non-natural amino acids containing the amine portions protected or masked on an aromatic portion have the structure of Formula (B), where: í r. - ^ / 'v -. ^ V ^ ^ is selected from the group consisting of a monocyclic aryl ring, a bicyclic aryl ring, a multicyclic aryl ring, a monocyclic heteroaryl ring, a bicyclic heteroaryl ring and a multicyclic heteroaryl ring; A is independently CRa or N; B is independently CRa, N, O or S; each Ra is independently selected from the group consisting of H, halogen, alkyl, -NO2, -CN, substituted alkyl, -N (R ') 2, -C (0) kR', -C (0) N (R ' ) 2, -OR 'and -S (0) kR', wherein k is 1, 2 or 3 and n is 0, 1, 2, 3, 4, 5, or 6; Ri is H, an amino protecting group, resin, amino acid, polypeptide or polynucleotide and R2 is OH, an ester protecting group, resin, amino acid, polypeptide or polynucleotide; each R3 and R is independently H, halogen, lower alkyl or substituted lower alkyl or R3 and R4 or two R3 groups optionally form a cycloalkyl or a heterocycloalkyl; Y is -NH-NH2, -NH-NHR ', -CR' = NR ', -N02 or -N3 and each R' is independently H, alkyl or substituted alkyl. Such protected non-natural amino acids may be in the form of a salt or they may be incorporated into an unnatural amino acid polypeptide, polymer, polysaccharide or polynucleotide. , »____ ,, • - ^ T. X / The structure \" ° (as presented in all examples here) does not present the relative orientations of "A", "B", "Y" and "Ra" Rather, these four aspects of this structure can be oriented in any chemically firm manner (together with other aspects of this structure), as illustrated by the examples herein. The non-natural amino acids that contain a portion of aromatic amine that has the structure of Formula (B) includes the non-natural amino acids having the structure of Formula (VIII), Formula (IX), Formula (X) and Formula: Such protected unnatural amino acids may be in the form of a =? Or they may be incorporated into an unnatural amino acid polypeptide, polymer, polysaccharide or polynucleotide. Such non-natural amino acids can also be translationally incorporated into an unnatural amino acid polypeptide and then post-translationally modified by reduction to form non-natural amino acids having the structure of Formula (A) as part of the non-natural amino acid polypeptide. further, non-natural amino acids having the structure of Formula (B) can also be incorporated into a polymer, polysaccharide, polynucleotide or chemically synthesized polypeptide and then reduced to form non-natural amino acids having the structure of Formula (A) as part of the polymer , polysaccharide, polymorphonucleotide or chemically synthesized polypeptide. Such incorporated non-natural amino acids having the structure of Formula (A) can then be reductively alkylated.

Other non-limiting examples of such non-natural amino acids containing protected or masked amine moieties are illustrated in Figure 15.

B. Structure and synthesis of non-natural amino acids: Alkylated aromatic amine groups Non-natural amino acids with reactive nucleophilic groups, such as, by way of example only, an aromatic amine group (in which secondary and tertiary amine groups are included), an amine group Masked aromatics (which can easily be converted to an aromatic amine group) or a protected aromatic amine group (which has similar reactivity to an aromatic amine group after deprotection) allow a variety of reactions to bind molecules via various reactions, in which they include but are not limited to reductive alkylation reactions with aldehyde-containing reagents. Such non-natural alkylated amino acids include amino acids having the structure of Formula (C): wherein: MI "<:,, s C '-'-- y) is selected from the group consisting of a monocyclic aryl ring, a bicyclic aryl ring, a multicyclic aryl ring, a monocyclic heteroaryl ring, a bicyclic heteroaryl ring and a multicyclic heteroaryl ring: A is independently CRa or N; B is independently CRa, N, 0 or S, each Ra is independently selected from the group consisting of H, halogen, alkyl, - (NR ' ) 2, -C (0) R ', C (0) N (R') 2, -OR and -S (0) 2R ', where k is 1, 2 or 3 and n is 0, 1, 2, 3, 4, 5, or 6, Ri is H, an amino protecting group, resin, amino acid, polypeptide or polynucleotide and R2 is OH, an ester, resin, amino acid, polypeptide or polynucleotide protecting group, each R and R4 is independently H , halogen, lower alkyl or substituted lower alkyl or R3 and R4 or two groups R3 optionally form a cycloalkyl or heterocycloalkyl, M is H or -CH2R5, or the MNC portion (R5) can form a structure of ring from 4 to 7 members; R5 is alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy substituted, alkylalkoxy, substituted alkylalkoxy, polyalkylene oxide, substituted polyalkylene oxide, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocycle, substituted heterocycle, alkaryl, substituted alkapl, aralkyl, substituted aralkyl,, -C ( 0) R ", -C (0) OR", -C (0) N (R ") 2, -C (0) NHCH (R") 2, - (alkylene or alkylene) -N (R ") 2 »- (alkenylene or substituted alkenylene) -N (R") 2, - (alkylene or substituted alkylene) - (substituted aryl or aryl), - (substituted alkenylene or alkenylene) - (substituted aryl or aryl), - (alkylene or substituted alkylane -ON (R ") 2, - (alkylene or substituted alkylene) -C (O) SR", - (alkylene or substituted alkylene) -SS- (aryl or substituted aryl), wherein each R "is independently hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocycle, substituted heterocycle, alkary ilo, substituted alkap, aralkyl, substituted aralkyl or C (0) OR '; or R5 is L-X, wherein X is selected from the group consisting of a label; a dye; a polymer, a water-soluble polymer, a polyethylene gl derivative, a photocrosslinker, a cytotoxic compound, a drug, an affinity tag, a photoprint tag, a reactive compound, a ream, a second protein or polypeptide or analogue polypeptide, an antibody or fragment of antibodies; a metal chelator; a co-factor; a fatty acid; a carbohydrate; a polynucleotide; a DNA; an RNA; an antisense polynucleotide; a saccharide, a water-soluble dendrimer, a cyclodextrm, a biomaterial; a nanoparticle; a spin marker; a fluorophore, a portion containing metal; a radioactive portion; a new functional group; a group that interacts covalently or non-covalently with other molecules; a photographed portion; an excitable portion by actinic radiation; a ligand; a photoisomerizable portion; biotin; a bioXna analogue; a portion incorporating a heavy atom; a group chemically cleaved it; a photo-compact group; an elongated side chain; a carbon-bonded sugar; a redox-active agent; an ammo thioacid; a toxic portion; an isotopically labeled portion; a biophysical probe; a phosphorescent group; a chemiluminescent group; a dense group of electrons; a magnetic group; an intercalary group; a chromophore; an energy transfer agent; a biologically active agent; a detectable marker; a small molecule; an inhibitory ribonucleic acid; a radionucleotide; an electron capture agent; a biotome derivative; quantum dot (s); a nannsmitter; a radio transmitter; an abzyme, an activator of activated complex, a virus, an adjuvant, an aglycan, an allergan, an angiostatma, an antihormone, an antioxidant, an aptamer, a guide RNA, a saponin, a vector of shuttle, a macromolecule, a mimotope, a receiver, a reverse micelle and any combination thereof and L is optional, and when present is a linker selected from the group consisting of alkylene, substituted alkylene, alkenylene, substituted alkenylene, -0- , -0- (alkylene or substituted alkylene), - (alkylene or substituted alkylene) -0-, -C (0) -, -C (0) - (alkylene or substituted alkylene), (alkylene or substituted alkylene) -C (0) -, -C (0) N (R ') -, -C (0) N (R') - (alkylene or substituted alkylene) -, - (alkylene or substituted alkylene) -0C (0) N ( R ') -, -N (R') CJ O) -, -N (R ') C (0) - (alkyl or substituted alkylene) -, (alkylene or substituted alkylene) -NR' C (0) - , -S-, -S- (alkylene or substituted alkylene) -, -S (0) k- wherein k is 1, 2 or 3, -S (0) - (alkylene or substituted alkylene) -, -C ( S) -, -C (S) - (alkylene or substituted alkylene) -, CSN (R ') -, CSN (R') - (alkylene or substituted alkylene) -, -N (R ') CO- (alkylene or substituted alkylene) -, -N (R ' ) C (0) 0-, - (alkylene or substituted alkylene) -0-N = CR '-, - (alkylene or substituted alkylene) -C (0) NR' - (alkylene or substituted alkylene) -, (alkylene or substituted alkylene) -S (0) k- (alkylene or substituted alkylene) -S-, - (alkylene or substituted alkylene) -SS-, S (0) kN (R ') -, -N (R') C ( 0) N (R ') -, - N (R') C (S) N (R ') -, - N (R') S (0) kN (R ') -, -N (R ') - N =, -C (R') = N-, -C (R ') = NN (R') -, -C (R ') = NN =, -C (R') 2-N = N and -C (R ') 2-N (R') -N (R ') -; or two R5 groups optionally form a cycloalkyl or heterocycloalkyl; or R5 and any Ra optionally form a cycloalkyl or a heterocycloalkyl and each R 'is independently H, alkyl or substituted alkyl. Such non-natural amino acids can also be in the form of a salt or can be incorporated into an unnatural amino acid polypeptide, a polymer, polysaccharide or a polynucleotide and optionally seduced alkylation. M I The structure., X j (as presented in all the examples herein) does not present the relative orientations of "A", "B", "N (M) CH2R5" and "Ra"; rather, these four aspects of this structure can be oriented in any chemically firm manner (together with other aspects of this structure), as illustrated by the examples herein. Unnatural amino acids containing a portion of aromatic amine having the structure of Formula (C) include unnatural amino acids having the structure of Formula (XII) Formula (XIII), Formula (XIV) Formula (XV) and Formula ( XVI): where, each A 'is independently selected from CR V M or C--. ^ - R »and up to two A' can be c- - .. ^ l :; with the remaining A1 selected from CR. or V or Such natural nc amino acids may also be in the form of a salt or may also be incorporated into a non-naturally occurring amino acid polypeptide, polymer, polysaccharide or a polynucleotide and optionally reductively alkylated. Other non-limiting examples of non-natural amino acids containing an aromatic amine moiety having the structure of Formula (C) are shown in Figures 8-10. Such non-natural amino acids can also be in the form of a salt or can be incorporated into a non-naturally occurring amino acid polypeptide, polymer, polysaccharide or a polynucleotide and optionally reductively alkylated. The compounds of formula (C) can be formed by reductive alkylation of compounds of formula (A) with carbonyl-containing reagents such as, for example, ketones, esters, thioesters and aldehydes. By way of example, such aldehyde containing reagents have the v > structure corresponding to * < R5 is alkyl, substituted alkyl, alkenyl, substituted alkenyl, alqumilo, substituted alkynyl, substituted alkoxy, alkylalkoxy, alkylalkoxy, substituted polyalkylene oxide, polyalkylene oxide substituted cycloalkyl, substituted cycloalkyl, aplo I aplo substituted heteroaplo I heteroaplo substituted heterocycle, substituted heterocycle, alkaryl, substituted alkapyl, aralkyl, substituted aralkyl,, -CFO) R ", -C (0) OR", -C (0) N (R ^ 2. ^ O) NHCH (R'J 2, - (alkylene-alkylene) -N (R ") 2" - (alkenylene or substituted alkenylene) -N (R ") 2, - (alkylene or substituted alkylene) - (aryl or substituted aryl), - (substituted alkenylene or alkenylene ) - (aryl or substituted aryl), - (alkylene or substituted alkylene) -ON (R ") 2, - (alkylene or substituted alkylene) -C (O) SR", - (alkylene or substituted alkylene) -SS- ( aplo or substituted aryl), wherein each R "is independently hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocycle, substituted heterocycle, alkaryl, substituted alkapyl, aralkyl, substituted aralkyl or C (0) OR '; or R5 is L-X, wherein X is selected from the group consisting of a label; a dye; a polymer ", a water soluble polymer, a polyethylene glycol derivative; photoreticulator; a cytotoxic compound; a drug; an affinity marker; a photoaffinity marker; a reactive compound; a resin; a second protein or polypeptide or polypeptide analogue; an antibody or antibody fragment; a metal chelator; a co-factor; a fatty acid; a carbohydrate; a polynucleotide; a DNA; an RNA; an antisense polynucleotide; a saccharide, a water-soluble dendrimer, a cyclodextrin, a biomaterial; a nanoparticle; a spin marker; a fluorophore, a portion containing metal; a radioactive portion; a new functional group; a group that interacts covalently or non-covalently with other molecules; a photoenaged portion; an excitable portion by actinic radiation; a ligand; a photoisomerizable portion; biotin; a biotin analogue; a portion that incorporates a heavy atom; a chemically cleavable group; a photocleavable group; an elongated side chain; a carbon-bonded sugar; a redox-active agent; an amino thioacid; a toxic portion; an isotopically labeled portion; a biophysical probe; a phosphorescent group; a chemiluminescent group; a dense group of electrons; a magnetic group; an intercalary group; a chromophore; an energy transfer agent; a biologically active agent; a detectable marker; a small molecule; an inhibitory ribonucleic acid; a radionucleotide; an electron capture agent; a biotin derivative; quantum dot (s); a nanotransmitter; a radio transmitter; an abzyme, an activator of activated complex, a virus, an adjuvant, an aqlicana, an allergana, an angiostatma, an antihormone, an antioxidant, an aptamer, a guide RNA, a saponin, a shuttle vector, a macromolecule, an mimotope, a receptor, a reverse micelle and any combination thereof and L is optional and when present is a linker selected from the group consisting of alkylene, substituted alkylene, alkenylene, substituted alkenylene, -0-, -0- (alkylene) or substituted alkylene), - (substituted alkylene or alkyl) -0-, -C (0) -, -C (0) - (alkylene or substituted alkylene), (alkylene or substituted alkylene) -C (0) ) -, -C (0) N (R ') -, -C (0) N (R') - (alkylene or substituted alkylene) -, - (alkylene or substituted alkylene) -0C (0) N (R ') ) -, -N (R ') C (0) -, -N (R') C (0) - (alkylene or substituted alkylene) -, (alkylene or substituted alkylene) -NR 'C (0) -, - S-, -S- (alkylene or substituted alkylene) -, -S (0) k- where k is 1, 2 or 3, -S (0) k- (alkyl) ene or substituted alkylene) -, -C (S) -, -C (S) - (alkylene or substituted alkylene) -, CSN (R ') -, CSN (R') - (alkylene or substituted alkylene) -, - N (R ') CO- (alkylene or substituted alkylene) -, -N (R') C (0) 0-, - (alkylene or substituted alkylene) -0-N = CR '-, - (alkylene or substituted alkylene) ) -C (0) NR '- (alkylene or substituted alkylene) -, (alkylene or substituted alkylene) -S (0) k- (alkylene or substituted alkylene) -S-, - (alkylene or substituted alkylene) -SS- , S (0) kN (R ') -, -N (R') C (0) N (R ') -, - N (R') C (S) N (R ') -, - N (R ') S (0) kN (R') -, -N (R ') - N =, -C (R') = N-, -C (R ') = NN (R') -, -C (R ') = NN =, -C (R') 2-N = N and -C (R ') 2-N (R') -N (R ') -; each R 'is independently H, alkyl or substituted alkyl. Other non-natural amino acids having the structure of formula (B) are non-limiting examples described herein: wherein each Ra is independently selected from the group consisting of H, halogen, alkyl, -N02, -CN, substituted alkyl, -N (R ') 2, -C (O) kR', -C (0) N ( R ') 2, -OR' and -S (0) kR ', wherein k is 1, 2 or 3; M is H or -CH2R5; or the portion M-N-C (R5) can form a ring structure of 4 to 7 members; R5 is alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, substituted alkoxy, alkylalkoxy, substituted alkylalkoxy, polyalkylene oxide, substituted polyalkylene oxide, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocycle, substituted heterocycle, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl, -C (0) R ", -C (0) OR", -C (0) N (R ") 2, -C (0) NHCH (R ") 2, - (alkylene or alkylene) -N (R'J 2 '- (alkenylene or substituted alkenylene) -N (R") 2, - (alkylene or substituted alkylene) - (aryl or substituted aryl), - (alkenylene or substituted alkenylene) - (aryl or substituted aryl), - (alkylene or substituted alkylene) -ON (R ") 2, - (alkylene or substituted alkylene) -C (0) SR", - (alkylene or alkylene) substituted) -SS- (aryl or substituted aryl), wherein each R "is independently hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocycle, substituted heterocycle , alkaryl, substituted alkaryl, aralkyl, substituted aralkyl or -C (0) 0R '; or Rs is L-X, wherein X is selected from the group consisting of a marker; a dye; a polymer ", a water-soluble polymer, a polyethylene glycol derivative, a photocrosslinker, a cytotoxic compound, a drug, an affinity tag, a photoaffinity tag, a reactive compound, a resin, a second protein or polypeptide or analogue polypeptide, an antibody or antibody fragment, a metal chelator, a co-factor, a fatty acid, a carbohydrate, a polynucleotide, a DNA, an RNA, an antisense polynucleotide, a saccharide, a soluble dendrimer in water, a cyclodextrin, a biomaterial; a nanoparticle; a spin marker; a fluorophore, a portion containing metal; a radioactive portion; a new functional group; a group that interacts covalently or non-covalently with other molecules; a photographed portion; an excitable portion by actinic radiation; a ligand; a photoisomerizable portion; biotin; a biotin analogue; a portion that incorporates a heavy atom; a chemically cleavable group; a photocleavable group; an elongated side chain; a carbon-bonded sugar; a redox-active agent; an amino thioacid; a toxic portion; an isotopically labeled portion; a biophysical probe; a phosphorescent group; a chemiluminescent group; a dense group of electrons; a magnetic group; an intercalary group; a chromophore; an energy transfer agent; a biologically active agent; a detectable marker; a small molecule; an inhibitory ribonucleic acid; a radionucleotide; an electron capture agent; a biotin derivative; quantum dot (s); a nanotransmitter; a radio transmitter; an abzyme, an activated complex activator, a virus, an adjuvant, an aglycan, an allergen, an angiostatin, an antihormone, an antioxidant, an aptamer, a guide RNA, a saponin, a shuttle vector, a macromolecule, an mimotope, a receptor, a reverse micelle and any combination thereof and L is optional and when present is a selected linker of the group consisting of alkylene, substituted alkylene, alkenylene, substituted alkenylene, -0-, -0- (alkylene or substituted alkylene), - (alkylene or substituted alkylene) -0-, -C (0) -, -C ( 0) - (alkylene or substituted alkylene), (alkylene or substituted alkylene) -C (0) -, -C (0) N (R ') -, -C (0) N (R') - (alkylene or alkylene substituted) -, - (alkylene or substituted alkylene) -0C (0) N (R ') -, -N (R') C (0) -, -N (R ') C (0) - (alkylene or alkylene substituted) -, (alkylene or substituted alkylene) -NR 'C (0) -, -S-, -S- (alkylene or substituted alkylene) -, -S (0) k- where k is 1, 2 c 3 , -S (0) v- (alkylene or alkane ne substituted) -, -C (S) -, -C (S) - (alkylene or substituted alkylene) -, CSN (R ') -, CSN (R') - (alkylene or substituted alkylene) -, -N (R ') CO- (alkylene or substituted alkylene) -, -N (R') C (0) 0-, - (alkylene or substituted alkylene) -0-N = CR '-, - (alkylene or substituted alkylene) -C (0) NR' - (alkylene or substituted alkylene) -, (alkylene or substituted alkylene) -S (0) k- (alkylene) or substituted alkylene) -S-, - (alkylene or substituted alkylene) -SS-, S (0) kN (R ') -, -N (R') C (0) N (R ') -, -N ( R ') C (S) N (R') -, -N (R ') S (0) kN (R') -, -N (R ') - N =, -C (R') = N- , -C (R ') = NN (R') -, -C (R ') = NN * =, -C (R') 2 -N = N and -C (R ') 2-N (R') ) -N (R ') -; or R5 and any Ra optionally form a cycloalkyl or heterocycloalkyl and each R 'is independently H, alkyl or substituted alkyl.

The following non-natural amino acids having the structure of Formula (C) are non-limiting examples of non-natural alkylated amino acids described herein: Such non-natural amino acids can also be in the form of a salt or can be incorporated into a non-naturally occurring amino acid polypeptide, polymer, polysaccharide or a polynucleotide and optionally reductively alkylated. Non-limiting exemplary syntheses of non-natural amino acid polypeptides containing Formula amino acids (C) are presented in Figure 15, wherein the masked amine portions of non-natural amino acids of Formula (B) contained in the polypeptides are initially reduced to give non-natural amino acids of Formula (A) containing incorporated aromatic amine moieties to non-natural amino acid polypeptides. Such aromatic amine portions are then seduced alkylated with carbonyl-containing reagents described above to give polypeptides containing non-natural amino acids of Formula (C). Such reactions can also be applied to non-natural amino acids incorporated into synthetic polymers, polysaccharides or polynucleotides. Additionally, such reactions can be applied to non-natural amino acids not incorporated. By way of example, the reducing agent used to allow masked portions of amine includes but is not limited to TCEP, Na2S, Na2S2? 4, LiAlH4, B2H6 and NaBH4. By way of example only, reductive alkylation can occur in aqueous pH buffer solutions with a pH of about 4 to about 7 and using a mild reducing agent, such as, by way of example only, sodium cyanoborohydride (NaBCNH3). In addition, other reducing agents can be used for reductive alkylation, which include but are not limited to TCEP, Na2S, Na2S204, LiAlH4, B2H6 and NaBH4. In Figure 16, non-limiting exemplary syntheses of non-natural amino acid polypeptides are presented. contain amino acids of Formula (C) by reductive alkylation of secondary aromatic amine portions contained in non-natural amino acids of Formula (A) with carbonyl-containing reagents described above. Such reductive alkylations give polypeptides containing non-natural amino acids with tertiary aromatic amine moieties. Such reactions may also be applied to non-natural amino acids incorporated into synthetic polymers, polysaccharides or polynucleotides. Additionally, such reactions can be applied to non-natural amino acids incorporated nc. By way of example only, reductive alkylation can occur in aqueous buffer solutions with a pH of from about 4 to about 7 and using a mild reducing agent, by way of example only, sodium cyanoborohydride (NaBCNHJ.) In addition, other reducing agents they can be used for reductive alkylation, which include but are not limited to TCEP, Na2 =, Na2S2? 4, LiAlH4, B2H6 and NaBH4 The compounds of formula (C) can also be formed by the reductive alkylation of formula (A) with reagents containing at least two carbonyl moieties, which include, but are limited to, the diketones, keto aldehydes and dialdehydes, For example, such reagents may have the structure corresponding to where; each Ri is independently selected from H, optionally substituted alkyl, optionally substituted alkene, optionally substituted alkyne, optionally substituted cycloalkyl, optionally substituted heterocycle, optionally substituted aryl, or optionally substituted heteroaryl. Rs is alkylene, substituted alkylene, substituted alkenylene, substituted alkenylene, alkynylene, substituted alkynylene, poyalkylene oxide, substituted polyalkylene oxide, cycloalkylene, substituted cycloalkylene, substituted arylene arylene, heteroarylene, substituted heteroarylene, heterocyclealkylene, substituted heterocyclealkylene, -C (0) ) R "-, - C (0) OR" -, - C (0) N (R ") -, - (substituted alkylene or alkylene) -ON (R") -, - (substituted alkenylene or alkenylene) -N (R ") -, - (substituted alkylene or alkylene) R", - (substituted alkylene or alkylene) -C (O) SR "-, wherein each R" is independently of hydroquin, alkyl, substituted alkyl, substituted alkenyl, alkenyl, alcoholic alkoxy, substituted alkylene, substituted alkenylene, alkynylene, substituted alkynylene, aryl, substituted aryl, heteroaryl, substituted heteroary, heterocycle, substituted heterocycle, alkaryl, substituted alkaryl, aralkyl, or substituted aralkyl or R5 is L-X, wherein X is selected from the group consisting of a label; a dye; a polymer ", a water-soluble polymer, a polyethylene glycol derivative, a photocrosslinker, a cytotoxic compound, a drug, an affinity tag, a photoaffinity tag, a reactive compound, a resin, a second protein or polypeptide or analogue polypeptide, an antibody or antibody fragment, a metal chelator, a co-factor, a fatty acid, a carbohydrate, a polynucleotide, a DNA, an NPC, an antisense polynucleotide, a saccharide, a water-soluble dendrimer, a cyclodextrin a biomaterial, a nanoparticle, a spin marker, a fluorophore, a metal-containing portion, a radioactive portion, a new functional group, a group that interacts covalently or non-covalently with other molecules, a photoenaged portion, a portion excitable by actinic radiation, a ligand, a photoisomerizable portion, biotin, a biotin analogue, a portion that incorporates a heavy atom, a chemical cleavable group mind, a photocleavable group; an elongated side chain; a carbon-bonded sugar; a redox-active agent; an amino thioacid; a toxic portion; an isotopically labeled portion; a biophysical probe; a phosphorescent group; a chemiluminescent group; a dense group of electrons; a magnetic group; a group interleaver a chromophore; an energy transfer agent; a biologically active agent; a detect marker; a small molecule; an inhibitory ribonucleic acid; a radionucleotide; an electron capture agent; a biotin derivative; quantum dot (s); a nanotransmitter; a radio transmitter; an abzyme, an activated complex activator, a virus, an adjuvant, an aglycan, an allergen, an angiostatin, an antihormone, an antioxidant, an aptamer, a guide RNA, a saponin, a shuttle vector, a macromolecule, an mimotope, a receptor, a reverse micelle and any combination thereof and L is optional and when present is a linker selected from the group consisting of a-alkylene, substituted alkylene, alkenylene, substituted alkenylene, -0-, -0- (alkylene) or substituted alkylene), - (alkylene or substituted alkylene) -0-, -C (0) -, -C (0) - (alkylene or substituted alkylene), (alkylene or substituted alkylene) -C (0) -, - C (0) N (R ') -, -C (0) N (R') - (alkylene or substituted alkylene) -, - (alkylene or substituted alkylene) -0C (0) N (R ') -, - N (R ') C (0) -, -N (R') C (0) - (alkylene or substituted alkylene) -, (alkylene or substituted alkylene) -NR 'C (0) -, -S-, - S- (alkylene or substituted alkylene) -, -S (0) k- where k is 1, 2 or 3, -S (0) k- (alkylene) or substituted alkylene) -, -C (S) -, -C (S) - (alkylene or substituted alkylene) -, CSN (R ') -, CSN (R') - (alkylene or substituted alkylene) -, -N (R ') C0- (alkylene or substituted alkylene) -, -N (R') C (0) 0-, - (alkylene or alkylene substituted) -0-N = CR '-, - (alkylene or substituted alkylene) -C (0) NR' - (alkylene or substituted alkylene) -, (alkylene or substituted alkylene) -S (0) k- (alkylene or substituted alkylene) -S-, - (alkylene or substituted alkylene) -SS-, S (0) kN (R ') -, -N (R') C (0) N (R ') -, -N (R ') C (S) N (R') -, -N (R ') S (0) kN (R') -, -N (R ') - N =, -C (R') = N-, -C (R ') = NN (R') -, -C (R ') = NN =, -C (R') 2 -N = N and -C (R ') 2-N (R') - N (R ') -; each R 'is independently H, alkyl or substituted alkyl; n is 1, 2 or 3.

C. Structure and synthesis of non-natural amino acids: Amino acids amino acids. Non-natural amino acids with electrophilic reactive groups, such as by way of example only, an aldehyde group, a masked aldehyde group (which is a functional group that can be easily converted to an aldehyde group) or a protected aldehyde group (which has reactivity similar to a group of the aldehyde in the deprotection) allow a variety of reactions to bind molecules via various reactions, which include but are not limited to reductive amination containing the reactants of the aromatic amine. Such amino-unnatural amino acids include amino acids having the structure of formula (D): wherein: L is optional, and when the present is lower alkylene, lower alkylene, substituted, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkylen, heteroalkylene mleruc, substituted hetero-alkylene, substituted lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroaplene, substituted heteroarylene, alkarylene, substituted alkylene, aralkylene, or substituted aralkylene; Q 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, -0-, (alkylene or substituted alkylene) -, -S -, -S- (alkylene or substituted alkylene) -, -S (0)? -where k is 1,2, or 3, -S (0)? (substituted alkylene or alkylene) -, -C (0) - (substituted alkylene or alkylene) -, C (0) - (substituted alkylene or alkylene) -C (S) -, -C (S) - (alkylene or alkylene substituted) -, -N (R '), R' (alkylene or alkylene) substituted) -, -C (0) N (R ') -, -CON (R ") - (alkylene or substituted alkylene) -, -CSN (R") -, -CS (R') - (alkylene or substituted alkylene) -, -N (R1) CO -, - N (R ') CO (alkylene or alkylene substitute) -, -N (R') C (0) 0-, -S (0) KN ( R ') -, -N (R') C (0) N (R ') -, -N (R') C (S) N (R ') -, -N (R ") S (0) N (R) -, -N (R) -N =, -C (R ") = N-, -C (R ') = N- (R') -, -C (R ') = NN =, - C (R ') 2-N = N-, and C (R') 2-N (R ') -N (R') -where each R is independently H, alkylene or substituted alkylene; Ri is H, a group of amino protector, ream, amino acid, polypeptide, or polynucleotide; and R? is OH, a group of proteccor ester, ream, amino acid, polypeptide, or polynucleotide; each of R3 and R4 is independently H, halogen, lower alkyl, or substituted lower alkyl, or R and R4 or two R3 groups optionally form a cycloalkyl or a heterocycloalkyl; each Ra is independently selected from the group consisting of H, halogen, alkyl, -N02, -CN, substituted alkyl, -N (R ') 2, -C (0) KR', -C (0) N (R ') 2, -0R'yS (O)? R 'where K is 1, 2, or 3; M is H or -CH2 R5; R5 is alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkyl, substituted alkyl, alkoxy, substituted alkoxy, alkylalcohoxy, substituted alkylalkoxy, polyalkylene oxide, substituted polyalkylene, substituted cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocycle, substituted heterocycle, substituted alkaryl alkalyl, aralkyl, substituted aralkyl, -C (0) R ", -C (0) OR", -C (0) N (R'J 2, -C (0) NHCH (R'J 2, - (substituted alkylene or alkylene) -N (R ") 2, - (substituted alkenylene or alkenylene) -N (R") 2, - (substituted alkylene or alkylene) - (substituted aryl or aryl), - (alkenylene or substituted alkenylene) - (substituted aryl or aryl), - (substituted alkylene or alkylene) -ON (R ") 2, - (alkylene) or substituted alkylene) -C (0) R ", - (substituted alkylene or alkylene) -SS- (substituted alkyl or aplo), wherein each R" is independently hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alcohol oxoyl , substituted alcohol, aryl, subtituted aryl, heteroaryl, substituted heteroaryl, heterocycle, substituted heterocycle, alkapyl, alkaryl subst ituido, aralkyl, substituted aralkyl, or -C (0) 0R '; or Rs is L-X, wherein X is selected from the group consisting of a marker; a dye; a polymer, a water-soluble polymer, a polyethylene glycol derivative, a photocrosslinker, a cytotoxic compound, a drug, an affinity tag, a photo-agility tag, a reactive compound, a ream, a second protein or polypeptide or analogue polypeptide, an antibody or fragment of antibodies, a chelator of metals, a co-factor, an acid fatty; a carbohydrate; a polynucleotide; a DNA; an RNA, an antisense polynucleotide; a saccharin, a water-soluble dendimer, a cyclodextrm, a biomaterial; a nanoparticle; a spm marker; a fluorophore, a portion containing metal; a radioactive portion; a new functional group; a group that interacts covalently or non-covalently with other molecules; a portion photographed aulada; an excitable portion by actmic radiation; a ligand; a photoisomectable portion; biotma; a biotin analogue; a portion that incorporates a heavy atom; a chemically cleavable group; a photo-compact group; a laceral chain alai aaa, a carbon-linked sugar; a redox-active agent; a thioacid ammo; a toxic portion; an isotopically labeled portion; a biophysical probe; a phosphorescent group; a chemiluminescent group; a dense group of electrons; a magnetic group; a misleading group; a chromophore; an energy transfer agent; a biologically active agent; a detectable marker; a small molecule; an inhibitory ribonucleic acid; a radionucleotide; an electron capture agent; a biotin derivative; quantum dot (s); a nanotransmitter; a radio transmitter; an abzyme, an activated complex activator, a virus, an adjuvant, an aglycan, an allergan, an angiostatma, an antihormone, an antioxidant, an aptamer, a guide RNA, a saponin, a shuttle vector, a macromolecule, an mimotope, a receiver, a reverse micelle and any combination thereof and L is optional and when present is a linker selected from the group consisting of alkylene, substituted alkylene, alkenylene, substituted alkenylene, -0-, -0- (alkylene or substituted alkylene), - (alkylene or substituted alkylene) -0-, -C (0) -, -C (0) - (alkylene or substituted alkylene), (alkylene or substituted alkylene) -C (0) -, -C (0) N ( R ') -, -C (O) N (R') - (alkylene or substituted alkylene) -, - (alkylene or substituted alkylene) -0C (0) N (R ') -, -N (R') C (0) -, -N (R ') C (0) - (alkylene or substituted alkylene) -, (alkylene or substituted alkylene) -NR' C (0) -, -S-, -S- (alkylene or alkylene substituted) -, -S (0) k- where k is 1, 2 or 3, -S (0) - (alkylene or substituted alkylene) -, -C (S) -, -C (S) - (alkylene or substituted alkylene) -, CSN (R ') -, CSN (R') - (alkylene or substituted alkylene) -, -N (R ') C0- (alkylene or substituted alkylene) -, -N (R') C (0) 0-, - (alkylene or substituted alkylene) -0-N = CR '-, - (alkylene or alkenyl) substituted (substituted)) -C (0) NR '- (alkylene or substituted alkylene) -, (alkylene or substituted alkylene) -S (0) k- (alkylene or substituted alkylene) -S-, - (alkylene or substituted alkylene) - SS-, S (0) kN (R ') -, -N (R') C (0) N (R ') -, -N (R') C (S) N (R ') -, -N (R ') S (0) kN (R') -, -N (R ') - N =, -C (R') = N-, -C (R ') = NN (R') -, - C (R ') = NN =, -C (R') 2 -N = N and -C (R ') 2-N (R') -N (R ') -; or R5 and any Ra optionally form a cycloalkyl or a heterocycloalkyl; each R 'is independently H, alkyl or alkyl replaced; is selected from the group consisting of a monocyclic aryl ring, a bicyclic aryl ring, a multicyclic aryl ring, a monocyclic heteroaryl ring, a bicyclic heteroaryl ring and a multicyclic heteroaryl ring; A is independently CRa c N; B is independently CRa, N, O or S; The structure, (as presented in all the examples herein) does not present the relative orientations of "A", "B", "Ra" and the other substituents represented by the line and wavy; rather these four aspects of this structure can be oriented in any chemically firm manner (together with other aspects of this structure) as illustrated in the examples herein. An embodiment of the non-natural amino acid containing a portion of protected aldehyde has a structure of formula (E) wherein: L is optional and when present is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylnene, substituted lower alkenylene, alkynene, heteroalkyl? lower, substituted heteroalkyl, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroaplene, alkylene, substituted alkarylene, substituted aralkylene or aralkylene; Q is optional and when present is a linker selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O- (alkylene or substituted alkylene) -, -S - (substituted alkylene or alkylene) -, wherein k is 1,2, or 3, -S- (O) k (substituted alkylene or alkylene) -, -C (O) - (substituted alkylene or alkylene) -, - C (S) - (substituted alkylene or alkylene) -, -NR '- (alkylene or substituted alkylene) -, -CON (R ") - (alkylene or substituted alkylene) -, -CSN (R ") -, -CSN (R ') - (substituted alkylene or alkylene) -, -N (R') CO- (substituted alkylene or alkylene) -, and wherein each R 'is independently H, alkyl or substituted alkyl, Ri is H an amino, amino acid, polypeptide or polynucleotide protecting group and R2 is OH, is a protecting group, ester, resin, amino acid, polypeptide or polynucleotide; each R3 and R4 is independently H, halogen, lower alkyl, substituted lower alkyl, or R3? R4, or two groups of R3 optionally form a cycloalkio or a heterocycioalkyl; Each Ra is independently selected from the group consisting of H, halogen, alkyl, -NO2, -CN, substituted alkyl, -N (R ') 2, -C (0) kR', -C (0) N (R ') 2, -0R', and -S (0) kR ', where k is 1, 2, or 3; R6 is an aldehyde, a protected aldehyde or a masked aldehyde, wherein the protecting group includes but is not limited to: wherein each Xx is independently selected from a group consisting of -0-, -S-, -N (H) -, -N (Ac) -, and -N (OMe) -; X2 is -OR, -OAc, -SR, -N (R) 2, -N (R) (Ac), -N (R) (0Me), or N3, Y where each R 'and R is independently H, alkyl or substituted alkyl. Such non-natural amino acids can also be in the form of a salt or can be incorporated into a non-natural amino acid polypeptide, polymer or a polynucleotide. Such non-natural amino acids can also be incorporated into a non-natural amino acid polypeptide and then modified post-translationally by deprotection to form an "in situ" aldehyde group followed by reductive amidation of aldehyde with an aromatic amine-containing reagent, forming these non-natural amino acids, which have the structure of formula (D) as part of the non-natural amino acid polypeptide. In addition, non-natural amino acids having the formula structure of (E) can also be incorporated into a polymer or a polynucleotide and are deprotected to form an "in-situ" aldehyde group followed by reductive amidation of the aldehyde with a reagent containing aromatic amine, thereby forming non-natural amino acids having a structure of formula (D) as part of the polymer or a polynucleotide. Non-limiting examples of such non-natural amino acids containing protected portions of aldehyde are shown in Figure 17. Compounds of formula (D) can be formed after deprotection of the aldehyde group of compounds of formula (E), followed by amidation reducing compounds of formula (D) with reagents containing aromatic amine. Such reagents containing aromatic amine have the structure corresponding to where is selected from the group consisting of a monocyclic aryl ring and a bicyclic aryl ring, a multicyclic aryl ring, a monocyclic heteroaryl ring, a bicyclic heteroaryl ring and a monocyclic heteroaryl ring. A is independently CRZ or N; and B is independently CRa, N, O or S; each Ra is independently selected from the group consisting of H, halogen, alkyl, -NO2, -CN, substituted alkyl, -N (R ') 2, -C (0)? R', -C (0) N (R ') ) 2, -0R ', and -S (0)? R', where k is 1,2, or 3, n is 0, 1, 2, 3, 4, 5 or 6; M is H or CH2R5; R5 is alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alcoholyl, substituted alkoxy, alkylalcohoxy, substituted alkylalcohoxy, polyalkylene oxide, substituted polyalkylene oxide, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocycle, substituted heterocycle, substituted alkaryl alkaryl, aralkyl, substituted aralkyl, -C (0) R ", - C (0) OR ", -C (0) N (R'J 2, -C (0) NHCH (R'J 2, - (alkylene or substituted alkylene) -N (R") 2, - (alkenylene or substituted alkenylene) -N (R ") 2, - (substituted alkylene or alkylene) - (substituted aryl or aplo), - (alkenylene or substituted alkenylene) - (substituted aryl or aryl), - (alkylene or alkylene suost? u? do) -ON F ") 2, - (substituted alkyl or alkylene) -C (0) SR", - (substituted alkylene or alkylene! -SS- (substituted aplo or aplo), wherein each R "is independently hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxyl, substituted alkoxyl, aplo, subtituted aryl, heteroaryl, substituted heteroaryl, hetero iclo, substituted heterocycle, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl, or -C (0) 0R '; or R5 is L-X, wherein X is selected from the group consisting of a label; a dye; a polymer, a water-soluble polymer, a polyethylene glycol derivative, a photocrosslinker, a cytotoxic compound, a drug, an affinity tag, a photoprint tag, a reactive compound, a ream, a second protein or polypeptide or analogue. polypeptide, an antibody or fragment of antibodies; a metal chelator; a co-factor; a fatty acid; a carbohydrate; a polynucleotide; a DNA; an RNA; an antisense polynucleotide; a saccharide, a water-soluble dendrimer, a cyclodextrin, a biomaterial; a nanoparticle; a spin marker; a fluorophore, a portion containing metal; a radioactive portion; a new functional group; a group that interacts covalently or non-covalently with other molecules; a photoenaged portion; an excitable portion by actinic radiation; a ligand; a photoisomerizable portion; biotin; a biotin analogue; a portion that incorporates a heavy atom; a chemically cleavable group; a photocleavable group; an elongated side chain; a carbon-bonded sugar; a redox-active agent; an amino thioacid; a toxic portion; an isotopically labeled portion; a biophysical probe; a phosphorescent group; a chemiluminescent group; a dense group of electrons; a magnetic group; an intercalary group; a chromophore; an energy transfer agent; a biologically active agent; a detectable marker; a small molecule; an inhibitory ribonucleic acid; a radionucleotide; an electron capture agent; a biotin derivative; quantum dot (s); a nanotransmitter; a radio transmitter; an abzyme, an activated complex activator, a virus, an adjuvant, an aglycan, an allergan, an angiostatin, an antihormone, an antioxidant, an aptamer, a guide RNA, a saponin, a vector of shuttle, a macromolecule, a mimotope, a receptor, a reverse micelle and any combination thereof and L is optional and when present is a linker selected from the group consisting of alkylene, substituted alkylene, alkenylene, substituted alkenylene, -0-, -0- (alkylene or substituted alkylene), - (alkylene or substituted alkylene) -0-, -C (0) -, -C (0) - (alkylene or substituted alkylene), (alkylene or substituted alkylene) -C (0) -, -C (0) N (R ') -, -C (0) N (R') - (alkylene or substituted alkylene) -, - ( alkylene or substituted alkylene) -0C (0) N (R ') -, -N (R') C (0) -, -N (R ') C (0) - (alkylene or substituted alkylene), (c' Icylene or alkylene isotiene) -NR 'C (O) -, -S-, -S- (alkylene or substituted alkylene) -, -S (0) k- where k is 1, 2 or 3, -S (0) y- (alkylene or substituted alkylene) -, -C (S) -, -C (S) - (alkylene or substituted alkylene) -, CSN (R ') -, CSN (R') - (alkylene or substituted alkylene) -, -N (R ') C0- (alkylene or substituted alkylene) -, -N (R') C (0) 0-, - (alkylene or substituted alkylene) -0-N = CR '-, - (alkylene or substituted alkylene) -C (0) NR '- (alkylene or substituted alkylene) -, (alkylene or substituted alkylene) -S (0) k- (alkylene or substituted alkylene) -S-, - (alkylene or substituted alkylene) -SS-, S (0) kN (R ') -, -N (R') C (0) N (R ') -, -N (R') C (S) N (R ') -, -N (R') S (0) kN (R ') -, -N (R') - N =, -C (R ') = N-, -C ( R ') = NN (R') -, -C (R ') = NN =, -C (R') 2 -N = N and -C (R ') 2-N (R') -N (R ') -; wherein each R 'is independently hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, alkylene substituted, alkenylene, substituted alkenylene, alkynylene, substituted alkynylene, aryl, substituted aryl, heteroaryl, substituted heteroaryl, substituted heterocycle, substituted alkaryl, substituted alkaryl, aralkyl or substituted aralkyl. An exemplary non-limiting synthesis of non-atural amino acid polypeptides of formula (D) as presented in Figure 18, wherein the aldehyde portions protected from non-natural amino acids of formula (E) contained in polypeptides are initially deprotected to give non-natural amino acids containing portions of aldehydes incorporated into non-native amino acid polypeptides. Such aldehyde portions are then reductively aminated with amino aromatic-containing active described above to give polypeptides containing non-natural amino acids of formula (D). Such reactions can be applied to non-natural amino acids incorporated into synthetic polymers, polysaccharides or polynucleotides. Additionally, such reactions can be applied to non-natural amino acids not incorporated. By way of example, the protected aldehyde is deprotected by acid-catalysed deprotection. By way of example only, reductive amination may occur in buffer solutions of a pH of about 4 of about 7 and using a mild reducing oil, such as, by way of example only, Sodium cyanoborohydride (NaBCNHJ) In addition, other reducing agents can also be used for reductive alkylation in which TCEP, NA2S, Na2S204, LiAIH4, B2H6 and NaBH4 are excluded but not limited.Figure 19 shows a non-limiting representation of the formation of non-natural amino acids of formula (C) by the reductive alkylation of the aromatic amine portion of non-natural amino acids of formula (A) and a non-limiting representation of the formation of the non-natural amino acids of formula (D) by reductive amination of a portion of the deprotected aldehyde on non-natural amino acids of formula (E) In Figure 19 the specificity and selectivity of the reductive alkylation reaction wherein only the aromatic amine portion of non-natural amino acids is illustrated. of formula (A) is reductively alkylated, while other portions of amine, thiols and disulfide bonds are reduced when they react under the reaction conditions used. Also shown in Figure 19 is the specificity and selectivity of the reductive amination reaction, wherein only the unprotected aldehyde portion of non-natural amino acids of formula (E) is reductively aminated, while other portions of caboxylate, thiols and linkages of disulfide are not reduced when they react under the reaction conditions used. Such specific derivations of selective site, alkylation Reductive or reductive amination reactions may allow the modification of polypeptides / proteins to designate agonists and / or antagonists, PEG-specific coating of the polypeptide / protein site, specific conjugation of the polypeptide / protein site, pro-drug design, polypeptide / protein glycosylacin , polypeptide / protein dimerization, small molecule polypeptide / protein drug conjugates and small molecule drug conjugates of antibodies. The modification of non-natural amino acids described herein using reductive alkylation or reductive amination reactions have any or all of the following advantages. First, the aromatic amines can be reductively alkylated with carbonyl-containing compounds, in which aldehydes and ketones are included, in a pH range of about 4 to about 10 (and in certain embodiments, in a pH range of about 4 to about 7) to generate substituted amine bonds, in which secondary and tertiary amine bonds are included. Secondly, under these reaction conditions, the chemistry is flexive for non-natural amino acids since the side chains of the amino acids that are concentrated in nature are not reactive. This allows site-specific derivation of polypeptides that have incorporated non-natural amino acids that contain aromatic amine or portions of protected aldehydes in which, by way of example, recombinant proteins are included. Such derived polypeptides and proteins can thereby be prepared as defined homogeneous products. Third, the mild conditions necessary to effect the reaction of a portion of an aromatic amine on an amino acid that has been incorporated into a polypeptide, with an aldehyde-containing reagent in general does not irreversibly destroy the tertiary structure of the polypeptide (except, for example, of course, where the purpose of the reaction is to destroy such a tertiary structure). Uniquely, the mild conditions necessary to effect the reaction of a portion of aldehyde on an amino acid, which has been incorporated into a polypeptide and deprotected, with an aromatic amine-containing reagent that generally does not irreversibly destroy the tertiary structure of the polypeptide (except, of course, where the purpose of the reaction is to destroy such a tertiary structure). Fourth, the reaction occurs rapidly at room temperature, which allows the use of many types of polypeptides or reagents that would otherwise be unstable at higher temperatures. Fifth, the reaction occurs easily under aqueous conditions, again allowing the use of incompatible polypeptides and reagents (to any extent) with non-aqueous solutions. Sixth, the reaction occurs easily even when the proportion of the polypeptide or The amino acid to the reagent is stoichiometric, stoichiometric or quasi-stoichiometric, in such a way that it is not necessary to add excess reagent or polypeptide to obtain a useful amount of reaction product. Seventh, the resulting amine can be produced regioselectively and / or regiospecifically, depending on the design of the amine and carbonyl positions of the reactants. Finally, the reductive alkylation of aromatic amines with aldehyde-containing reagents and the reductive amination of aldehydes with reagents containing aromatic amine, general amine, in which secondary and tertiary amine bonds are included which are stable under biological conditions. Figure 20 shows non-limiting examples of the specificity and selectivity of the reductive alkylation of aromatic amine portions on amino acid residues close to a cysteine residue. Reductive alkylation of reduced urotensin-II (UT-II-SH) with propionaldehyde (I) or benzaldehyde (II) demonstrates that a product is formed predominantly from the respective reactions and that the free thiol group did not react. Also shown in Figure 20 are the speed of the reactions, which was about 2 hours and the stoichiometric or near stoichiometric ratio of the reduced urotensin-II (UT-II-SH) and the respective aldehyde-containing residues. Additionally, MS data for UT-II-SH, I and II are presented.

Figure 21 shows non-limiting examples of the specificity and selectivity of the reductive alkylation of aromatic amine portions on amino acid residues close to a cysteine residue. Reductive alkylation of urotensin-II (UT-II) with propionaldehyde (I) or benzaldehyde (II) demonstrates that a product was formed predominantly from the respective reactions and that the disulfide bond did not react. Also shown in Figure 21 are the reaction rates, which were approximately 2 hours and the stoichiometric or quasi-stoichiometric ratio between urotensin-II (UT-II) and the respective aldehyde-containing reagents. Additionally, the MS data for UT-II-SH, I and II are presented. Further non-limiting examples of the specificity and selectivity of the reductive alkylation regions of aromatic amine moieties on terminal amino acid residues are shown in Figure 22, wherein the reductive alkylation of the XT-8 peptide with propionaldehyde (I), benzaldehyde ( II), isobutaldehyde (III) and pivaldehyde (IV), demonstrate that a product was formed predominantly from the respective reactions and that the disulfide bond did not react. Also shown in Figure 22 are the speed of the reactions, which was about 2 hours and the stoichiometric or near stoichiometric ratio between the XT-8 and the respective aldehyde-containing residues.

Additionally, MS data for XT-8 I, II, III and IV are presented. Further non-limiting examples of the specificity and selectivity of reductive alkylation reactions of aromatic amine portions on amino acid residues that are close to the various N-terminal amino acids are shown in Figures 23-27. In Figure 23, the reductive alkylation of the peptide SXT-9 (N-terminal serine) with propionaldehyde (I), benzaldehyde (II), isobutaldehyde (III'1 and pivaldehyde (IV), shows the rate of reactions, which was approximately 2 hours and the stoichiometric or near stoichiometric ratio between the SXT-9 and the respective aldehyde-containing reagents In addition, Figure 23 demonstrates that a product was formed predominantly from the respective reactions, and that the disulfide bond did not react and In addition, the MS data for SXT-9, I, II, III, and IV are presented, and in Figure 24, reductive alkylation of the HXT-9 peptide (histidine N). -terminal) with propionaldehyde (I), benzaldehyde (II), isobutaldehyde (III) and pivaldehyde (IV), shows the speed of the reactions, which was about 2 hours and the stoichiometric or almost stoichiometric ratio between the HXT-9 and the reagents containing respective aldehyde. In addition, Figure 24 shows that a The product was formed predominantly from the respective reactions, and that the disulfide bond did not react and that the N-His residue has minimal effect on the selectivity. Also, the MS data for HXT-9, I, II, III and IV are shown. In Figure 25, the reductive alkylation of peptide XT-9 (N-terminal tryptophan) with propionaldehyde (I), benzaldehyde (II) and isobutaldehyde (III), shows the speed of the reactions, which was approximately 2 hours and the stoichiometric or quasi-stoichiometric ratio between XT-9 and the respective aldehyde-containing reagents. In addition, Figure 25 demonstrates that a product was formed predominantly from the respective reactions, and that the disulfide bond did not react and that the N-Trp residue has minimal effect on the selectivity. Also, the MS data for XT-9, I, II and III are shown. In Figure 26, the reductive alkylation of the peptide NXT-9 (N-terminal asparagine) with propionaldehyde (I) and benzaldehyde (II), shows the speed of the reactions, which was about 2 hours and the stoichiometric or almost stoichiometric ratio between NXT-9 and the respective aldehyde-containing reagents. In addition, Figure 26 demonstrates that a product was formed predominantly from the respective reactions, and that the disulfide bond did not react and that the N-Asn residue has minimal effect on the selectivity.

Also, the MS data for NXT-9, I and II are shown. In Figure 27, the reductive alkylation of the peptide RXT-10 (N-terminal arginine) with propionaldehyde (I) and benzaldehyde (II), shows the speed of the reactions, which was about 2 hours and the stoichiometric or almost stoichiometric ratio between the RXT-10 and the respective aldehyde-containing reagents. In addition, Figure 27 demonstrates that a product was formed predominantly from the respective reactions, and that the disulfide bond did not react and that the N-Arq residue has minimal effect on the selectivity. Also, the MS data for RXT-10, I and II are shown. A further non-limiting example of the specificity and selectivity of the reductive alkylation reactions of aromatic amine portions on amino acid residues in a peptide is shown in Figure 28, wherein the reductive alkylation of the peptide AXT-11 with propionaldehyde (I) and benzaldehyde (II), shows the speed of the reactions, which was about 2 hours and the stoichiometric or almost stoichiometric ratio between the AXT-11 and the respective aldehyde-containing reagents. In addition, Figure 28 demonstrates that a product was formed predominantly from the respective reactions, that the disulfide bond did not react and that the N-Ala residue has minimal effect on the selectivity. Also, the MS data for AXT-11, I and II are shown. Additional non-limiting examples of Specificity and selectivity of the reductive alkylation reactions of aromatic amine portions on amino acid residues in a peptide with various aldehyde-containing reagents are shown in Figures 29-30. In Figure 29, the reductive alkylation of the AXT-11 peptide with various aldehyde-containing reagents shows the rate of reactions, which was about 5 hours and the stoichiometric or near-stoichiometric ratio between AXT-11 and aldehyde-containing reagents respective. In addition, Figure 29 demonstrates that a product was formed predominantly from the respective reactions, that the disulfide bond did not react. The MS data for AXT-11, I, II and III are also presented. In Figure 30, the reductive alkylation of the AXT-11 peptide with various heteroaromatic aldehyde-containing reagents shows the rate of reactions, which was about 5 hours and the stoichiometric or near stoichiometric ratio between AXT-11 and the reagents containing aldehyde respectively. In addition, Figure 30 demonstrates that a product was formed predominantly from the respective reactions and that the disulfide bond did not react. Also, the MS data for AXT-11, IV, V and VI are presented. Figures 31-33 show non-limiting examples of the specificity and selectivity of the reductive alkylation reactions of the aromatic amine moieties on amino acid residues in a peptide, wherein the Aromatic amine portions are reacted with either a reagent containing aldehyde, a reagent containing ketone or a mixture thereof. In Figure 31, the reductive alkylation of the AXT-11 peptide with an aldehyde-containing reagent, a ketone-containing reagent or a mixture thereof shows the rate of the reactions, which was about 2 hours and the stoichiometric or near-ratio stoichiometric between AXT-11 and the respective aldehyde-containing reagents. In addition, Figure 31 demonstrates under the reaction conditions used, substantially the aldehyde-containing reagent reacted with the aromatic amine portion and only one product was predominantly formed. Again, the disulfide bond did not react. In Figure 32, the reductive alkylation of the peptide NXT-9 with an aldehyde-containing reagent, a ketone-containing reagent or a mixture thereof shows the rate of the reactions, which was about 2 hours and the stoichiometric ratio or nearly stoichiometric between NXT-10 and the respective aldehyde-containing reagents. In addition, Figure 32 demonstrates that under the reaction conditions used, only the aldehyde-containing reagent reacted with the aromatic amine portion and only one product was predominantly formed. Again, the disulfide bond did not react. In Figure 33, reductive alkylation of the MXT-9 peptide with an aldehyde-containing reagent, a reagent containing ketone or a mixture thereof demonstrates that under the reaction conditions used, only the aldehyde-containing reagent reacted with the aromatic amine portion and only one product was predominantly formed. Again, the disulfide bond did not react. A non-limiting example of the deprotection (or unmasking) of a protected (or masked) aromatic amine moiety on an amino acid in a peptide, followed by reductive alkylation of the resulting aromatic amine moiety with various aldehyde-containing reagents is shown in the Figure 34. The reduction of azide on the peptide MXT-9-N3 with TCEP gave the primary aromatic amine portion on the peptide MXT-9NH2 (I). Subsequent reductive alkylations of MXT-9NH2 (I) with propionaldehyde (II) or benzaldehyde (III) then produces the corresponding alkylated peptides. Figure 34 demonstrates the ability to form aromatic amine portions after incorporation of an unnatural amino acid containing a protected or masked amine group. The stoichiometric or near stoichiometric ratio between the MXT-9NH2 (I) and the respective aldehyde-containing reagents is also shown in the rate of reactions that was approximately one hour, only one product was predominantly formed and the thiol groups did not react .

D. Absorption of cellular non-natural amino acids Absorption of non-natural amino acids by a cell is a matter considered when designing and selecting non-natural amino acids, including, but not limited to, the incorporation of a protein. For example, the high charge density of the α-amino acids suggests that these compounds are unlikely to be cell-permeable. The natural amino acids are absorbed into the eukaryotic cell via a collection of the protein-based transport system. A quick selection can be made that determines which unnatural amino acids, if any, are absorbed by the cells. See, for example, toxicity analyzes in, for example, U.S. Patent Publication No. 2004/198637 entitled "Protein Arrays", which is incorporated herein by reference in its entirety and Liu, D.R. and Schultz, P.G. (1999) Progress towards the evolution or fan organism wi th an expanded genetic code. PNAS United States 96: 4780-4785. Although absorption is easily analyzed with several analyzes, an alternative to designing non-natural amino acids that are prone to cellular absorption pathways and provide biosynthetic pathways to create amino acids in vivo.

E. Biosynthesis of non-natural amino acids There are already many biosynthetic pathways in cells for the production of amino acids and other compounds. While a biosynthetic method for a non-natural amino acid may not exist in nature, in which, but not limited to, in a cell, the methods and compositions described herein provide such methods. For example, biosynthetic pathways for non-natural amino acids are optionally generated in a host cell by adding new enzymes or modifying existing host cell notes. New additional enzymes are optionally enzymes that are stably present in nature or artificially evolved enzymes. For example, the biosynthesis of p-aminophenylalanine (as presented in an example in WO 2002/085923 entitled "In vivo incorporation of unnatural amino acids") depends on the addition of a combination of known enzymes from other organisms. The genes for these enzymes can be introduced into a eukaryotic cell by transforming the cells with a plasmid comprising the genes. Genes, when expressed in the cell, provide an enzymatic route to synthesize the desired compound. Examples of types of enzymes that are additionally added are provided in the examples below. Sequences of additional enzymes are found, for example, in Genbank. Artificially evolved enzymes are also optionally added to a cell in the same way. In this way, the machinery and cellular resources of A cell is manipulated to produce non-natural amino acids. A variety of methods are available to produce new enzymes for use in biosynthetic pathways or for evolution of existing routes. For example, recursive recombination, in which but not limited to, as developed by Maxygen, Inc. (available on the World Wide Web at www.maxygen.com) is optionally used to develop new enzymes and routes. See, for example, Stemmer (1994), Rap id evolution of a protein in Vi tro by DNA shuffling, Nature 370 (4): 389-391 and Stemmer (1994), FNA shuffling by random fragmentation and reassembly: In vi recombine trope for molecular evolution, Proc. Nati Acad. Sci. USA., 91: 10747-10751. Similarly, DesignPath ™, developed by Genencor (available on the World Wide Web at genencor.com) is optionally used for the design of metabolic pathways, including but not limited to, to design a route to create O-methyl- L-tyrosine in a cell. This technology reconstructs existing routes in host organisms using a combination of new genes, including, but not limited to, those identified through functional genomics and molecular evolution and design. Diversa Corporation (available on the World Wide Web at diversa.com) also provides technology to quickly select gene libraries and genetic routes, which include but are not limited to, to create new routes. Commonly, the non-natural amino acid produced with a biostatic route designed as described herein is produced in sufficient concentration for efficient protein biosynthesis, in which, but not limited to, a natural cellular amount is included, but not to such an extent to affect the concentration of the other amino acids or deplete cellular resources. Typical concentrations produced m vivo in this manner are from about 10 mM to about 0.05 mM. Once a cell is transformed with a plasmid q comprises the genes used to produce desired enzymes for a specific route and an unnatural amino acid is generated, selections m vivo are optionally used to further optimize the production of the non-natural amino acid for both the synthesis of ribosomal protein as cell growth.

F. Additional Synthesis Methodology The non-natural amino acids described herein can be synthesized using methodologies described in the art or using the techniques described herein or by a combination thereof. As an auxiliary, the following Table provides several starting electrophiles and nucleotides that can be combined to create a desired functional group. The information provided is intended to be illustrative and not limiting of the synthesis techniques described herein.

Table. Examples of covalent bonds and precursors thereof In general, carbon electrophiles are susceptible to attack by complementary nucleophiles, in which carbon nucleotides are included, where an attacking nucleophile brings a pair of electrons to the carbon electrophile in order to form a new bond between the nucleophile and the carbon electrophile. Suitable carbon nucleophiles include but are not limited to alkyl, alkenyl, aphenyl and alkynyl Grignard, organolithium, organocmc, alkyl-, alkenyl-, aryl- and alkyl-1-tin (organocstanan) reagents, alkyl-, alkenyl- reagents, apl- and alqumil-borano (organoboranos and organoboronates); These carbon nucleophiles have the advantage of being synthetically stable in water or polar organic solvents. Other carbon nucleophiles include phosphorus iodides, enol and enolate reagents; these carbon nucleophiles have the advantage of being relatively easy to generate from well-known precursors for those skilled in the art of synthetic organic chemistry. Carbon nucleophiles, when used in conjunction with carbon electrophiles, generate new carbon-carbon bonds between the carbon nucleotide and the carbon electrophile. Carbonless nucleophiles suitable for coupling to carbon electrophiles include but are not limited to primary and secondary amines, thiols, thiolates and thioethers, alcohols, alkoxides, azides, semicarbazides and the like. These non-carbon nucleophiles, when used in conjunction with carbon electrophiles, commonly generate heteroatom bonds (C-X-C), wherein X is a heteroatom, for example, oxygen or nitrogen.

SAW . Non-natural Amino Acid Polypeptides The compositions and methods described herein provide for the incorporation of at least one non-natural amino acid to a polypeptide. The non-natural amino acid may be present at any site in the polypeptide in which any terminal position or any internal position of the polypeptide is included. Preferably, the non-natural amino acid does not destroy the activity and / or the tertiary structure of the polypeptide in relation to the amino acid polypeptide that is stably present in the homologous nature, unless such destruction of activity and / or tertiary structure was one of the purposes of incorporating the non-natural amino acid into the polypeptide. In addition, the incorporation of the non-natural amino acid into the polypeptide can modify the activity to some extent (eg, manipulate the therapeutic effectiveness of the polypeptide, improve the safety profile of the polypeptide, adjust the pharmacokinetics, pharmacology and / or pharmacodynamics of the polypeptide (eg. example, increase water solubility, bioavailability, increase the half-life in the serum, increase the therapeutic half-life, modulate the immunogenicity, modulate the biological activity or prolong the circulation time), provide additional functionality to the polypeptide, incorporate a label, marker or detectable signal to the polypeptide, use the properties of polypeptide isolation and any combination of the aforementioned modifications) and / or tertiary structure of the polypeptide in relation to the amino acid polypeptide that is stably exhibited in the homologous nature without fully causing destruction of the activity and / or tertiary structure . Such modifications of activity and / or tertiary structure are frequently one of the objectives of making such incorporations, although of course, incorporation of the non-natural amino acid into the polypeptide may also have little effect on the activity and / or tertiary structure of the polypeptide in relation to with the amino acid polypeptide that occurs stably in the homologous nature. Correspondingly, non-natural amino acid polypeptides, compositions comprising unnatural amino acid polypeptides, methods for making such polypeptides and polypeptide compositions, methods for purifying, isolating and characterizing such polypeptides and polypeptide compositions and methods for using such polypeptides and compositions of polypeptide are considered within the scope of the present disclosure. In addition, The non-natural amino acid polypeptides described herein may also be linked to another polypeptide (in which are included, by way of example, an unnatural amino acid polypeptide or an amino acid polypeptide that occurs stably in nature). The non-natural amino acid polypeptides described herein may be biosynthetically or non-biosynthetically produced. Biosynthetically means any method that uses a translation system (cellular or non-cellular), which includes the use of at least one of the following components: a polynucleotide, a cdton, a tRNA and a ribosome. Not biosynthetically means any method that does not use a translation system: this procedure can be further divided into methods using solid state peptide synthesis methods, solid phase peptide synthesis methods, methods using at least one enzyme and methods that do not use at least one enzyme; in addition any of these sub-divisions can be superimposed and many methods can use a combination of these sub-divisions. The methods, compositions, strategies and techniques described herein are not limited to a particular type, class or family of polypeptides or proteins. Of course, virtually any polypeptides can include at least one non-natural amino acid described herein. TO exemplary only, the polypeptide can be homologous to a therapeutic protein selected from the group consisting of: alpha-1 antitrypsin, angiostatin, antihemolytic factor, antibody, apolipoprotein, apoprotein, atrial natriuretic factor, atrial natriuretic polypeptide, atrial peptide, chemokine CXC , T39765, NAP-2, ENA-78, gro-a, gro-b, gro-c, IP-10, GCP-2, NAP-4, SDF-I, PF4, MIG, calcitonin, c-kit ligand, cytokine, CC chemokine, monocyte chemoattractant protein-1, monocyte chemoattractant protein-2, monocyte chemoattractant protein-3, onocito inflammatory alpha-1 protein, monocyte inflammatory beta-1 pro-enzyme, 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, peptide-78 activator of epithelial neutrophil, MIP-16, MCP-I, growth factor ep Erythropoietic peptide (EGF), neutrophil activating peptide, erythropoietin (EPO), exfoliating toxin, Factor IX, Factor VII, Factor VIII, Factor X, fibroblast growth factor (FGF), fibrinogen, fibronectin, four-helix bundle protein, G-CSF, glp-1, GM-CSF, glucocerebrosidase, gonadotropin, growth factor, growth factor receptor, grf, hedgehog protein, hemoglobin, hepatocyte growth factor (hGF), hirudin, human growth hormone ( hGH), human serum albumin, ICAM-I,ICAM-1 receptor, LFA-I, LFA-1 receptor, insulin, insulin-like growth factor (IGF), IGF-I, IGF-II, interferon (IFN), IFN-alpha, IFN-beta, IFN -gamma, any molecule similar to inferred or member of the IFN family, methylleukin (IL), IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL -8, IL-9, IL-10, IL-11, IL-12, keratinocyte growth factor (KGF), lactoferpna, leukemia inhibitory factor, luciferase, neurturm, neutrophil inhibiting factor (NIF), oncostat a M , osteogenic protein, oncogene product, paracytoma, parathyroid hormone, PD-ECSF, PDGF, peptide hormone, pleiotropin, pj_otein A, protein G, pth, exotoxma A pyrogenic, exotoxma B pyrogenic, exotoxma C pyrogenic, pyy, relaxin, reniña , SCF, small biosthetic protein, soluble complement receptor I, soluble I-CAM 1, soluble metalleucine receptor, soluble TNF receptor, somatomedma, somatostatin, somatotropy, streptoemase, superantigens, enterotoxin filococal, SEA, SEB, SEC1, SEC2, SEC3, SED, SEE, steroid hormone receptor, superoxide dismutase, toxic shock syndrome toxin, alpha 1 tymosta, 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) , uroemasa, mos, ras, raf, met, p53, tat, fos, myc, jun, myb, laugh, estrogen receptor, progesterone receptor, testosterone receptor, aldosterone receptor, LDL receptor and corticosterone. The non-natural amino acid polypeptide can also be homologous to any polypeptide member of the ++++ superfamily of the growth hormone supergene. Non-limiting examples of peptides / proteins that can incorporate non-natural amino acids containing an aromatic amine moiety are shown in Figures 20-34. The non-natural amino acid polypeptides can be further modified as described elsewhere in this disclosure or the non-natural amino acid polypeptide can be used without further modification. Incorporation of a non-natural amino acid into a polypeptide can be done for a variety of purposes, including, but not limited to, making changes in protein structure and / or function, changing size, acidity, nucleophilicity, linkage of hydrogen, hydrophobicity, access to the target sites of protease, targeting a portion (in which they include but not limited to, for an array of proteins), etc. Proteins that include an unnatural amino acid may have improved or even completely new catalytic or biophysical properties. By way of example only, the following properties are optionally modified by inclusion of a non-natural amino acid to a protein: toxicity, biodistribution, properties structural, spectroscopic properties, chemical and / or photochemical properties, catalytic ability, half-life (including, but not limited to, serum half-life), ability to react with other molecules, including but not limited to a, covalent or non-covalent and the like. Compositions that include proteins that include at least one non-natural amino acid are useful for, including, but not limited to, novel therapeutics, diagnostics, catalytic enzymes, industrial enzymes, binding proteins (in which but are not included) limited to, antibodies) and in which is included but not limited to, the study of protein structure and function. See, for example, Dougherty, (2000) Unna tural Amino Acids to Probes of Protein Structure and Function, Current Opinion in Chemical Biology. 4: 645-652. In addition, the lateral chain of the non-natural amino acid component (s) of a polypeptide can provide a wide range of additional functionality to the polypeptide; by way of example only and not as limitation, the side chain of the unnatural amino acid portion of a polypeptide can include any of the following: a label; a dye; a polymer; a water soluble polymer; a polyethylene glycol derivative; a photo-reticulator; a cytotoxic compound; a drug; an affinity marker; a photoaffinity marker; a reactive compound; a resin; a second protein or polypeptide or polypeptide analogue; an antibody or antibody fragment; a metal chelator; a co-factor; a fatty acid; a carbohydrate; a polmucleotide; a DNA; an RNA; an antisense polynucleotide; a saccharide, a water-soluble dendrimer, a cyclodextrm, a biomaterial; a nanoparticle; a spin marker; a fluorophore, a portion containing metal; a radioactive portion; a new functional group; a group that interacts covalently or non-covalently with other molecules; a photoengineered portion; an excitable portion by actinic radiation; a ligand; a photo-discernable portion; Diotn.a; a biotin analogue; a portion that incorporates a heavy atom; a group chemically cleaved it; a photo-escmdible group; an elongated side chain; a carbon-bonded sugar; a redox-active agent; an ammo thioacid; a toxic portion; an isotopically labeled portion; a biophysical probe; a phosphorescent group; a chemiluminescent group; a dense group of electrons; a magnetic group; a misleading group; a chromophore; an energy transfer agent; a biologically active agent; a detectable marker; a small molecule; an inhibitory ribonucleic acid; a radionucleotide; a neutron capture agent; a biotome derivative; quantum dot (s); a nanotransmitter; a radio transmitter; an abzyme, an activated complex activator, a virus, an adjuvant, an aglycan, an allergan, an angiostatin, an antihormone, an antioxidant, an aptamer, a guide RNA, a saponin, a shuttle vector, a macromolecule, a mimotope, a receptor, a reverse micelle and any combination thereof. In one aspect, the composition includes at least one protein with at least one, which includes but is not limited to, at least two, at least three, at least four, at least five, so minus six, at least seven, at least eight, at least nine or at least ten or more non-natural amino acids. The non-natural amino acids may be the same or different, in which it is included but not limited to, there may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more different sites in the protein comprising 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 another aspect, a composition includes a protein with at least one, but less than all, of a particular amino acid present in the protein is substituted with the non-natural amino acid. For a given protein with more than one non-natural amino acid, the non-natural amino acids may be identical or different (in which they include but are not limited to, the protein may include two or more different types of non-natural amino acids or may include two of the same non-natural amino acid). For a given protein with more than two non-natural amino acids, the Non-natural amino acids can be the same or different. Although embodiments of the non-natural amino acid polypeptides described herein can be chemically synthesized via solid phase peptide synthesis methods (e.g., on a solid resin), by peptide synthesis methods in solution phase and / or without the aid of enzymes, other embodiments of the non-natural amino acid polypeptides described herein allow synthesis via a cell membrane, cell extract or lysate system or via a system in vivo, that is, using the cell machinery of a prokaryotic cell or eukaryotic In d more embodiments or additional modalities, one of the key aspects of the non-natural amino acid polypeptides described herein is that they can be synthesized using ribosomes. In other embodiments or additional embodiments of the non-natural amino acid polypeptides described herein, the polypeptides of non-natural amino acids can be synthesized by a combination of solid resins, without the aid of enzymes, with the help of ribosomes, via a system in vitro, via an in vivo system or any combination thereof. The synthesis of non-natural amino acid polypeptides via ribosomes and / or in an in vivo system has distinct and characteristic advantages of an unnatural amino acid polypeptide synthesized on a solid resin or without the help of enzymes. These advantages or characteristics include different impurity profiles, a system that uses ribosomes and / or an in vivo system will have impurities that are derived from the biological system used, in which host cell proteins, membrane portions and lipids are included, while the impurity profile of a system using a solid resin and / or without the aid of enzymes will include organic solvents, protecting groups, resin materials, coupling reagents and other chemical compounds used in the synthesis processes. In addition, the isotopic pattern of the non-natural amino acid polypeptide synthesized via the use of ribosomes and / or an in vivo system will reflect the isotopic pattern of the feedstock used for the cells; on the other hand, the isotopic pattern of the non-natural amino acid polypeptide synthesized on a solid resin and / or without the aid of enzymes will reflect the isotopic pattern of the amino acids used in the synthesis. In addition, the non-natural amino acid synthesized via the use of ribosomes and / or an in vivo system will be substantially free of the D-isomers of the amino acids and / or be able to readily incorporate internal cysteine amino acids into the structure of the polypeptide, and / or will rarely provide internal amino acid cancellation polypeptides. On the other hand, an unnatural amino acid polypeptide synthesized via a solid resin and / or without the use of enzymes will have a higher content of D isomer of the amino acids and / or a lower content of internal cysteine amino acids and / or a higher percentage of internal amino acid cancellation polypeptides. In addition, one skilled in the art will be able to differentiate an unnatural amino acid polypeptide synthesized by the use of a ribosome and / or an in vivo system of an unnatural amino acid polypeptide synthesized via a solid resin and / or without the use of enzymes In certain embodiments, there is found a method of making a compound or salt thereof that contains at least one unnatural amino acid selected from the group consisting of: wherein the compound is formed by a reductive alkylation of an aromatic amino moiety on at least one non-natural amino acid comprising the aromatic amino moiety with at least one reagent comprising at least one portion of aldehyde; each Ra is independently selected from the group consisting of H, halogen, alkyl, - (NR ') 2, -C (0) R', C (0) N (R ') 2, -OR and -S (0) 2R ', where k is 1, 2 or 3; M is H or -CH2R5; or the portion M-N-C (R5) can form a ring structure of 4 to 7 members; Ri is H, an amino protecting group, resin, amino acid, polypeptide or polynucleotide; R2 is OH, an ester, resin, amino acid, polypeptide or polynucleotide protecting group; R5 is alkyl, substituted alkyl, ayanyl, substituted alkenyl, alkynyl, substituted alkynyl, substituted alkoxy, alkylalkoxy, substituted alkylalkoxy, polyalkylene oxide, substituted polyalkylene oxide, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocycle , substituted heterocycle, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl,, -C (0) R ", -C (0) 0R", -C (0) N (R ") 2, -C (0) NHCH ( R'J 2, - (alkylene or alkylene) -N (R ") 2 '- (alkenylene or substituted alkenylene) -N (RJ 2 / - (alkylene or substituted alkylene) - (aryl or substituted aryl), - (alkenylene or substituted alkenylene) - (aryl or substituted aryl), - (alkylene or substituted alkylene) -ON (R ") 2, - (alkylene or substituted alkylene) -C (0) SR", - (alkylene or substituted alkylene) - SS- (aryl or substituted aryl), wherein each R "is independently hydrogen, alkyl, alkyl substituted, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocycle, substituted heterocycle, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl or -C (0) 0R '; or R5 is L-X, wherein X is selected from the group consisting of a label; a dye; a polymer ", a water-soluble polymer, a polyethylene glycol derivative, a photocrosslinker, a cytotoxic compound, a drug, an affinity label: a photoaffinity marker, a reactive compound, a resin, a second protein or polypeptide or analogue polypeptide, an antibody or antibody fragment, a metal chelator, a co-factor, a fatty acid, a carbohydrate, a polynucleotide, a DNA, an RNA, an antisense polynucleotide, a saccharide, a water-soluble dendrimer, a cyclodextrin a biomaterial, a nanoparticle, a spin marker, a fluorophore, a metal-containing portion, a radioactive portion, a new functional group, a group that interacts covalently or non-covalently with other molecules, a photoenaged portion, a portion excitable by actinic radiation, a ligand, a photoisomerizable portion, biotin, a biotin analogue, a portion that incorporates a heavy atom, a chemical cleavable group mind, a photocleavable group; an elongated side chain; a carbon-bonded sugar; a redox-active agent; an amino thioacid; a toxic portion; an isotopically labeled portion; a biophysical probe; a phosphorescent group; a chemiluminescent group; a dense group of electrons; a magnetic group; an intercalary group; a chromophore; an energy transfer agent; a biologically active agent; a detectable marker; a small molecule; an inhibitory ribonucleic acid; a radionucleotide; an electron capture agent; a biotin derivative; quantum dot (s); a nanotransmitter; a radio transmitter; an abzyme, an activated complex activator, an ius, an adjuvant, an aglycan, an allergan, an angiostatin, an antihormone, an antioxidant, an aptamer, a guide RNA, a saponin, a shuttle vector, a macromolecule, a mimotope, a receptor, a reverse micelle and any combination thereof and L is optional and when present is a linker selected from the group consisting of alkylene, substituted alkylene, alkenylene, substituted alkenylene, -0-, -0- ( alkylene or substituted alkylene), - (alkylene or substituted alkylene) -0-, -C (0) -, -C (0) - (alkylene or substituted alkylene), (alkylene or substituted alkylene) -C (0) -, -C (0) N (R ') -, -C (0) N (R') - (alkylene or substituted alkylene) -, - (alkylene or substituted alkylene) -0C (0) N (R ') -, -N (R ') C (0) -, -N (R') C (0) - (alkylene or substituted alkylene) -, (alkylene or substituted alkylene) -NR 'C (0) -, -S-, -S- (alkylene or substituted alkylene) -, -S (0) k- where k is 1, 2 or 3, -S (O) and- (alkylene) no or substituted alkylene) -, -C (S) -, -C (S) - (alkylene or substituted alkylene) -, CSN (R ') -, CSN (R') - (alkylene or substituted alkylene) -, -N (R ') CO- (alkylene or substituted alkylene) - , -N (R ') C (0) 0-, - (alkylene or substituted alkylene) -0-N = CR' -, - (alkylene or substituted alkylene) -C (0) NR '- (alkylene or substituted alkylene) ) -, (alkylene or substituted alkylene) -S (0) k- (alkylene or substituted alkylene) -S-, - (alkylene or substituted alkylene) -SS-, S (0) kN (R ') -, N ( R ') C (0) N (R') -, -N (R ') C (S) N (R') -, -N (R ') S (0) kN (R') -, -N (R ') - N =, -C (R') = N-, -C (R ') = NN (R') -, -C (R ') = NN =, -C (R') 2- N = N and -C (R ')? -N (R') -N (PJ) - or R5 and any Ra optionally form a cycloalkyl or heterocycloalkyl and each R 'is independently H, alkyl or substituted alkyl. In other embodiments, there is found a method of manufacturing a compound or salt thereof, wherein Ri and R2 of at least one non-natural amino acid is H and OH respectively. In some embodiments, a method of making a compound is found, wherein Ra of at least one non-natural amino acid is a halogen. In other embodiments, there is found a method of making a compound or salt thereof, wherein both Ri and R2 of at least one non-natural amino acid are polypeptides. In some embodiments, there is found a method of making a compound or salt thereof, wherein X of at least one non-natural amino acid is a biologically active agent selected from the group consisting of a peptide, protein, enzyme, antibody, drug. , dye, lipid, nucleoside, oligonucleotide, cell, virus, liposome, microparticle and micelle. In some embodiments, there is found a method of making a compound or salt thereof, wherein X of at least one non-natural amino acid is a drug selected from the group consisting of an antibiotic, fungicide, antiviral agent, anti-inflammatory agent, anti-inflammatory agent, anti-inflammatory agent, anti-inflammatory agent, anti-inflammatory agent, anti-inflammatory agent. -tumor, cardiovascular agent, anti-anxiety agent, hormone, growth factor and spheroidal agent. In other embodiments, a method of making a compound c salt thereof is found, and X of at least one non-natural amino acid is an enzyme selected from the group consisting of horseradish peroxidase, alkaline phosphatase, β-galactosidase and glucose oxidase. In some embodiments, there is found a method of making a compound or salt thereof, wherein X of at least one unnatural amino acid is a detectable marker selected from the group consisting of a fluorescent, phosphorescent, chemiluminescent, chelating moiety, group electron dense, magnetic portion, spider, radioactive, chromophobic and energy transfer portion. In further embodiments there is found a method of manufacturing a compound or salt thereof, wherein X of the non-natural amino acid is a polymer comprising alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkyl, substituted alkyl, alkoxy, substituted alkoxy, alkylalkoxy, substituted alkyloxy, oxide of polyalkylene, substituted polyalkylene oxide, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkaryl, substituted alkaryl, aralkyl or substituted aralkyl. In other embodiments, there is found a method of making a compound or salt thereof, wherein the polymer comprises a polyalkylene oxide or substituted polyalkylene oxide. In some embodiments, there is found a method of manufacturing a compound or salt thereof, wherein the polymer comprises - [(alkylene or substituted alkylene) -O- (hydrogen, alkyl or substituted alkyl)] x, wherein x is 20 -10,000. In other embodiments, there is found a method of making a compound or salt thereof, wherein the polymer is m-PEG having a molecular weight ranging from about 2 to about 40 KDa. In further embodiments, there is found a method of making a compound or salt thereof, wherein at least one non-natural amino acid is selected from the group: where x is a halogen. In some modalities there is a method of making a compound or salt thereof wherein at least one non-natural amino acid is selected from the group: In further embodiments, there is found a method of making a compound or salt thereof, wherein the at least one non-natural amino acid is selected from the group it consists of where x is a halogen, In other embodiments, there is found a method of making a compound or salt thereof that contains at least one unnatural amino acid selected from the group consisting of: wherein the compound is formed by a reductive alkylation of an aromatic amino moiety on at least one non-natural amino acid comprising the aromatic amino moiety with at least one reactive comprising at least a portion of aldehyde; each Ra is independently selected from the group consisting of H, halogen, alkyl, - (NR ') 2, -C (0) R', C (0) N (R ') 2, -OR and -S (0) 2R ', where k is 1, 2 or 3; Ri is H, an amino protecting group, resin, amino acid, polypeptide or polynucleotide and R2 is OH, an ester, resin, amino acid, polypeptide or polynucleotide protecting group; each R3 and R4 is independently H, halogen, lower alkyl or substituted lower alkyl or R3 and R4 or two R3 groups optionally form a cycloalkyl or heterocycloalkyl; M is H or -CH2R5; or the portion M-N-C (R5) can form a ring structure of 4 to 7 members; R5 is alkyl, substituted alkyl, ayl, substituted ayl, alkynyl, substituted alkynyl, substituted alkoxy, alkylalkoxy, substituted alkylalkoxy, polyalkylene oxide, substituted polyalkylene oxide, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocycle, substituted heterocycle , alkaryl, substituted alkaryl, aralkyl, substituted aralkyl,, -C (0) R ", -C (0) OR", -C (0) N (R ") 2, -C (0) NHCH (R'J 2, - (alkylene or alkylene) -N (R ") 2 '- (aylene or substituted aylene) -N (R") 2, - (alkylene or substituted alkylene) - (aryl or substituted aryl), - (aylene or substituted aylene) - (substituted aryl or aryl), - (alkylene or substituted alkylene) -ON (R ") 2, - (alkylene or substituted alkylene) -C (0) SR", - (alkylene or substituted alkylene) -SS - (aryl or substituted aryl), wherein each R "is independently hydrogen, alkyl, substituted alkyl, ayl, substituted ayl, alkoxy, substituted alkoxy, ryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocycle, substituted heterocycle, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl or -C (0) OR '; or R5 is L-X, where X is selected from the group that consists of a marker; a dye; a polymer; a water soluble polymer; a polyethylene glycol derivative; a photocrosslinker; a cytotoxic compound; a drug; an affinity marker; a photoaffinity marker; a reactive compound; a resin; a second protein or polypeptide or polypeptide analogue; an antibody or antibody fragment; a metal chelator; a co-factor; a fatty acid; a carbohydrate; a polynucleotide; a DNA; an RNA; an antisense polynucleotide; a saccharide, a water soluble dendrimer, a clodextrin, a biomaterial; a nanoparticle; a spin marker; a fluorophore, a portion containing metal; a radioactive portion; a new functional group; a group that interacts covalently or non-covalently with other molecules; a photoenaged portion; an excitable portion by actinic radiation; a ligand; a photoisomerizable portion; biotin; a biotin analogue; a portion that incorporates a heavy atom; a group chemically cleaved it; a photocleavable group; an elongated side chain; a carbon-bonded sugar; a redox-active agent; an amino thioacid; a toxic portion; an isotopically labeled portion; a biophysical probe; a phosphorescent group; a chemiluminescent group; a dense group of electrons; a magnetic group; an intercalary group; a chromophore; an energy transfer agent; a biologically active agent; a detectable marker; a small molecule; a ribonucleic acid inhibitor; a radionucleotide; an electron capture agent; a biotin derivative; quantum dot (s); a nanotransmitter; a radio transmitter; an abzyme, an activated complex activator, a virus, an adjuvant, an aglycan, an allergen, an angiostatin, an antihormone, an antioxidant, an aptamer, a guide RNA, a saponin, a shuttle vector, a macromolecule, an mimotope, a receptor, a reverse micelle and any combination thereof and L is optional and when present is a linker selected from the group consisting of alkylene, substituted alkylene, alkenylene, substituted alkenylene, -O-, -O- (alkylene) or substituted alkylene), - (alkylene or substituted alkylene) -0-, -C (0) -, -C (0) - (alkylene or substituted alkylene), (alkylene or substituted alkylene) -C (0) -, - C (0) N (R ') -, -C (0) N (R') - (alkylene or substituted alkylene) -, - (alkylene or substituted alkylene) -0C (0) N (R ') -, - N (R ') C (0) -, -N (R') CJ 0) - (alkylene or substituted alkylene) -, (alkylene or substituted alkylene) -NR 'C (0) -, -S-, -S - (alkylene or substituted alkylene) -, -S (0) k- where k is 1, 2 or 3, -S (0) - (alkyl) ene or substituted alkylene) -, -C (S) -, -C (S) - (alkylene or substituted alkylene) -, CSN (R ') -, CSN (R') - (alkylene or substituted alkylene) -, - N (R ') C0- (alkylene or substituted alkylene) -, -N (R') C (0) 0-, - (alkylene or substituted alkylene) -0-N = CR '-, - (alkylene or substituted alkylene) ) -C (0) NR '- (alkylene or substituted alkylene) -, (alkylene or substituted alkylene) -S (0) k- (alkylene or substituted alkylene) - S-, - (alkylene or substituted alkylene) -S-S-, S (0) kN (R ') -, N (R ') C (0) N (R') -, - N (R ') C (S) N (R') -, - N (R ') S (O) kN (R') -, -N (R ') - N =, -C (R') = N-, -C (R ') = NN (R') -, -C (R ') = NN =, -C (R') 2-N = N and - C (R ') 2-N (R') -N (R ') -; or two R5 groups optionally form a cycloalkyl or a heterocycloalkyl; or R5 and any Ra optionally form a cycloalkyl or a heterocycloalkyl; with the proviso that when Ri is H, then R2 is not OH or when R2 is OH then Ri is not H; and G is an amine protecting group. In some embodiments, a method of fab_ing a compound or salt thereof is found wherein the amine protecting group is selected from the group consisting of: In other embodiments, there is found a method of making a compound or salt thereof wherein both Ri and R2 are polypeptides. In some embodiments, there is found a method of manufacturing a compound or salt thereof, wherein at least a portion of the aldehyde has the structure corresponding to: where; R5 is alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, alkylalkoxy, substituted alkylalkoxy, polyalkylene oxide, substituted pclyalkylene oxide, cycloalkyl, substituted cycloaicyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl , heterocycle, substituted heterocycle, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl, -C (0) R ", -C (0) OR", -C (0) N (R ") 2, -C (0) NHCH (R'J 2, - (alkylene or substituted alkylene) -N (R'J 2, - (alkenylene or substituted alkenylene) -N (R ") 2, - (alkylene or substituted alkylene) - (aryl or substituted aryl) , - (alkenylene or substituted alkenylene) - (aryl or substituted aryl), - (alkylene or substituted alkylene) -ON (R ") 2, - (alkylene or substituted alkylene) -C (O) SR", - (alkylene or substituted alkylene) -SS- (aryl or substituted aryl), wherein each R "is independently hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, coxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocycle, substituted heterocycle, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl or C (0) OR '; or R5 is L-X, wherein, X is selected from the group consisting of a label; a dye; a polymer; a water soluble polymer; a polyethylene glycol derivative; a photocrosslinker; a cytotoxic compound; a drug; an affinity marker; a photoaffinity marker; a reactive compound; a resin; a second protein or polypeptide or polypeptide analogue; an antibody or antibody fragment; a metal chelator; a co-factor; a fatty acid; a carbohydrate; a polynucleotide; a DNA; an RNA; an antisense polynucleotide; a liquid, a water-soluble dendrimer, a cyclodextrin, a biomaterial; a nanoparticle; a spin marker; a fluorophore, a portion containing metal; a radioactive portion; a new functional group; a group that interacts covalently or non-covalently with other molecules; a photoenaged portion; an excitable portion by actinic radiation; a ligand; a photoisomerizable portion; biotin; a biotin analogue; a portion that incorporates a heavy atom; a chemically cleavable group; a photocleavable group; an elongated side chain; a carbon-bonded sugar; a redox-active agent; an amino thioacid; a toxic portion; an isotopically labeled portion; a biophysical probe; a phosphorescent group; a chemiluminescent group; a dense group of electrons; a magnetic group; an intercalary group; a chromophore; an energy transfer agent; a biologically active agent; a marker detectable; a small molecule; an inhibitory ribonucleic acid; a radionucleotide; an electron capture agent; a biotin derivative; quantum dot (s); a nanotransmitter; a radio transmitter; an abzyme, an activated complex activator, a virus, an adjuvant, an aglycan, an allergan, an angiostatin, an antihormone, an antioxidant, an aptamer, a guide RNA, a saponin, a shuttle vector, a macromolecule, an mimotope, a receptor, a reverse micelle and any combination thereof and L is optional and when a linker selected from the group consisting of alkylene, substituted alkylene, alkenylene, substituted alkenylene, -0-, -0- (alkylene) is present. or substituted alkylene), - (alkylene or substituted alkylene) -0-, -C (0) -, -C (0) - (alkylene or substituted alkylene), (alkylene or substituted alkylene) -C (0) -, -C (0) N (R ') -, -C (0) N (R') - (alkylene or substituted alkylene) -, - (alkylene or substituted alkylene) -0C (0) N (R ') -, -N (R ') C (0) -, -N (R') C (0) - (alkylene or substituted alkylene) -, (alkylene or substituted alkylene) -NR 'C (0) -, -S-, -S- (alkylene or substituted alkylene) -, -S (0) k- where k is 1, 2 or 3, -S (0) k- (alkyl nickel or substituted alkylene) -, -C (S) -, -C (S) - (alkylene or substituted alkylene) -, CSN (R ') -, CSN (R') - (alkylene or substituted alkylene) -, - N (R ') C0- (alkylene or substituted alkylene) -, -N (R') C (0) 0-, - (alkylene or substituted alkylene) -0-N = CR '-, - (alkylene or substituted alkylene) ) -C (0) NR '- (alkylene or substituted alkylene) -, (alkylene or substituted alkylene) -S (O) k- (alkylene or substituted alkylene) -S-, - (alkylene or substituted alkylene) -SS-, S (0) kN (R ') -, -N (R') C ( 0) N (R ') -, - N (R') C (S) N (R ') -, - N (R') S (0) kN (R ') -, - N (R') - N =, -C (R ') = N-, -C (R') = NN (R ') -, -C (R') = NN =, -C (R ') 2 -N = N and - C (R ') 2"N (R') - N (R ') - In other embodiments, there is found a method of manufacturing a compound or salt thereof having the structure of: wherein L 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 heterocyclealkylene, substituted lower heterocyclealkylene, arylene, substituted arylene , heteroarylene, substituted heteroarylene, 'alkarylene, substituted alkarylene, aralkylene or substituted aralkylene; Q 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, -0- (alkylene or substituted alkylene) -, -S- (alkylene or substituted alkylene) -, -S (0) k (alkylene or substituted alkylene) -, where k is 1, 2 or 3, -C (0) - (alkylene or substituted alkylene) -, -C (S) - (alkylene or substituted alkylene) -, -NR '- (alkylene or substituted alkylene) -, -CON (R ") - (alkylene or substituted alkylene) -, -CSN (R ') - (alkylene or substituted alkylene) -, -N (R') C0- (alkylene or substituted alkylene) -, wherein each R 'is independently H, alkyl or substituted alkyl; Rx is H, an amino, resin, amino acid, polypeptide or polynucleotide protecting group, and R2 is OH, an ester, resin, amino acid, polypeptide or polynucleotide protecting group, each of R3 and R4 is independently H, halogen, lower alkyl or substituted lower alkyl or R3 and R4 or two R3 groups optionally form a cycloalkyl or a heterocycloalkyl, each Ra is independently selected from the group consisting of H, halogen, alkyl ilo, -N02, -CN, substituted alkyl, -N (R ') 2, -C (0) kR', -C (0) N (R ') 2, -OR' and -S (0) kR ' , where k is 1, 2 or 3; R5 is a protected aldehyde or a masked aldehyde, wherein the protective group includes, but is not limited to, where each Xi is independently selected from the group consisting of -O-, -S-, -N (H) -, -N (R) -, -N (Ac) - and -N (OMe) -; X2 is -OR, -OAc, -SR, -N (R) 2, -N (R) (Ac), -N (R) (OMe) or N3, and wherein each R 'and R is independently H, alkyl or substituted alkyl. In other embodiments, there is found a method of making a compound or salt thereof wherein Xi is O. In some embodiments, there is found a method of making a compound or salt thereof wherein both Ri and R2 are polypeptides.

VII. Compositions and methods comprising ncs acids and oligonucleotides A. General methods of recombinant nucleic acid for use herein In numerous embodiments of the methods and compositions described herein, nucleic acids encoding a polypeptide of interest (in which are included by way of example a GH polypeptide) will be isolated, cloned and frequently altered using recombinant methods. Such modalities are used, in which but not limited to, for expression of protein or during the generation of variants, derivatives, expression cassettes or other sequences derived from a polypeptide. In some embodiments, the sequences encoding the polypeptides are operably linked to a promoter heterologous Also described herein are cells that produce non-natural amino acid polypeptides, wherein at least one unnatural amino acid on the polypeptide comprises a side chain having an aromatic amine moiety or a masked or protected aldehyde moiety. Cells that biosynthesize at least one unnatural amino acid polypeptide can be produced using the techniques, methods, compositions and strategies described herein or variants thereof. A nucleotide sequence that encodes a polypeptide comprising an unnatural amino acid can be synthesized based on the amino acid sequence of the original polypeptide and then change the nucleotide sequence to effect the introduction (i.e., incorporation or substitution) or removal ( is, cancellation or substitution) of the relevant amino acid residue (s). The nucleotide sequence can be conveniently modified by site-directed mutagenesis according to conventional methods. Alternatively, the nucleotide sequence can be prepared by chemical synthesis, which include but are not limited to, by using an oligonucleotide synthesizer, wherein the oligonucleotides are designed based on the amino acid sequence of the desired polypeptide and preferably selecting those codons that are favored in the host cell in which the recombinant polypeptide will be produced. For example, several small oligonucleotides encode portions of the desired polypeptide can be synthesized and assembled by PCR, ligation or ligation chain reaction. See, for example, Barany, et al., Proc. Nati Acad. Sci. 88: 189-193 (1991); US Patent 6,521,427 which are incorporated by reference herein. The methods and compositions of non-natural amino acids described herein utilize routine techniques in the field of recombinant genetics. Basic texts that disclose the general methods of use for the non-natural amino acid methods and compositions described herein include Sambrook et al., Molecular Cloning, A La bora tory Manua l (3rd ed., 2001); Kriegler, Gene Transfer and Expression: A Labora tory Manual (1990); and Curren t Protocol s in Molecular Biology (Ausubel et al., eds., 1994)). General texts describing molecular biology techniques include Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology Volume 152 Academic Press, Inc., San Diego, CA. (Berger); Sambrook et al., Molecular Cloning - A Laboratory Manual (2nd Ed-). Vol. 1-3. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1989 ("Sambrook") and Current Protocols in Molecular Biology, F.M. Ausubel et al., Eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (++++ supplemented through 1999) ("Ausubel")). These texts describe mutagenesis, the use of vectors, promoters and many other relevant topics, including, but not limited to, the generation of genes or polynucleotides that include codons selectors for production of proteins that include non-natural amino acids, orthogonal tRNAs , orthogonal synthetases and pairs of them. Various types of mutagenesis are used in the non-natural amino acid methods and compositions described herein for a variety of purposes, including, but not limited to, producing new synthetases or tRNA, for mutating tRNA molecules, for mutating polynucleotides that encode synthetases, to produce tRNA libraries, to produce libraries of synthetases, to produce selector codons, to insert selector codons that encode non-natural amino acids into a protein or polypeptide of interest. They include but are not limited to site-directed point mutagenesis, homologous recombination, DNA intermixing or other methods of recursive mutagenesis, chimeric construction, mutagenesis using uracil-containing templates, oligonucleotide-directed mutagenesis, phosphorothioate-modified DNA mutagenesis, mutagenesis using DNA duplex with spaces or the like, or any combination of the same. Additional appropriate methods include repair of point mismatch, mutagenesis using host strains deficient in repair, restriction-selection and restriction-purification, cancellation mutagenesis, mutagenesis by total gene synthesis, double-strand rupture repair and the like. Mutagenesis, in which but not limited to, which involves chimeric constructs, are also included in the methods and compositions of non-natural amino acids described herein. In one embodiment, mutagenesis can be guided by knowing information of the molecule that is stably presented in the nature or molecule that is stably present in the mutated nature, in which it includes, but is not limited to, sequence, comparisons of sequence, physical properties, crystalline structure or the like. The texts and examples found herein describe these procedures. Additional information can be found in the following publications and references cited therein: Ling et al., Approa ches to DNA mutagenesis: an overview, Anal Biochem. 254 (2): 157-178 (1997); Dale et al., Oligonucleotide-directed random mutagenesis using the phosphorothioa te method, Methods Mol. Biol. 57: 369-374 (1996); Smith, In Vi tro mutagenesis, Ann. Rev. Genet. 19: 423-462 (1985); Botstein & Shortle, Stra tegies and appli cations of in vi tro mu tagenesis, Science 229: 1 193-1201 (1985); Carter, Site-directed mutagenesis, Biochem. J. 237: 1-7 (1986); Kunkel, The efficiency of oligonucleotide directed mutagenesis, m Nucleic Acids & Molecular Biology (Eckstem, F. and Lilley, D.M.J, eds., Sprmger Veilag, Berlin)) (1987); Kunkel, Rapid and efficient site-specific mutagenesis without phenotypic selection, Proc. Nati Acad. Sci. USA 82: 488-492 (1985); Kunkel et al., Rapid and efficient site-specific mutagenesis without phenotypic selection, Methods m Enzymol. 154, 367-382 (1987); Bass et al., Mutant Trp repressors with new DNA-bmding specificities, Science 242: 240-245 (1988); Zoller & Smith, Oligonucleotide-directed mutagenesis using M13-der? Ved vectors: an efficient and general procedure lor the production of point mutations m any DNA fragment, Nucleic Acids Res. 10: 6487-6500 (1982); Zoller & Smith, Oligonucleotide-directed mutagenesis of DNA fragments cloned into M13 vectors, Methods in Enzymol. 100: 468-500 (1983); Zoller & Smith, Oligonucleotide-directed mutagenesis: a simple method using two oligonucleotide ppmers and a smgle-stranded DNA template, Methods m Enzymol. 154: 329-350 (1987); Taylor et al., The use of phosphorothioate-modified DNA m restriction enzyme reactions to prepare nicked DNA, Nucí. Acids Res. 13: 8749-8764 (1985); Taylor et al., The rapid generation of oligonucleotide-directed mutations at high frequency using phosphorothioate-modified DNA, Nucí. Acids Res. 13: 8765-8785 (1985); Nakamaye & Eckstem, Inhibition of restriction endonuclease Net 1 cleavage by phosphorothioate groups and application to oligonucleotide-directed mutagenesis, Nucí. Acids Res. 14: 9679-9698 (1986); Sayers et al., 5 '-3' Exonucleases m phosphorothioate-based oligonucleotide-directed utagenesis, Nuci. Acids Res. 16: 791-802 (1988); Sayers et al., Strand specific cleavage of phosphor othioate-containmg DNA by reaction with restriction endonucleases in the presence of ethidium bromide, (1988) Nucí. Acids Res. 16: 803-814; Kramer et al., The gapped duplex DNA approach to oligonucleotide-directed mutation construction, Nucí. Acids Res. 12: 9441-9456 (1984); Kramer & Fritz Oligonucleotide-directed construction of mutations via gapped duplex 5 DNA, Methods in Enzymol. 54: 350-367 (1987); Framer et al., Improved enzymatic m vitro reactions m the gapped duplex DNA approach to oligonucleotide-directed construction of mutations, Nucí. Acids Res. 16: 7207 (1988); Fptz et al., Oligonucleotide-directed construction of mutations: a gapped duplex DNA procedure without enzymatic reactions m vitro, Nucí. Acids Res. 16: 6987-6999 (1988); Kramer et al., Point Mismatch Repair, Cell 38: 879-887 (1984); Cárter et al., Improved oligonucleotide site-directed mutagenesis using M13 vectors, Nucí. Acids Res. 13: 4431-4443 (1985); Crankcase, Improved oligonucleotide-directed mutagenesis using M13 vectors, Methods m Enzymol. 154: 382-403 (1987); Eghtedarzadeh & Henikoff, Use of oligonucleotides to genérate large deletions, Nucí. Acids Res. 14: 5115 (1986); Wells et al., Importance of hydrogen-bond formation m stabilizmg the transition state of subtilism, Phil. Trans. R. Soc. Lond. A 317: 415-423 (1986); Nambiar et al., Total synthesis and cloning of a gene codon for the pbonuclease S protein, Science 223: 1299-1301 (1984); Sakmar and Khorana, Total synthesis and expression of a gene for the alpha subunit of bovme rod outer segment guanine nucleotide-b Lndmg protein (transducm), Nucí. Acids Res. 14: 6361-6372 (1988); Wells et al., Cassette mutagenesis: an efficient method for generation of multiple mutations at defined sites, Gene 34: 315-323 (1985); Grundstrom et al., Oligonucleotide-directed mutagenesis by microscale Xhot-gun 'gene synthesis, Nucí. Acids Res. 13: 3305-3316 (1985); Mandecki, Oligonucleotide-directed double-strand break repair plasmids of Eschepchia coli: a method for site-specific mutagenesis, Proc. Nati Acad. Sci. USA. 83: 7177-7181 (1986); Arnold, Protein engmeepng for unusual environments, Current Opinion in Biotechnology 4: 450-455 (1993); Sieber, et al., Nature Biotechnology, 19: 456-460 (2001). W. P. C. Stemmer, Nature 370, 389-91 (1994); and, I. Lorimer, I. Pastan, Nucleic Acids Res. 23, 3067-8 (1995). Additional details regarding many of the above methods can be found in Methods m Enzymology Volume 154, which also describes useful controls for solving problems with various methods of mutagenesis. The methods and compositions described herein also include the use of eukaryotic host cells, non-eukaryotic host cells and organisms for the in vivo incoporation of a non-natural amino acid via orthogonal tRNA / RS pairs. Host cells are genetically engineered (in which they include but are not limited to, transformed, transduced or transfected) with the polynucleotides corresponding to the polypeptides described herein or constructs that include a polynucleotide corresponding to the polypeptides described herein, in those which include but are not limited to a vector corresponding to the polypeptides described herein, which may be, for example, a cloning vector or an expression vector. For example, the coding regions for the orthogonal tRNA, the orthogonal tRNA synthetase and the protein to be derived are operably linked to gene expression control elements that are functional in the desired host cell. The vector can be, for example, in the form of a plasmid, a cosmid, a phage, a bacterium, a virus, a naked polynucleotide or a conjugated polynucleotide. The vectors are introduced into the cells and / or microorganisms by standard methods in which electroporation is included (Fromm et al., Proc. Nati, Acad. Sci. USA 82, 5824 (1985), infection by viral vectors, ballistic penetration of high speed by small particles with the nucleic acid either within the matrix of beads or small particles or on the surface (Klein et al., Nature 327.70-73 (1987)), and / or the like.

The engineered host cells can be cultured in modified conventional nutrient media as appropriate for needs such as for example selection steps, promoter activation or selection of transformants. These cells can optionally be cultured in transgenic organisms. Other useful references, which include but are not limited to for cell culture and isolation (eg, for subsequent nucleic acid isolation) include Freshney (1994) Culture of Animal Cells, a Manual of Basic Technique, third edition, Wiley- Liss, New York, and the references cited therein; Payne et al. (1992) Plant Cell and Tissue Culture in Liquid Systems John Wiley & Sons, Inc. New York, NY; Gamborg and Phillips (eds) (1995) Plant Cell, Tissue and Organ Culture; Fundamental Methods Springer Lab Manual, Springer-Verlag (Berlin Heidelberg New York) and Atlas and Parks (eds) The Handbook of Microbiological Media (1993) CRC Press, Boca Raton, FL. Several well-known methods for introducing target nucleic acids into cells are available, any of which can be used in the methods and compositions described herein. These include: fusion of the recipient cells with bacterial protoplasts containing the DNA, electroporation, projectile bombardment and infection with viral vectors (discussed further below in FIG. present), etc. Bacterial cells can be used to amplify the number of plasmids containing DNA constructs corresponding to the polypeptides described herein. The bacteria are grown to logarithmic phase and the plasmids within the bacteria can be isolated by a variety of methods known in the art (see, for example, Sambrook). In addition, kits are commercially available for the purification of bacterial plasmids, (see, for example, EasyPrep ™, FlexiPrep ™, both from Pharmacia Biotech, StrataClean ™, from Stratagene, and QIAprep ™ from Qiagen). Then the isolated and purified plasmids are further manipulated to produce other plasmids, used to transfect cells or incorporated into related vectors to infect organisms. Typical vectors contain transcription and translation thermometers, transcription and translation initiation sequences and promoters useful for the regulation of the expression of the particular target nucleic acid. The vectors optionally comprise generic expression cassettes containing at least one independent thermometer sequence, sequences that allow replication of the cassette in eukaryotes or procapone, or both, (in which, but not limited to, shuttle vectors) and selection markers for both prokaryotic and eukaryotic systems. The vectors are suitable for replication and integration in procapontes, eucapone or both of them. See, Gillam & Smith, Gene 8:81 (1979); Roberts, et al., Nature. 328: 731 (1987); Schneider, E., et al., Protein Expr. Purif. 6 (1) -10-14 (1995); Ausubel, Sambrook, Berger (all above). A catalog of bacteria and bacteriophages useful for cloning is provided for example by the ATCC, for example, The ATCC Catalog of bacteria and bacteriophage (1992) Gherna et al. (eds) published by the ATCC. Additional basic procedures for sequencing, cloning and other aspects of molecular biology and fundamental theoretical considerations are also found in Watson et al. (19D2) Recombinant DNA Second Edition Scientific American Books, NY. In addition, essentially any nucleic acid (and virtually any labeled nucleic acid, either standard or non-standard) can be ordered upon request or standard order from any of a variety of commercial sources, such as the Midland Certified Reagent Company (Midland, TX mcrc.com), The Great American Gene Company (Ramona, CA available on the world wide web at genco.com), ExpressGen Inc. (Chicago, IL available on the world wide web at expressgen.com), Operon Technologies Inc. ( Alameda, CA) and many others.

B. Selector Codes Sequence codons encompassed within the methods and compositions described herein expand the genetic codon structure of the protein biosynthetic machinery. For example, a selector codon includes, but is not limited to, a single three-codon, a nonsense codon, such as a retention codon, including, but not limited to, an amber codon (UAG), an ocher codon or an opal codon (UGA), an unnatural codon, a codon of four or more bases, a rare codon or the like. There is a wide range in the number of selector codons that can be introduced to a desired gene or polynucleotide, which includes but is not limited to, one or more, two or more, three or more, 4, 5, 6, 7 , 8, 9, 10 or more in a single poiinucleotide encoding at least a portion of a polypeptide of interest. In one embodiment, the methods involve the use of a selector codon that is a retention codon for the incorporation of one or more non-natural amino acids in vivo. For example, an O-tRNA is produced that recognizes the retention codon, in which it is included but not limited to, UAG and is aminoacylated by an ORS with a desired unnatural amino acid. This O-tRNA is not recognized by the aminoacyl-tRNA synthetase of the host that occurs stably in nature. Conventional site-directed mutagenesis can be used to introduce the retention codon, in which, but not limited to, TAG, is included in the site of interest in a polypeptide of interest. See, for example, Sayers, J.R., et al. (1988), 5 '-3' Exonuclease in phosphorothioa te-ba sed ol i gonucleotide-directed mutagenesis. Nucleic Acids Res. 16: 791-802. When the O-RS, the O-tRNA and the nucleic acid encoding the polypeptide of interest are combined in vivo, the unnatural amino acid is incorporated in response to the UAG codon to give a polypeptide containing the unnatural amino acid in the position specified Non-natural amino acids can also be encoded with rare codons. For example, when the concentration of argmam in an in vitro protein synthesis reaction is reduced, the rare argmma codon AGG, has proved efficient for the insertion of Ala by a synthetic acrylated tRNA with allanma. See, for example, Ma et al., Biochemistry. 32: 7939 (1993). In this case, synthetic tRNA competes with the tRNAArg, which occurs stably in nature, which exists as a minor species in Escherichia coli. Some organisms do not use all triplet codons. An unassigned codon AGA in Micrococcus luteus has been used for the insertion of amino acids into a m vitro transcription / translation extract. See, for example, Kowal and Oliver, Nucí. Acid Res., 25: 4685 (1997). The components of the present invention can be generated to use these rare codons m alive. The incorporation of non-natural amino acids in vivo can be done without significant alteration of the eukaryotic host cell. For example, because the efficiency The deletion for the UAG codon depends on the competition between the O-tRNA, which includes but is not limited to, the amber suppressor tRNA and a eukaryotic release factor (in which, but not limited to, eRF) is included ( which binds to a retention codon and initiates the release of the growing peptide from the ribosome), the suppression efficiency can be modulated by including, but not limited to, increasing the level of expression of O-tRNA and / or suppressor tRNA. Selector codons also comprise extended codons, which include but are not limited to, codons of four or more bases, such as codons of four, five, six or more bases. Examples of four base codons include, but are not limited to, AGGA, CUAG, UAGA, CCCU and the like. Examples of five base codons include, but are not limited to, AGGAC, CCCCU, CCCUC, CUAGA, CUACU, UAGGC and the like. One aspect of the methods and compositions described herein includes using extended codons based on deletion of frame shift. Codons of four or more bases can insert, in which is, but not limited to, one or multiple unnatural amino acids to the same protein. For example, in the presence of mutated O-tRNA, which includes, but is not limited to, a special frame shift suppressor, with anticodon loops, for example, with at least 8-10 anticodon loops nt, the codon of four or more bases is read as a single amino acid. In others embodiments, the anticodon loops may decode, which includes but is not limited to, at least one codon of four bases, at least one codon of five bases or at least one codon of six bases or more. Since there are 256 codons of four possible bases, multiple non-natural amino acids can be encoded in the same cell using a codon of four or more bases. See, Anderson et al., (2002) Exploring the Limits of Codon and An ticodon Si ze, Chemistry and Biology. 9: 237-244; Magliery, (2001) Expanding the Geneti c Code: Selection of Effi hundred t Suppressors of Four-base Codons and Iden tifi cation of "Shifty" Four-base Codons wi th a Library Approa ch ín Escheri chia col i, J. Mol. Biol. 307: 755-769. For example, four-base codons have been used to incorporate non-natural amino acids into proteins using in vitro biosynthetic methods. See, for example, Ma et al., (1993) Biochemistry. 32: 7939; and Hohsaka et al., (1999) J. Am. Chem. Soc. 121: 34. CGGG and AGGU were used to simultaneously incorporate 2-naphthylalanine and a NBD derivative of lysine to streptavidin in vitro with two chemically-acylated table-displacement suppressor tRNAs. See, for example, Hohsaka et al., (1999) J. Am. Chem. Soc. 121: 12194. In an in vivo study, Moore et al. examined the ability of tRNALeu derivatives with NCUA anticodons to suppress the UAGN codons (N can be U, A, G or C) and found that the UAGA quadruplet can be decoded by a tARNLeu with a UCUA anticodon with an efficiency of 13 to 26% with little decoding in frame 0 or -1. See, Moore et al., (2000) J. Mol. Biol., 298: 195. In one embodiment, extended codons based on rare codons or nonsense codons can be used in the methods and compositions described herein, which can reduce meaningless misreading and deletion of frame shift in other undesirable sites. For a given system, a selector codon may also include one of the codons of three natural bases, wherein the endogenous system does not use (or rarely uses) the natural base codon. For example, this includes a system that lacks a tRNA that recognizes the three-base natural codon and / or a system where the three-base codon is a rare codon. The selector codons optionally include unnatural base pairs. These unnatural base pairs further expand the existing genetic alphabet. An extra pair of bases increases the number of triplet codons from 64 to 125. Peer properties of third base include stable and selective base pairing, efficient enzymatic incorporation to DNA with high fidelity by a polymerase and efficient continuous primer extension after of the synthesis of the nascent non-natural base pair. Descriptions of unnatural base pairs that can be adapted for methods and compositions include, for for example, Hirao, et al., (2002) An unna tura l base pair for incorporates ting amino acid analogues into protein. Nature Biotechnology, 20: 177-182 and see also, Wu, Y., et. to the. (2002) J. Am. Chem. Soc. 124: 14626-14630. Other relevant publications are listed below in the present. For in vivo use, the non-natural nucleoside is permeable to the membrane and is phosphorylated to form the corresponding triphosphate. In addition, the increased genetic information is stable and is not destroyed by cellular enzymes. Previous efforts by Benner and others took advantage of hydrogen bonding patterns that are different from those in canonical Watson-Crick pairs, the most notable example of which is the iso-C: iso-G pair. See, for example, Switzer et al., (19S9) J. Am. Chem. Soc, 111: 8322; and Piccirilli et al., (1990) Nature, 343: 33; Kool, (2000) Curr. Opin. Chem. Biol., 4: 602. These bases in general are poorly matched to some degree with natural bases and can not be replicated enzymatically. Kool et al. Demonstrated that hydrophobic packaging interactions between bases can replace the hydrogen bond to drive base pair formation. See, Kool, (2000) Curr. Opin. Chem. Biol., 4: 602; and Guckian and Kool, (1998) Angew. Chem. Int. Ed. Engl., 36, 2825. In an effort to develop a non-natural base pair that satisfies all the above requirements, Schultz, Romesberg and collaborators have synthesized systematically and studied a series of non-natural hydrophobic bases. We found that a PICS: PICS self-pair is more stable than the natural base pairs and can be efficiently incorporated into DNA by the Klenow fragment of Escherichia coli (KF) DNA polymerase I. See, for example, McMinn et al., (1999) J. Am. Chem. Soc. 121: 11585-6; and Ogawa et al., (2000) J. Am. Chem. Soc. 122: 3274. A 3MN: 3MN self-pair can be synthesized by KF with sufficient efficiency and selectivity for biological function. See, for example, Ogawa et al., (2000) J. Am. Chem. Soc, 122: 8803. However, both bases act as a chain terminator for additional replication. A mutant DNA polymerase has been recently evolved that can be used to replicate the self-pair of PICS. In addition, a 7AI auto-par can be replicated. See, for example, Tae et al., (2001) J. Am. Chem. Soc. 123: 7439. A new pair of metallobase, Dipic: Py, has also been developed, which forms a stable pair in the Cu (II) bond. See, Meggers et al., (2000) J. Am. Chem. Soc 122: 10714. Because extended codons and unnatural codons are intrinsically orthogonal to natural codons, the non-natural amino acid methods described herein can take advantage of this property to generate orthogonal tRNAs for them. A translation deviation system can also be used to incorporate a non-natural amino acid into a desired polypeptide. In a translation deviation system, a large sequence is incorporated into a gene but is not translated into proteins. The sequence contains a structure that serves as a clue to induce the ribosome to jump over the sequence and resume translation downstream of the insertion. In certain embodiments, the protein or polypeptide of interest (or portion thereof) in the methods and / or compositions described herein is encoded by a nucleic acid. Commonly, the nucleic acid comprises at least one selector codon, at least two selector codings, at least three codons selectors, at least four codons selectors, at least five codons selectors, at least six codons selectors, so minus seven codons selectors, at least eight codons selectors, at least nine codons selectors, ten or more codons selectors. Genes coding for proteins or polypeptides of interest can be mutagenized using methods well known to those of skill in the art and described herein under "Mutagenesis and other Molecular Biology techniques" to include, for example, one or more codons selectors. for the incorporation of a non-natural amino acid. For example, a nucleic acid for a protein of interest is mutagenized to include one or more codons selectors, providing the incorporation of one or more non-amino acids. natural The methods and compositions described herein include any such variants, which include, but are not limited to, mutant, versions of any protein, for example, in which at least one non-natural amino acid is included. Similarly, the methods and compositions described herein include corresponding nucleic acids, that is, any nucleic acid with one or more selector codons that encode one or more non-natural amino acids. Nucleic acid molecules encoding an isolate protein, which is included by way of example only, can be easily mutated to introduce a cistern at any desired position of the polypeptide. Cysteine is widely used to introduce reactive molecules, water-soluble polymers, proteins or a wide variety of other molecules, onto an isolate protein. Appropriate methods for the incorporation of cysteine to a desired position of a polypeptide are well known in the art, such as those described in U.S. Patent No. 6,608,183, which is incorporated by reference herein and standard mutagenesis techniques.

VII I. In vivo generation of polypeptides comprising unnatural amino acids The polypeptides described herein can be generated in vivo using modified tRNA and tRNAs. to add or replace amino acids that are not encoded in systems that occur in a stable manner in nature. Methods for generating smttasas tRNA and tRNA that use amino acids that are not encoded in systems that occur stably in nature are described, for example, in U.S. Patent No. 7,045,337, entitled "In vivo mcorporation of unnatural ammo acids" and U.S. Pat. No. 7,083,970, entitled "Methods and compositions for the production of orthogonal tRNA-aminoacl tRNA synthetase pairs", which are incorporated by reference herein. These methods involve generating a translation machinery that functions independently of endogenous smtetases and tRNAs in the translation system (and are therefore sometimes referred to as "orthogonal"). In one embodiment, the translation system comprises a polynucleotide that encodes the polypeptide; the polynucleotide may be mRNA that was transcribed from the corresponding DNA or the mRNA may arise from a viral RNA vector; in addition, the polynucleotide comprises a selector codon corresponding to the predesignated incorporation site for the non-natural amino acid. The translation further comprises a tRNA that comprises the non-natural amino acid, wherein the tRNA is specific to the selector codon mentioned above; In additional embodiments, the non-natural amino acid is ammoacylated. In other modalities or additional modalities, the translation system comprises a aminoacyl synthetase specific for tRNA and in other embodiments or additional embodiments, the translation system comprises an orthogonal tRNA and an orthogonal aminoacyl tRNA synthetase. In other embodiments or additional embodiments, the translation system comprises at least one of the following: a plasmid comprising the aforementioned polynucleotide (commonly in the form of DNA), genomic DNA comprising the aforementioned polynucleotide (commonly in the form of DNA ) or genomic DNA to which the aforementioned polynucleotide has been integrated (in additional embodiments, the integration is stable integration). In other modalities or additional modalities of the translation system, the selector codon is selected from the group consisting of an amber codon, ocher codon, opal codon, single codon, rare codon, an unnatural codon, a five-base codon and a codon of four bases. In other modalities or additional modalities of the translation system, the tRNA is a suppressor tRNA. In other embodiments or additional embodiments, the non-natural amino acid polypeptide is synthesized by a ribosome. In other embodiments or additional modalities, the translation system comprises an orthogonal tRNA (O-tRNA) and an orthogonal aminoacyl tRNA N synthetase (O-RS). Commonly, O-RS preferably aminoates O-tRNA with at least one non-natural amino acid in the translation system and O-tRNA. recognizes at least one selector codon that is not recognized by other tRNAs in the system. Thus, the translation system inserts the unnatural amino acid into a protein produced in the system, in response to a coding selector codon, thereby "substituting" an amino acid at a position in the encoded polypeptide. A wide variety of orthogonal tRNAs and aminoacyl tRNAs synthetases have been described in the art for inserting particular synthetic amino acids into polypeptides and are generally suitable for use in the methods described herein to produce the non-natural amino acid polypeptides described herein. For example, 0-tRNA / keto-specific aminoacyl-tRNA synthetases are described in Wang, L., et al., Proc. Nati Acad. Sci. USA 100: 56-61 (2003) and Zhang, Z. et al., Biochem. 42 (22): 6735-6746 (2003). 0-RS examples or portions thereof, are encoded by polynucleotide sequences and include amino acid sequences disclosed in the publications of US patent applications 2003/0082575 and 2003/0108885, each incorporated herein by reference. Corresponding O-tRNA molecules for use with the O-RSs are also described in U.S. Patent No. 7,045,337, entitled "In vivo incorporation of unnatural amino acids" and U.S. Patent No. 7,083,970, entitled "Methods and compositions for the production of orthogonal tRNA-aminoacyl tRNA synthetase pairs "which are incorporated by reference herein, in addition, Mehl et al., in J. Am. Chem. Soc. 2003; 125: 935-939 and Santoro et al., Nature Biotechnology 2002 Oct; 20: 1044-1048. , which are incorporated by reference herein in their entirety, discuss selection methods and molecules of ammoacyl tRNA synthetase and tRNA for the incorporation of p-ammophenylalanine into polypeptides.Examples of exemplary O-tRNAs suitable for use in the methods described in present include, but are not limited to, nucleotide sequences SEQ ID NOs: 1-3 as disclosed in U.S. Patent Application Publication 200370108885 (Serial No. 10 / 126,931) which is incorporated by reference herein. examples of 0-tRNA / aminoacyl-tRNA-smtetase pairs specific to particular non-natural amino acids are described in U.S. Patent Application Publication 2003/0082575 (Serial No. 10 / 126,927) which is incorporated by reference at the moment. O-RS and O-tRNA that incorporate both amino acids containing keto and amino acids containing azide in S. cerevisiae are described in Chin, J. W., et al., Science 301: 964-967 (2003). The use of O-tRNA / ammoacyl-tRNA synthetases involves the selection of a specific codon that encodes the unnatural amino acid. While any codon can be used, it is generally desirable to select a codon that is rarely used or never used in the cell in which the O-tRNA / ammoacyl-tRNA Synthetase is expressed. For example, exemplary codons include a nonsense codon such as retention codons (amber, ocher and opal), codons of four or more bases and other codons of three natural bases that are rarely used or not used. Specific codon (s) selector (s) can be (are) introduced to appropriate positions in the polynucleotide coding sequence using mutagenesis methods known in the art (including, but not limited to, site-specific mutagenesis, cassette mutagenesis, restriction selection mutagenesis, etc.). Methods for generating components of the protein biosynthetic machinery, such as O-RSs, O-tRNA and orthogonal O-tRNA / O-RS pairs that can be used to incorporate a non-natural amino acid are described in Wang, L., et. to the. Science 292: 498-500 (2001); Chin, J. W., et al., J. Am. Chem. Soc. 124: 9026-9027 (2002); Zhang, Z. et al., Biochemistry 42: 6735-6746 (2003). Methods and compositions for the in vivo incorporation of non-natural amino acids are described in U.S. Patent Application Publication 2003/0082575 (Serial No. 10 / 126,927) which is incorporated by reference herein. Methods for selecting a pair of orthogonal tRNA-tRNA synthetase for use in an in vivo translation system of an organism are also described in U.S. Patent No. 7,045,337, entitled "In vivo incorporation". of unnatural amino acids "and U.S. Patent No. 7,083,970, entitled" Methods and compositions for the production of orthogonal tRNA-aminoacyl tRNA synthetase pairs "which are incorporated by reference herein, In addition PCT Publication No. WO 04/035743 entitled "Site Specific Incorporation of Keto Amino Acids into proteins", which is incorporated by reference in its entirety, discloses orthogonal RS and tRNA pairs for the incorporation of keto amino acids PCT Publication No. WO 04/094593 entitled "Expanding the Eukaryotic Genetic Code ", which is incorporated by reference herein in its entirety, discloses orthogonal RS and tRNA pairs for the incorporation of naturally uncoded amino acids into eukaryotic host cells .. Methods for producing at least one orthogonal aminoacyl-tRNA synthetase (OR -RS) recombinant comprise: (a) generating a library of RS (optionally mutant) derived from at least one aminoacyl-tRNA without tetase (RS) of a first organism, including, but not limited to, a prokaryotic organism, such as Me thanococcus j anna schi i, Methanoba cteri um thermoa u totrophi cum, Ha l oba cteri um, Escheri chia col i, A. fulgidus, P. furi osus, P. horikoshi i, A. pernix, T. thermophi l us, or the like or a eukaryotic organism; (b) select (and / or filter) the RS library (optionally RS mutants) in terms of members that aminoacylate an orthogonal tRNA (O-tRNA) in the presence of a non-natural amino acid and a natural amino acid, thereby providing an accumulation of active RS (optionally mutant); and / or, (c) selecting (optionally by means of negative selection) the accumulation as to active RSs (where, but not limited to, mutant RS) that aminoacylize preferably O-tRNA in the absence of the non-amino acid natural, thereby providing the at least one recombinant O-RS; wherein the at least one recombinant O-RS preferably aminoates the O-tRNA with the non-natural amino acid. In one modality, the RS is an inactive RS. Inactive RS can be generated by mutating an active RS. For example, inactive RS can be generated by mutating at least about 1, at least about 2, at least about 3, at least about 4, at least about 5, at least about 6 or at least about 10 or more amino acids to different amino acids, including, but not limited to, alanine. SR libraries of mutants can be generated using various techniques known in the art, in which it is included but not limited to rational design based on the structure of id T-dimensional protein or mutagenesis of nucleotides of RS in a random or randomized technique. rational design. For example, mutant RSs can be generated by site-specific mutagenesis, random mutations, diversity generation combination mutations, chimeric constructs, rational design and by other methods described herein or known in the art. In one embodiment, the selection (and / or filtering) of the RS library (optionally RS mutant) in terms of members that are active, including, but not limited to, aminoacylating an orthogonal tRNA (O-tRNA) in the presence of a non-natural amino acid and a natural amino acid, includes: introducing a selection or positive binding marker, in which is included but not limited to, an antibiotic resistance gene or the like and the RS library (optionally mutant ) to a plurality of cells, wherein the selection and / or positive binding marker comprises at least one selector codon, including, but not limited to, an amber codon, an ocher codon, an opal codon, a single codon , a rare codon, an unnatural codon, a five-base codon and a four-base codon; cultivate the plurality of cells in the presence of a selection agent; identify cells that survive (or show a specific response) in the presence of the selection and / or filtering agent by suppressing the at least one selector codon in the selection or positive filtering marker, thereby providing a subset of positively selected cells containing the accumulation of active RS (optionally mutant).

Optionally, the agent concentration of the selection and / or filtering agent can be varied. In one aspect, the positive selection marker is a chloramphenicol acetyltransferase (CAT) gene and the selector codon is an amber stop codon in the CAT gene. Optionally, the positive selection marker is a β-lactamase gene) and the selector codon is an amber retention codon in the β-lactamase gene. In another aspect, the positive selection marker comprises a fluorescent or luminescent selection marker or an affinity-based selection marker (in which, but not limited to, a cell surface marker). In one embodiment, the selection or negatively filtering of the cluster of active (optionally mutant) RSs that preferentially aminoacylates the O-tRNA in the absence of the non-natural amino acid includes: introducing a negative selection or filtration marker with the accumulation cluster of active RSs ( optionally mutant) of the selection or positive filtration to a plurality of cells of a second organism, wherein the negative selection or filtration marker comprises at least one selector codon (in which, but not limited to, a resistance gene is included) antibiotic, which includes but is not limited to, a chloramphenicol acetyltransferase (CAT) gene); and identify cells that survive or show a selection response positive in a first medium supplemented with the non-natural amino acid and a selection or filtering agent, but fail to survive or show the specific response in a second medium not supplemented with the non-natural amino acid and the selection or filtering agent, providing this surviving cells or cells selected with the at least one recombinant O-RS. For example, a CAT identification protocol optionally acts as a positive selection and / or a negative filtration in the determination of appropriate recombinant O-RSs. For example, a cluster of clones is optionally replicated on culture plates containing CAT (comprising at least one selector codon) with or without one or more non-natural amino acids. Colonies that grow exclusively on plates containing non-natural amino acids are thus considered to contain recombinant O-RS. In one aspect, the concentration of the selection agent (and / or filtration) is varied. In some aspects the first and second organisms are different. Thus, the first and / or second organism optionally comprises: a prokaryote, a eukaryote, a mammal, an Escherichia coli, a fungus, a yeast, an archaebacterium, a eubacterium, a plant, an insect, a protist, etc. In other embodiments, the selection marker comprises a fluorescent or luminescent selection marker or an affinity-based selection marker. In another modality, the filtration or selection (in the which include but are not limited to, select negatively) the active RS cluster (optionally mutant) includes: isolating the cluster of RS active mutants from the positive selection step (b); introducing a selection or negative filtration marker, wherein the selection or negative filtration marker comprises at least one selector codon (in which, but not limited to, a toxic marker gene is included, but not limited to) , a ribonucleasease barnase gene, comprising at least one selector codon) and the cluster of active RSs (optionally mutants) to a plurality of cells of a second organism; and identifying cells that survive or show a specific selection response in a first medium not supplemented with the non-natural amino acid, but fail to survive or show a specific selection response in a second medium supplemented with the non-natural amino acid, thereby providing cells surviving or selected cells with the at least one recombinant O-RS, wherein the at least one recombinant O-RS is specific for the non-natural amino acid. In one aspect, the at least one selector codon comprises approximately two or more selector codons. Such modalities may optionally include wherein the at least one selector codon comprises two or more selector codons, and wherein the first and second organisms are different (in which they include but are not limited to, each organism is optionally, in which HE include, but are not limited to, a prokaryote, a eukaryote, a mammal, an Escherichia coli, a fungus, a yeast, an archaebacter, a eubactepa, a plant, an insect, a protist, etc.). Also, some aspects include wherein the negative selection marker comprises a pBaseasease barnase gene (comprising at least one selector codon). Other aspects include wherein the selection marker optionally comprises a fluorescent or luminescent selection marker or an affinity-based selection marker. In the embodiments herein, sections and / or filtering optionally include variation of the severity of selection and / or filtering. In one embodiment, the methods for producing an ammoacyl-tRNA orthogonal (O-RS) recombinant ammoacyl can further comprise: (d) isolating the at least one recombinant O-RS; (e) generating a second set of O-RS (optionally mutated) derived from at least one recombinant O-RS; and (f) repeating steps (b) and (c) until a mutated O-RS is obtained comprising the ability to ammoacrylate preferably the O-tRNA. Optionally, steps (d) - (f) are repeated, in which but not limited to, at least approximately twice. In one aspect, the second set of mutated O-RSs derived from at least one recombinant O-RS can be generated by mutagenesis, in which but not limited to, random mutagenesis, site-specific mutagenesis, recombination or a combination thereof. The severity of the selection / filtration stages, which include but are not limited to the selection / positive filtration stage (b), the selection / negative filtration stage (c) or both of the selection / positive filtration stages and negative (b) and (c), in the methods described above, optionally includes varying the selection / filtering severity. In another embodiment, the positive filtering / selection step (b), the negative filtering / selection step (c) or both of the positive and negative filtering / selection steps (b) and (c) involve using a reporter, in where the reporter is detected by cell sorting activated by fluorescence (FACS) or where the reporter is detected by luminescence. Optionally, the reporter is displayed on a cell surface, on a display of phage or the like and selected on the basis of affinity or catalytic activity that involves the non-natural amino acid or an analogue. In one embodiment, the mutated synthetase is displayed on a cell surface, on a display of phage or the like. Methods to produce a recombinant orthogonal tRNA (O-tRNA) include: (a) generating a library of tRNA mutants derived from at least one tRNA, in which it is included but not limited to, a suppressive tRNA, of a first organism; (b) select (in those that include but not limited to, select negatively) or filter the library as to tRNA (optionally mutants) that are aminoacylated by an aminoacyl-tRNA synthetase (RS) of a second organism in the absence of a RS of the first organism, thereby providing a tRNA cluster (optionally mutants); and, (c) selecting or filtering the tRNA cluster (optionally mutants) for members that are aminoacylated by an orthogonal RS (O-RS) introduced, thereby providing at least one recombinant O-tRNA; wherein the at least one recombinant C-tRNA recognizes a selector codon and is not efficiently recognized by the RS of the second organism and is preferably aminoacylated by the O-RS. In some embodiments the at least one tRNA is a suppressor tRNA and / or comprises a unique three-base codon of natural and / or unnatural bases or is a nonsense codon, a rare codon, an unnatural codon, a codon that it comprises at least 4 bases, an amber codon, an ocher codon or an opal retention codon. In one embodiment, the recombinant O-tRNA has an orthogonality enhancement. It will be appreciated that in some embodiments, the 0-tRNA is optionally imported into a first organism of a second organism without the need for modification. In several embodiments, the first and second organisms are either the same or different and are optionally chosen from, in the which includes but is not limited to prokaryotes (including, but not limited to, Methanococcus jannaschi i, Methanoba cteri um thermoa utotrophi cum, Escheri chia col i, Halobacterium, etc.), eukaryotes, mammals, fungi, yeasts, archaebacteria, eubacteria, plants, insects, protists, etc. Additionally, the recombinant tRNA is optionally aminoacylated by an unnatural amino acid, wherein the unnatural amino acid is biosynthesized in vivo either naturally or by means of genetic manipulation. The non-natural amino acid is optionally added to a culture medium for at least the first or second organism. In one aspect, the selection (in which, but not limited to, negative selection) or filtration of the library as to tRNA (optionally mutants) that are aminoacylated by an aminoacyl-tRNA synthetase (step (b)) includes: introducing a toxic marker gene, wherein the toxic marker gene comprises at least one of the codons selectors (or a gene that leads to the production of a toxic or static agent or a gene essential to the organism wherein said marker gene comprises at least minus a selector codon) and the tRNA library (optionally mutants) to a plurality of cells of the second organism; and selecting surviving cells, wherein the surviving cells contain the tRNA cluster (optionally mutants) comprising at least one orthogonal tRNA or non-functional tRNA. For example, Surviving cells can be selected by using a comparative ratio cell density analysis. In another aspect, the toxic marker gene may include two or more selector codons. In another embodiment of the methods, the toxic marker gene is a ribonuclease barnase gene, wherein the ribonuclease barnase gene comprises at least one amber codon. Optionally, the ribonuclease barnase gene may include two or more amber codons. In one embodiment, the selection or filtration of the tRNA pool (optionally mutants) in terms of members that are aminoac-ladDS by an orthogonal KS (O-RS) introduced can include: introducing a selection marker gene or positive filtration, in wherein the positive marker gene comprises a drug resistance gene (in which there is included but not limited to a β-lactamase gene, comprising at least one of the selector codons, such as at least one retention codon amber) or a gene essential to the organism or a gene that leads to the detoxification of a toxic agent, together with the O-RS and the tRNA cluster (optionally mutants) to a plurality of cells of the second organism; and identifying surviving cells or selected cells cultured in the presence of a screening or filtering agent, in which is included but not limited to an antibiotic, thereby providing a cluster of cells possessing the at least one recombinant tRNA, in wherein the at least one recombinant tRNA is aminoacylated by the O-RS and inserts an amino acid into a translation product encoded by the positive marker gene, in response to at least one selector codon. In another embodiment, the concentration of the selection and / or filtering agent is varied. Methods for generating specific 0-tRNA / 0-RS pairs are provided. The methods include: (a) generating a tRNA library of mutants derived from at least one tRNA of a first organism; (b) screening or negatively screening the library for tRNAs (optionally mutants) that are aminoacylated by an aminoacyl-tRNA synthetase (RS) of a second organism in the absence of an RS of the first organism, thereby providing a tRNA pool ( optionally mutants); (c) selecting or screening the tRNA library (optionally mutants) for members that are aminoacylated by an orthogonal RS (O-RS) introduced, thereby providing at least one recombinant O-tRNA. The at least one recombinant O-tRNA recognizes a selector codon and is not efficiently recognized by the RS of the second organism and is preferably aminoacylated by the O-RS. The method also includes (d) generating a library of RS (optionally mutants) derived from at least one aminoacyl-tRNA synthetase (RS) from a third organism; (e) selecting or screening the library of mutant SRs for members that preferably aminoacylate the at least one O- recombinant tRNA in the presence of an unnatural amino acid and a natural amino acid, thereby providing a cluster of active RS (optionally mutants); and, (f) select or negatively filter the cluster for active RS (optionally mutants) that aminoacylates the at least one recombinant O-tRNA in the absence of the non-natural amino acid, thereby providing at least a pair of 0 -tRNA / O-RS specific, wherein the at least one specific 0-tRNA / 0-RS pair comprises at least one recombinant O-RS that is specific for the non-natural amino acid and the at least one 0- t7 Recombinant RN. Specific O-tRNA / 0-RS pairs produced by the methods are included. For example, the specific O-tARN / 0-RS pair may include, but not limited to, a mutRNATyr-mutTyrRS pair, such as a mutRNATyr-SS12TyrRS pair, a mutRNALeu-mutLeuRS pair, a mutRNAThr pair. -mutThrRS, a mutRNAGlu-mutGluRS pair, or the like. Additionally, such methods include where the first and the third organisms are the same (in which, but not limited to, Methanococcus jannaschii). Also included herein are methods for selecting a pair of orthogonal tRNA-tRNA synthetase for use in an in vivo translation system of a second organism in the methods described herein. The methods include: introducing a marker gene, a tRNA and an aminoacyl-tRNA synthetase (RS) isolated or derived from a first organism to a first set of cells of the second organism; introduce the marker gene and the tRNA to a set of cells in duplicate of a second organism; and selecting for surviving cells in the first set that fail to survive in the set of cells in duplicate or select as to cells that show a specific selection response that fails to give such a response in the set of cells in duplicate, in wherein the first set and the set of cells in duplicate are cultured in the presence of a screening or filtering agent, wherein the surviving cells or selected cells comprise the pair of tRNA-tRNA orthogonal tRNA for use in the live traction system m of the second organism. In one embodiment, the comparison and selection or filtering includes a live m complementation analysis. The concentration of the selection or filtering agent can be varied. The organisms described herein comprise a variety of organisms and a variety of combinations. In one embodiment, the organisms are optionally a prokaryotic organism, including, but not limited to, Methanococcus janna schu, Methanoba cteri um thermoa utotrophicum, Haloba cterium, Escheri chia coli, A. fulgidus, P. fuposus, P. horikoshn, A. pernix, T. thermophilus, or the like. Alternatively, the organisms comprise a eukaryotic organism, which includes but is not limited to, plants (including, but not limited to, complex plants such as monocots or dicots), algae, protists, fungi (including, but not limited to, yeast, etc.), animals (which include but not limited to, mammals, insects, arthropods, etc.), or the like.

A. Expression in Non-Eukaryotes and Eukaryotes The techniques disclosed in this section can be applied to the expression in non-eukaryotes and eukaryotes of the non-natural amino acid poltides described herein. To obtain high level expression of a cloned polynucleotide, polynucleotides encoding a desired poltide are commonly subcloned to an expression vector that contains a strong promoter to direct transcription, a transcription / translation terminator and if it is to a nucleic acid encoding a protein, a ribosome binding site for the initiation of traction. Suitable bacterial promoters are described in, for example, Sambrook et al. and Ausubel et al. Bacterial expression systems for expressing poltides are available from, including, but not limited to, E. col i, Ba cill us sp. , Pseudomonas fl uorescens, Pseudomonas aeruginosa, Pseudomonas s putida and Salmonella (Palva et al., Gene 22: 229-235 (1983); Mosbach et al., Nature 302: 543-545 (1983). Equipment for such expression systems are commercially available. Eukaryotic expression systems for mammalian cells, yeast and insect cells are also commercially available. In cases where the orthogonal tRNA and ammoacyl tRNA synthetases (described elsewhere herein) are used to express the poltides, the host cells for expression are selected based on their ability to use the orthogonal components. Exemplary host cells include yram-positive bacteria (in which but not limited to B. brevis or B. subti l i s or Streptomyces) and gram-negative bacteria (E. coli or Pseudomonas fl uorescens, Pseudomonas a erugmosa, Pseudomonas s putida), also as yeast and other eucaponeous cells. Cells comprising pairs of O-tRNA / O-RS as described herein can be used. A eukaryotic host cell or non-eukaryotic host cell as described herein provides the ability to synthesize poltides comprising unnatural amino acids in large useful amounts. In one aspect, the composition optionally includes, but is not limited to, at least 10 micrograms, at least 50 micrograms, at least 75 micrograms, at least 100 micrograms, at least 200 micrograms, at least 250 micrograms , at least 500 micrograms, at least 1 milligram, at least 10 milligrams, at least 100 milligrams, at least one gram, or more of the polypeptides comprising an unnatural amino acid or an amount that can be obtained with polypeptide production methods in vivo (details as to the production of recombinant protein and purification are provided herein). In another aspect, the protein is optionally present in the composition at a concentration of, in which but not limited to, at least 10 micrograms of polypeptide per liter, at least 50 micrograms of polypeptide per liter, pjr least 75 micrograms of polypeptide per liter, at least 100 micrograms of polypeptide per liter, at least 200 micrograms of polypeptide per liter, at least 250 micrograms of polypeptide per liter, at least 500 micrograms of polypeptide per liter, at least 1 milligram of polypeptide per liter or at least 10 milligrams of polypeptide per liter or more, including, but not limited to, a cell lysate, a pH buffer, a pharmaceutical pH buffer, or other liquid suspension (in which, but not limited to, in a volume of anywhere from about 1 to about 100 L or more). The production of large quantities (including, but not limited to, greater than those commonly possible by other methods, including, but not limited to, in vitro translation) of a protein in a eukaryotic cell that includes at least one unnatural amino acid is an aspect of the methods, techniques and compositions described herein. A eukaryotic host cell or non-eukaryotic host cells as described herein provides the ability to biosynthesize proteins comprising unnatural amino acids in large useful amounts. For example, proteins comprising an unnatural amino acid can be produced at a concentration of, in which but not limited to, at least 10 μg / liter, at least 50 μg / liter, at least 75 μg / liter. liter, at least 100 μg / liter, at least 200 μg / liter, at least 250 μg / liter or at least 500 μg / liter, at least 1 mg / liter, at least 2 mg / liter, at least 3 mg / liter, at least 4 mg / liter, at least 5 mg / liter, at least 6 mg / liter, at least 7 mg / liter, at least 8 mg / liter, so at least 9 mg / liter, at least 10 mg / liter, at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900 mg / liter, 1 g / liter, 5 g / liter, 10 g / liter or more of protein in a cell extract, cell lysate, culture medium, pH regulating solution and / or the like. 1. Expression, Cultivation and Isolation Systems The techniques revealed in this section can be applied to the expression, cultivation and isolation systems of the non-natural amino acid polypeptides described herein. Non-natural amino acid polypeptides can be expressed in any number of appropriate expression systems, which include, but are not limited to, yeast, insect cells, mammalian cells and bacteria. A description of exemplary expression systems is provided herein. Yeast . As used herein, the term "yeast" includes any of the various yeasts capable of expressing a gene encoding the non-natural amino acid polypeptide. Such yeasts include, but are not limited to, ascosporogenous yeasts (Endomycetales), basidiosporogeneas yeasts and yeasts belonging to the Fungi imperfecti group. { Blastomycetes). The ascosporogenous yeasts are divided into two families, Spermophthoraceae and Saccharomycetaceae. The latter consists of four sub-families, Schizosaccharomycoideae (for example, genus Schizosaccharomyces), Nadsonioideae, Lipomycoideae and Saccharomycoideae (for example, genera Pichia, Klvyveromyces and Saccharomyces). Basidiosporogenous yeasts include the genera Leucosporidium, Rhodosporidium, Sporidiobolus, Filobasidium and Filobasidiella. The yeasts belonging to the Fungi Imperfecti group. { Blastomycetes) are divided into two families, Sporobolomycetaceae (for example, genera Sporobolomyces and Bullera) and Cryptococcaceae (for example, Candida genus). In certain embodiments, species within the genera Pichia, Kluyveromyces, Saccharomyces, Schizosaccharomyces, Hansenula, Torulopsis and Candida, which include, but are not limited to, P. pastoris, P. guillerimondii, S. cerevisiae, S. carlsbergensis, S. diastaticus, S. douglasii, S. kluyveri, S, norbensis, S. oviformis, K. lactis, K. fragilis, C. albicans, C. maltosa and H. polymorpha are used in the methods, techniques and compositions described in the present. The selection of appropriate yeast for expression of the non-natural amino acid polypeptide is within the skill of that of ordinary skill in the art. In the selection of yeast hosts for expression, appropriate hosts may include, but are not limited to, those shown to have good secretory capacity, low proteolytic activity and overall robustness. Yeasts are generally available from a variety of sources including, but not limited to, the Yeast Genetic Stock Center, Department of Biophysics and Medical Physics, University of California (Berkeley, CA), and the American Type Culture Collection ( "ATCC") (Manassas, VA). The term "yeast host" or "yeast host cell" includes yeast which can be or has been used as a receptor for recombinant vectors or other DNA of transfer. The term includes the progeny of the original yeast host cell that has received the recombinant vectors or other transfer DNA. It will be understood that the progeny of a single parental cell may not necessarily be completely identical in morphology or in genomic or total DNA complement to the original parent, due to accidental or deliberate mutation. Progeny of the parental cell that are sufficiently similar to the parent to be characterized by the relevant property, such as the presence of a nucleotide sequence encoding a non-naturai amino acid polypeptide, are included in the progeny proposed by this definition. Expression and transformation vectors, in which extrachromosomal replicons or integration vectors are included, have been developed for transformation to many yeast hosts. For example, expression vectors have been developed for S. cerevisiae (Sikorski et al., Genetics (1998) 122: 19; Ito et al., J. Bacteriol. (1983) 153: 163; Hinnen et al., Proc. Nati Acad. Sci. USA (1978) 75: 1929); C. albicans (Kurtz et al., Mol Cell Cell Biol. (1986) 6: 142); C. maltose (Kunze et al., J. Basic Microbiol. (1985) 25: 141); H. polymorpha (Gleeson et al., J. Gen. Microbiol. (1986) 132: 3459; Roggenkamp et al., Mol. Gen. Genet. (1986) 202: 302); K. fragilis (Das et al., J. Bacteriol. (1984) 158: 1165); K. lactis (De Louvencourt et al., J. Bacteriol. (1983) 154: 737; Van den Berg et al., Bio / Technology (1990) 8: 135); P. guillerimondii (Kunze et al., J. Basic Microbiol. (1985) 25: 141); P. pastoris (U.S. Patent Nos. 5,324,639; 4,929,555; and 4,837,148; Cregg et al., Mol. Cell. Biol. (1985) 5: 3376); Schizosaccharomyces pombe (Beach et al., Nature (1982) 300: 706); A. nidulans (Ballance et al., Biochem. Biophys., Res. Commun. (1983) 112: 284-89; Tilburn et al., Gene (1983) 26: 205-221; and Yelton et al., Proc. Nati Acad. Sci. USA (1984) 81: 1470-74); A. niger (Kelly and Hynes, EMBO J. (1985) 4: 475-479); T. reesia (EP 0244 234); and filamentous fungi such as, for example, Neurospora, Penicillium, Tolypocladium (WO 91/00357), each incorporated herein by reference in its entirety. Control sequences for yeast vectors include, but are not limited to, gene promoter regions such as alcohol dehydrogenase (ADH) (EP 0 284 044); enolasa; glucokinase; glucose-6-phosphate isomerase; glyceraldehyde-3-phosphate dehydrogenase (GAP or GAPDH); hexokinase; phosphofructokinase; 3-phosphoglycerate mutase; and pyruvate kinase (PyK) (EP 0 329 203). Yeast PH05, which codes for acid phosphatase, can also provide useful promoter sequences (Miyanohara et al., PROC.NATL.

ACAD. SCI. USA (1983) 80: 1). Other sequences suitable for use with yeast hosts may include promoters for 3-phosphoglycerate kinase (Hitzeman et al., J.

BIOL. CHEM. (1980) 255: 12073); and other glycolytic enzymes, such as pyruvate decarboxylase, triosephosphate isomerase and phosphoglucose isomerase (Holland et al., BIOCHEMISTRY (1978) 17: 4900; Hess et al., J. ADV. ENZYME REG. (1969) 7: 149). Inducible yeast promoters that have the additional advantage of transcription controlled by culture conditions can include the promoter regions for alcohol dehydrogenase 2; isocytochrome C; acid phosphatase; metallothionein; glyceraldehyde-3-phosphate dehydrogenase; degrading enzymes associated with nitrogen metabolism; and enzymes responsible for the use of maltose and galactose. Suitable vectors and promoters for use in the expression of yeast are further described in EP 0 073 657. Yeast improvers can also be used with yeast promoters. In addition, synthetic promoters can also function as yeast promoters. By way of example, the upstream activation (UAS) sequences of a yeast promoter can be linked to the transcription activation region of another yeast promoter, creating a synthetic hybrid promoter. Examples of such hybrid promoters include the ADH regulatory sequence linked to the transcription activation region of GAP. See U.S. Patent Nos. 4,880,734 and 4,876,197, which are incorporated by reference herein in their entirety. Other examples of hybrid promoters include promoters that consist of the regulatory sequences of the ADH2, GAL4, GALIO or PH05 genes, combined with the transcriptional activation region of a glycolytic enzyme gene such as GAP or PyK. See EP 0 164 556. In addition, a yeast promoter can include promoters that are stably present in the naturally yeast-free state that have the ability to bind to yeast RNA polymerase and initiate transcription. Other control elements that may comprise part of the yeast expression vectors include terminators, for example, of CAPDn or the enolase genes (Holland et al., J. BIOL.CHEM. (1981) 256: 1385). In addition, the origin of replication of the 2μ plasmid origin is appropriate for yeast. A suitable selection gene for use in yeast is the trpl gene present in the yeast plasmid. See Tschumper et al., GENE (1980) 10: 157; Kmgsman et al., GENE (1979) 7: 141. The trpl gene provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan. Similarly, yeast Leu2-deficient strains (ATCC 20,622 or 38,626) are complemented by known plasmids carrying the Leu2 gene. Methods for introducing exogenous DNA to yeast hosts include, but are not limited to, either spheroplast or host cell transformation.

Intact yeast treated with alkaline cations. By way of example, the yeast transformation can be carried out according to the method described in Hsiao et al., PROC. NATL. ACAD. SCI. USA (1979) 76: 3829 and Van Solingen et al., J. BACT. (1977) 130: 946. However, other methods for introducing DNA into cells such as by nuclear injection, electroporation or protoplast fusion can also be used as described generally in SAMBROOK ET AL., MOLECULAR CLONING: A LAB. MANUAL (2001). Then the yeast host cells can be cultured using standard techniques known to those of ordinary skill in the art. Other methods for expressing heterologous proteins in yeast host cells are well known to those of ordinary skill in the art. See generally U.S. Patent Publication No. 20020055169, U.S. Patent Nos. 6,361,969; 6,312,923; 6,183,985; 6,083,723; 6,017,731; 5,674,706; 5,629,203; 5,602,034; and 5,089,398; U.S. Reexamined Patents Nos. RE37,343 and RE35,749; published patent applications of PCT WO 99/07862; WO 98/37208; and WO 98/26080; European patent applications EP 0 946 736; EP 0 732 403; EP 0 480 480; WO 90/10277; EP 0 340 986; EP 0 329 203; EP 0 324 274; and EP 0 164 556. See also Gellissen et al., ANTONIE VAN LEEUWENHOEK (1992) 62 (1-2): 79-93; Romanos et al., YEAST (1992) 8 (6): 423-488; Goeddel, METHODS IN ENZYMOLOGY (1990) 185: 3-7, each incorporated by reference herein in its entirety. The yeast host strains can be cultured in fermenters during the amplification step using standard feed batch fermentation methods. The fermentation methods can be adapted to take into account differences in the carbon utilization path of the particular yeast host or expression control mode. By way of example, the fermentation of a Sa ccharomyces yeast host may require a single complex nitrogen feed source of glucose (eg, casein hydrolyzate) and multiple vitamin supplementation, where the methylotropic strain P. pas tori s may require glycerol, methanol and trace mineral feeds, but only simple ammonia salts (nitrogen) for optimal growth and expression. See, for example, U.S. Patent No. 5,324,639; Elliott et al., J. PROTEIN CHEM. (1990) 9:95; and Fieschko et al., BIOTECH. BIOENG (1987) 29: 1 113, each incorporated by reference herein in its entirety. Such fermentation methods, however, may have certain common aspects independent of the yeast host strain employed. By way of example, a growth-limiting nutrient, commonly carbon, can be added to the fermenter during the amplification phase to allow maximum growth. In addition, the methods of Fermentation generally employs a fermentation medium designed to contain appropriate amounts of carbon, nitrogen, basal salts, phosphorus and other minor nutrients (vitamins, trace minerals and salts, etc.). Examples of suitable fermentation media for use with Pi chia are described in U.S. Patent Nos. 5,324,639 and 5,231,178, each incorporated by reference herein in its entirety. Baculovirus infected insect cells. The term "insect host" or "insect host cell" refers to an insect that can be or has been used as a receptor for recombinant vectors or other transfer DNA. The term includes the progeny of the original insect host cell that has been transfected. It will be understood that the progeny of a single parental cell may not necessarily be completely identical in morphology or in complement of genomic DNA or complement of total DNA to the original parent, due to accidental or deliberate mutation. Progeny of the parental cell that are sufficiently similar to the parent to be characterized by the relevant property, such as the presence of a nucleotide sequence encoding an unnatural amino acid polypeptide, are included in the progeny proposed by this definition. The selection of appropriate insect cells for Expression of a polypeptide is well known to those of ordinary skill in the art. Various species of insect are well described in the art and are commercially available including, but not limited to, Aedes aegypti, Bombyx mor i, Drosophila melanogas ter, Spodoptera frugiperda and Tri chopl usia ni. In selecting insect hosts for expression, appropriate hosts may include, but are not limited to, those shown to have, inter alia, good secretory ability, low proteolytic activity and overall robustness. Insects are generally available from a variety of sources including, but not limited to, the Insect Genetic Stock Center, Department of Biophysics and Medical Physics, University of California (Berkeley, CA); and the American Type Culture Collection ("ATCC") (Manassas, VA). In general, the components of a baculovirus-infected insect expression system include a transfer vector, usually a bacterial plasmid, that contains both a fragment of the baculovirus genome and a convenient restriction site for insertion of the heterologous gene to be expressed; a wild-type baculovirus with a sequence homologous to the baculovirus-specific fragment in the transfer vector (this allows homologous recombination of the heterologous gene to the baculovirus genome); and insect host cells and appropriate culture medium. The materials, methods and techniques used in vector construction, cell transfection, plate chopping, cell culture and the like are known in the art and manuals describing these techniques are available. After inserting the heterologous gene into the transfer vector, the wild-type viral vector and genome are transfected into an insect host cell, where the vector and the viral genome recombine. The packaged recombinant virus is expressed and the recombinant plaques are identified and purified. Materials and methods for baculovirus / insect cell expression systems are commercially available in the form of equipment for example from, Invitrogen Corp. (Carlsbad, CA). These techniques are described in SUMMERS AND SMITH, TEXAS AGRICULTURAL EXPERIMENT STATION BULLETIN No. 1555 (1987), incorporated herein by reference. See also, RICHARDSON, 39 METHODS IN MOLECULAR BIOLOGY: BACULOVIRUS EXPRESSION PROTOCOLS (1995); AUSUBEL ET AL., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY 16.9-16.11 (1994); KING AND POSSEE, THE BACULOVIRUS SYSTEM: A LABORATORY GUIDE (1992); and O'REILLY ET AL., BACULOVIRUS EXPRESSION VECTORS: A LABORATORY MANUAL (1992). The production of several heterologous proteins using baculovirus / insect cell expression systems is described in the following references and such techniques can be adapted to produce the polypeptides of non-natural amino acids described herein. See, for example, U.S. Patent Nos. 6,368,825; 6,342,216; 6,338,846; 6,261,805; 6,245,528, 6,225,060; 6,183,987; 6,168,932; 6,126,944; 6,096,304; 6,013,433; 5,965,393; 5,939,285; 5,891,676; 5,871,986; 5,861,279; 5,858,368; 5,843,733; 5,762,939; 5,753,220; 5,605,827; 5,583,023; 5,571,709; 5,516,657; 5,290,686; WO 02/06305; WO 01/90390; WO 01/27301; WO 01/05956; WO 00/55345; WO 00/20032 WO 99/51721; WO 99/45130; WO 99/31257; WO 99/10515; WO 99/09193; WO 97/26332; WO 96/29400; WO 96/25496; WO 96/06161; WO 95/20672; WO 93/03173; WO 92/16619; WO 92/02628; WO 92/01801; WO 90/14428; WO 90/10078; WO 90/02566; WO 90/02186; WO 90/01556; WO 89/01038; WO 89/01037; WO 88/07082, each incorporated by reference herein in its entirety. Vectors that are useful in the baculovirus / insect cell expression systems are known and include, but are not limited to, insect expression and transfer vectors derived from insect expression and transfer vectors derived from the baculovirus Autographa ca li fornica virus. nuclear polyhedrosis (AcNPV), which is an auxiliary-independent viral expression vector. Viral expression vectors derived from this system usually use the strong viral polyhedrin gene promoter to drive the expression of heterologous genes. See In general, O'Reilly ET AL., BACULOVIRUS EXPRESSION VECTORS: A LABORATORY MANUAL (1992).

Prior to inserting the foreign gene into the baculovirus genome, the components described above, comprising a promoter, leader (if desired), coding sequence of interest and transcription termination sequence, are commonly assembled into an intermediate translocation construct ( transfer vector). Intermediate translocation constructs are frequently maintained in a replicon, such as an extrachromosomal element (eg, plasmids) capable of stable maintenance in a host, such as bacteria. The replicon will have a replication system, thus allowing it to be maintained in an appropriate host for cloning and amplification. More specifically, the plasmid can contain the polyhedrin polyadenylation signal (Miller, ANN MICROBIOL REV. (1988) 42: 177) and a prokaryotic ampicillin (amp) resistance gene and origin of replication for E-selection and propagation. coli. A transfer vector commonly used to introduce foreign genes to AcNPV is pAc373. Many other vectors, known to those of skill in the art, have also been designed which include, for example, pVL985, which alters the polyhedrin start codon from ATG to ATT and which introduces a BamHI cloning site. base pairs downstream of the ATT. See Luckow and Summers, VIROLOGY 170: 31 (1989). Other commercially available vectors include, for example, PBlueBac4.5 / V5-His; pBlueBacHis2; pMelBac; pBlueBac4.5 (Invitrogen Corp., Carlsbad, CA). After insertion of the heterologous gene, the transfer vector and wild-type baculoviral genome are co-transfected into an insect cell host. Illustrative methods for introducing heterologous DNA to the desired site in the baculovirus virus are described in SUMMERS AND SMITH, TEXAS AGRICULTURAL EXPERIMENT STATION BULLETIN NO. 1555 (1987); Smith et al., MOL. CELL. BIOL. (1983) 3: 2156; Luckow and Summers, VIROLOGY (1989) 170: 31. By way of example, the insertion can be to a gene such as the polyhedra gene, by homologous double cross-over recombination; the insertion may also be to a restriction enzyme site designed to the desired baculovirus gene. See Miller et al., BIOESSAYS (1989) 11 (4): 91. Transfection can be effected by electroporation using the methods described in TROTTER AND WOOD, 39 METHODS IN MOLECULAR BIOLOGY (1995); Mann and King, J. GEN. VIROL. (1989) 70: 3501. Alternatively, liposomes can be used to transfect the insect cells with the recombinant expression vector and the baculovirus. See, for example, Liebman et al., BIOTECHNIQUES (1999) 26 (1): 36; Graves et al., BIOCHEMISTRY (1998) 37: 6050; Nomura et al., J. BIOL. CHEM. (1998) 273 (22): 13570; Schmidt et al., Schmidt et al., PROTEIN EXPRESSION AND PURIFICATION (1998) 12: 323; Siffert et al., NATURE GENETICS (1998) 18:45; TILKINS ET AL., CELL BIOLOGY: A LABORATORY HANDBOOK 145-154 (1998); Cai et al., PROTEIN EXPRESSION AND PURIFICATION (1997) 10: 263; Dolphin et al., NATURE GENETICS (1997) 17: 491; Kost et al., GENE (1997) 190: 139; Jakobsson et al., J. BIOL. CHEM. (1996) 271: 22203; Rowles et al., J. BIOL. CHEM. (1996) 271 (37): 22376; Reverey et al., J. BIOL. CHEM. (1996) 271 (39): 23607-10; Stanley et al., J. BIOL. CHEM. (1995) 270: 4121; Sisk et al., J. VIROL. (1994) 68 (2): 766; and Peng et al., BIOTECHNIQUES (1993) 14 (2): 274. Commercially available liposomes include, for example, Cellfectin® and Lipofectin® (Invitrogen, Corp., Carlsbad, CA).

In addition, calcium phosphate transfection can be used. See TROTTER AND WOOD, 39 METHODS IN MOLECULAR BIOLOGY (1995); Kitts, NAR (1990) 18 (19): 5667; and Mann and King, J. GEN. VIROL. (1989) 70: 3501. Baculovirus expression vectors usually contain a baculovirus promoter. A baculovirus promoter is any DNA sequence capable of binding a baculovirus RNA polymerase and initiating transfection downstream (3 ') of a coding sequence (eg, structural gene) to mRNA. A promoter will have a transcription initiation region that is usually placed near the 5 'end of the coding sequence. This transcription initiation region commonly includes an RNA polymerase binding site and a transcription initiation site. A baculovirus promoter can also have a second domain called an enhancer, which, if present, is usually distant from the structural gene. In addition, the expression can be either regulatory or constitutive. Structural genes, transcribed abundantly at later times in the infection cycle, provide prornotopic sequences: particularly useful. Examples include sequences derived from the gene encoding the viral polyhedron protein (FRIESEN ET AL., The Regulation of Ba cuovirus Gene Expression in THE MOLECULAR BIOLOGY OF BACULOVIRUSES (1986); EP 0 127 839 and 0 155 476) and the gene that encodes the plO protein (VJak et al., J. Gen. VIROL. (1988) 69: 765. The newly formed baculovirus expression vector is packaged in an infectious recombinant baculovirus and subsequently culture plates can be purified by techniques such as those described in Miller et al., BIOESSAYS (1989) 11 (4) 91; SUMMERS AND SMITH, TEXAS AGRICULTURAL EXPERIMENT STATION BULLETIN No. 1555 (1987). Expression vectors of recombinant baculoviruses have been developed for infection to several insect cells For example, recombinant baculoviruses have been developed for, inter alia, Aedes aegypti (ATCC No. CCL-125), Bombyx mori (ATCC No. CRL-8910), Drosophi la melanogaster (ATCC No. 1963), Spodoptera frugiperda and Tri chopl usia ni. See Wright, NATURE (1986) 321: 718; Carbonell et al., J. VIROL. (1985) 56: 153; Smith et al., MOL. CELL. BIOL. (1983) 3: 2156.

See generally, Fraser et al., IN VITRO CELL. DEV. BIOL. (1989) 25: 225. More specifically, cell lines used for baculovirus expression vector systems commonly include, but are not limited to, Sf9 (Spodoptera frugiperda) (ATCC No. CRL-1711), Sf21. { Spodoptera frugiperda) (Invitrogen Corp., Cat. No. 11497-013 (Carlsbad, CA)), Tr? -368 . { Tp chopulsia ni) and High-Five ™ BTI-TN-5B1-4. { Tp chopulsia ni). Cells and culture media are commercially available for both direct expression and fusion expression of heterologous polypeptides in a baculovirus / expression. E. Coli Pseudomonas, and other Prokaryotes: Bacterial expression techniques are well known in the art. A wide variety of vectors are available for use in bacterial hosts. The vectors can be of a single copy or vectors of low or high quantity of multicopies. The vectors can serve for cloning and / or expression. In view of the extensive literature concerning vectors, the commercial availability of many vectors, and even manuals describing vectors and their restriction and feature maps, no extensive discussion is required in the present. As is well known, vectors normally involve markers that allow selection, such markers can provide resistance to cytotoxic agent, protrusion or immunity. Frequently, a plurality of Markers are present, which provide different characteristics. A bacterial promoter is any DNA sequence capable of binding to bacterial RNA polymerase and initiating transcription downstream (3") of a coding sequence (eg, structural gene) to mRNA.A promoter will have a transcription initiation region that it is usually placed near the 5 'end of the coding sequence This transcription initiation region commonly includes an RNA polymerase binding site and a transcription initiation site A bacterial promoter may also have a second domain called an operator, which can be superimposed on an adjacent RNA polymerase binding site at which RNA synthesis begins.The operator allows negative regulated transcription (mducible), since a gene repressor protein can bind to the operator and thereby inhibit the transcription of a specific gene Constitutive expression can occur in the absence of negative regulatory elements, such as In addition, positive regulation can be obtained by a gene activator protein binding sequence, which if present, is usually close (5 ') to the RNA polymerase binding sequence. An example of a gene-activating protein is the catabolite-enhancing protein (CAP), which aids the initiation of transcription of the lac operon in Eschep chia coli (E. coli) [Raibaud et al., ANNU. REV. GENET (1984) 18: 173]. Accordingly, regulated expression can be either positive or negative, improving by this or by reducing transcription. Sequences encoding metabolic pathway enzymes provide particularly useful promoter sequences. Examples include promoter sequences derived from enzymes that metabolize sugar, such as galactose, lactose (lac) [Chang et al., NATURE (1977) 198: 1056] and maltose. Additional examples include promoter sequences derived from such biosynthetic enzymes as tryptophan (trp) [Goeddel et al., NUC. ACIDS RES. (1980) 8: 4057; Yelverton et al., NUCL. ACIDS RES. (1981) 9: 731; U.S. Patent No. 4,738,921; IFN Pub. Nos. 036 776 and 121 775], each of which is incorporated herein by reference in its entirety. The β-galactosidase promoter system (bla) [Weissmann (1981) "The cloning of interferon and other mistakes" in Inferieron 3 (Ed. 1. Gresser)], bacteriophage lambda PL systems [Shimatake et al., NATURE ( 1981) 292: 128] and T5 [U.S. Patent No. 4,689,406], each of which is incorporated by reference in its entirety, also provide useful promoter sequences. Preferred methods encompassed herein utilize strong promoters, such as the T7 promoter to induce polypeptide production at high levels. Examples of such vectors include but are not limited to the pET29 series of Novagen and the pPOP vectors described in WO99 / 05297, which is incorporated herein by reference in its entirety. Such expression systems produce high levels of polypeptide in the host without compromising host cell viability or growth parameters. In addition, synthetic promoters that do not occur in nature also function as bacterial promoters. For example, transcriptional activation sequences of a bacterial or bacteriophage promoter can be linked to the operon sequences of another bacterial or bacteriophage promoter, creating a synthetic hybrid promoter [U.S. Patent No. 4,551,433, which is incorporated herein by reference in its total]. For example, the tac promoter is a hybrid trp-lac promoter consisting of both trp and laperon operon promoter sequences that is regulated by the lac repressor [Amann et al., GENE (1983) 25: 167; de Boer et al., PROC. NATL. ACAD. SCI. (1983) 80:21]. In addition, a bacterial promoter can include promoters that are stably present in the non-bacterial nature of origin that have the ability to bind to bacterial RNA polymerase and initiate transcription. A promoter that occurs stably in the non-bacterial nature of origin can also be coupled with compatible RNA polymerase to produce high levels of expression of some genes in prokaryotes. He bacteriophage T7 RNA polymerase / promoter is an example of a coupled promoter system [Studier et al., J. MOL. BIOL. (1986) 189: 113; Tabor et al., Proc Nati. Acad. Sci. (1985) 82: 1074]. In addition, a hybrid promoter may also consist of a bacteriophage promoter and an E. coli operator region (IFN Pub. No. 267 851). In addition to a functional promoter sequence, an efficient ribosome binding site is also useful for the expression of foreign genes in prokaryotes. In E. coli, the ribosome binding site is called the Shine-Dalgarno (SD) sequence and includes a start codon (ATG) and a sequence of 3-9 nucleotides in length located 3-11 nucleotides upstream of the codon [Shine et al., NATURE (1975) 254: 34]. It is considered that the SD sequence promotes the binding of mRNA to the ribosome by base pairing between the SD and 3 'sequences and rRNA of E. coli 16S Steitz et al. "Genetic signals and nucleotide sequences in messenger RNA", in Biological Regulation and Development: Gene Expression (Ed. R. F. Goldberger, 1979)]. To express eukaryotic genes and prokaryotic genes with weak ribosome binding site [Sambrook et al. "Expression of cloned genes in Escherichia coli", Molecular Cloning: A Laboratory Manual, 1989]. The term "bacterial host" or "bacterial host cell" refers to a bacterium that can be or has been used as a receptor for recombinant vectors or other transfer DNA. The term includes the progeny of the original bacterial host cell that has been transfected. It will be understood that the progeny of a single parental cell may not necessarily be completely identical in morphology or in complement of genomic or total DNA to the original parent, due to accidental or deliberate mutation. Progeny of the parenteral cell that are sufficiently similar to the parent to be characterized by the relevant property, such as the presence of a nucleotide sequence encoding a polypeptide, are included in the progeny proposed by this definition. The selection of appropriate host bacteria for expression of a polypeptide is well known to those of ordinary skill in the art. In selecting bacterial hosts for expression, appropriate hosts may include those shown to have, inter alia, good inclusion body formation ability, low proteolytic activity, good secretory ability, good soluble protein production capacity and overall robustness. Bacterial hosts are generally available from a variety of sources including, but not limited to, the Bacterial Genetic Stock Center, Department of Biophysics and Medical Physics, University of California (Berkeley, CA); and the American Type Culture Collection ("ATCC") (Manassas, VA). The Industrial / pharmaceutical fermentation generally use bacteria derived from K strains (for example, W3110) or from bacteria derived from strains B (for example, BL21). These strains are particularly useful because their culture parameters are extremely well known and robust. In addition, these strains are non-pathogenic, which is commercially important for safety and environmental reasons. In one embodiment of the methods described and encompassed herein, the host of E. coli includes, but is not limited to, strains BL21, DH10B or derivatives thereof. In another embodiment of the methods described and encompassed herein, the host of E. coli is a lesser protease strain in those that include, but are not limited to, OMP- and LON-. In another embodiment, the bacterial host is a species of Pseudomonas, such as P. fluorescens, P. aeruginosa and P. putida. An example of a strain of Pseudomonas is P. fluorescens biovar I, strain MB101 (Dow-Chemical). Once a recombinant host cell strain has been established (ie, the expression construct has been introduced into the host cell and host cells with the appropriate expression construct are isolated), the recombinant host cell strain is cultured under suitable for the production of polypeptides. The culture method of the recombinant host cell strain will be dependent on the nature of the expression construct used and the identity of the host cell. Recombinant host strains are normally cultured using methods that are well known in the art. Recombinant host cells are commonly cultured in liquid medium containing assimilable sources of carbon, nitrogen and inorganic salts and optionally containing vitamins, amino acids, growth factors and other proteinaceous culture supplements well known in the art. Liquid media for culturing host cells may optionally contain antibiotics or anti-fungal to prevent the growth of microorganisms and / or undesirable compounds in which they include but are not limited to, antibiotics to select host cells containing the expression vector. Recombinant host cells can be grown in batch or continuous formats, either with cell harvesting (in the case where the polypeptide is accumulated intracellularly) or harvesting the culture supernatant in batch or continuous formats. For production in prokaryotic host cells, batch culture and cell harvesting are preferred. In one embodiment, the non-natural amino acid polypeptides described herein are then purified in recombinant systems. The polypeptides can be purified from host cells or culture medium by a variety of methods known in the art. Usually, many polypeptides produced in bacterial host cells may be deficiently soluble or insoluble (in the form of inclusion bodies). In one embodiment, amino acid substitutions can be readily made in polypeptides that are selected for the purpose of increasing the solubility of the recombinantly produced polypeptide using the methods disclosed herein, also as those known in the art. In the case of insoluble polypeptide, the polypeptide can be collected from used host cells by centrifugation or filtration and can further be followed by homogenization of the cells. In the case of sparingly soluble polypeptides, compounds in which, but not limited to, polyethylene imine (PEI) can be added to induce the precipitation of the partially soluble polypeptide. Then the precipitated protein can be conveniently collected by centrifugation or filtration. The recombinant host cells can be disrupted or homogenized to release the inclusion bodies from within the cells using a variety of methods well known to those of ordinary skill in the art. Host cell disruption or homogenization can be effected using well known techniques which include but are not limited to enzymatic cell disruption, sonification, dounce homogenization or high pressure release disruption. In one modality of the methods described and encompassed herein, the high-pressure release technique is used to subject the E. coli host cells to disruption to rid the inclusion bodies of the polypeptides. When handling polypeptide inclusion bodies, it is advantageous to minimize the homogenization time in the repeats in order to maximize the yield of inclusion bodies without loss due to factors such as solubilization, mechanical cutting or proteolysis. The insoluble or precipitated polypeptides can then be solubilized using any of a variety of suitable solubilization agents known in the art. By way of example, the polypeptides are solubilized with urea or guanidine hydrochloride. The volume of the solubilized polypeptides must be minimized in such a way that large batches can be produced using conveniently manageable batches sizes. This factor can be significant in a large-scale commercial facility, where the recombinant host can be grown in batches that are thousands of liters in volume. In addition, when polypeptides are manufactured in a large-scale commercial facility, in particular, for human pharmaceutical uses, the avoidance of harmful chemical compounds that may damage the machinery and container or the protein product itself, should be avoided, if possible. It has been demonstrated in the methods described and covered in the present that the softer denaturant urea agent can be used to solubilize the polypeptide inclusion bodies in place of the stronger denaturing agent guanidine hydrochloride. The use of urea significantly reduces the risk of damage to the stainless steel equipment used in the manufacture and purification process of a polypeptide while efficiently solubilizing the polypeptide inclusion bodies. In the case of soluble polypeptides, the peptides can be secreted in the periplasmic space or in the culture medium. In addition, soluble peptides may be present in the cytoplasm of host cells. The soluble peptide can be concentrated before carrying out the purification steps. Standard techniques in which but not limited to those described herein can be used to concentrate the soluble peptide of, for example, cell lysates or culture media. In addition, standard technique in which but not limited to those described herein can be used to subject host cells to disruption and release the soluble peptide from the cytoplasm or periplasmic space of the host cells. When the polypeptide is produced as a fusion protein, the fusion sequence is preferably removed. The removal of a fusion sequence can be effected by methods which include but are not limited to enzymatic cleavage or chemical excision. Enzymatic removal of fusion sequences can be effected using methods well known to those skilled in the art. The choice of enzyme for removal of the fusion sequence will be determined by the identity of the fusion and the reaction conditions will be specified by the choice of enzyme. Chemical cleavage can be effected using reagents which include but are not limited to cyanogen bromide, TEV protease and other reagents. The cleaved polypeptide is optionally purified from the cleaved fusion sequence by well-known methods. Such methods will be determined by the identity and properties of the fusion sequence and the polypeptide. Methods for purification may include, but are not limited to, size exclusion chromatography, hydrophobic interaction chromatography, ion exchange chromatography or dialysis or any combination thereof. The polypeptide is also optionally purified to remove the DNA from the protein solution. The DNA can be removed by any suitable method known in the art, in which are included but not limited to precipitation or ion exchange chromatography. In one embodiment, the DNA is removed by precipitation with a nucleic acid precipitating agent, such as but not limited to protamine sulfate. The polypeptide can be separated from DNA precipitated using standard well known methods in which are included but not limited to centrifugation or filtration. The removal of host nucleic acid molecules is an important factor in an installation where the polypeptide is to be used to treat humans and the methods described herein reduce the host cell DNA to pharmaceutically acceptable levels. Methods for small scale or large scale fermentation can also be used in protein expression, which include but are not limited to fermentors, shake flasks, fluidized bed bioreactors, hollow fiber bioreactors, bottle culture systems, roller and agitated tank bioreactor systems. Each of these methods can be carried out in a batch process, batch feeding or a continuous process. The human forms of the non-natural amino acid polypeptides described herein can generally be recovered using standard methods in the art. For example, the culture medium or cell lysate can be centrifuged or filtered to remove cell debris. The supernatant can be concentrated or diluted to a desired volume or diafiltered to an appropriate pH buffer solution to condition the preparation for further purification. Further purification of the non-natural amino acid polypeptides described herein include but they are not limited to separating the deamidated and reported forms of a polypeptide variant from the corresponding intact form. Any of the following exemplary procedures may be employed for the purification of a non-natural amino acid polypeptide described herein: affinity chromatography; anionic or cation exchange chromatography (using, in which but not limited to, DEAE SEPHAROSE); chromatography on silica; Reverse phase HPLC; gel filtration (using, in which but not limited to, SEPHADEX G-75); hydrophobic interaction chromatography; size exclusion chromatography, metal-chelate chromatography; ultrafiltration / diafiltration; ethanol precipitation; precipitation of ammonium sulfate; chromatofocusing; displacement chromatography; electrophoretic procedures (in which but not limited to preparative isoelectric focusing), differential solubility (in which, but not limited to, ammonium sulfate precipitation), SDS-PAGE or extraction are included. Polypeptides encompassed in the methods and compositions described herein, wherein but not limited to polypeptides comprising unnatural amino acids, antibodies to polypeptides comprising non-natural amino acids, binding partners for polypeptides comprising non-natural amino acids, etc. ., They may be purified, either partially or substantially homogeneously, according to standard procedures known to and used by those skilled in the art. Thus, the polypeptides described herein can be recovered and purified by any of a variety of methods well known in the art, including but not limited to precipitation of ammonium sulfate or ethanol, extraction with acid or base, chromatography in column, affinity column chromatography, anionic or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, hydroxyl apatite chromatography, lectin chromatography, gel electrophoresis and any combination thereof. Protein re-folding steps can be used, as desired, to make mature proteins folded correctly. High performance liquid chromatography (HPLC), affinity chromatography or other appropriate methods can be employed in final purification steps where high purity is desired. In one embodiment, antibodies made against non-natural amino acids (or polypeptides comprising non-natural amino acids) are used as purification reagents, including, but not limited to, affinity-based purification of polypeptides comprising one or more unnatural amino acid (s). Once purified, partially or homogeneously, as desired, the polypeptides are optionally used for a wide variety of utilities, which include but are not limited to, as components of analysis, therapy, prophylaxis, diagnostics, research reagents, and / or as immunogens for antibody production. In addition to other references indicated herein, a variety of protein purification / folding methods are well known in the art, including, but not limited to, those summarized in R. Scopes, Protein Purification. Springer-Verlag, N.Y. (1982); Deutscher, Methods in Enzymology Vol. 182: Guide to Protein Purification. Academic Press, Inc. N.Y. (1990); = swarm (1997) Bioseparation of Proteins. Academic Press, Inc .; Bollag et al. (1996) Protein Methods. 2nd Edition Wiley-Liss, NY; Walker (1996) The Protein Protocols Handbook Humana Press, NJ, Harris and Angal (1990) Protein Purification Applications: A Practical Approach IRL Press at Oxford, Oxford, England; Harris and Angal Protein Purification Methods: A Practical Approach IRL Press at Oxford, Oxford, England; Scopes (1993) Protein Purification: Principies and Practice 3rd Edition Springer Verlag, NY; Janson and Ryden (1998) Protein Purification: Principies. High Resolution Methods and Applications. Second Edition Wiley-VCH, NY; and Walker (1998) Protein Protocols on CD-ROM Humana Press, NJ; and the references cited therein. An advantage of producing polypeptides comprising at least one non-natural amino acid in a host cell eukaryotic or non-eukaryotic host cell is that polypeptides will commonly be folded in their natural conformations. However, in certain embodiments of the methods and compositions described herein, after synthesis, expression and / or purification, the polypeptides may possess a different conformation from the desired confirmations of the relevant polypeptides. In one aspect of the methods and compositions described herein, the expressed protein is optionally denatured and then renatured. This optional denaturation and renaturation is carried out using methods known in the art, including but not limited to, by adding a chaperonin to the polypeptide of antibodies and by solubilizing the proteins in a chaotropic agent in which include but not limited to guanidma HCl and using proterne isomerase disulfide. In general, it is occasionally desirable to denature and reduce the expressed polypeptides and then cause the polypeptides to re-fold to the preferred conformation. By way of example, such re-folding can be effected with the addition of guanidma, urea, DTT, DTE and / or a chaperonma to a translation product of interest. Methods for reducing, denaturing and re-naturalizing proteins are well known to those of skill in the art (see references above and Debmski, et al. (1993) J. Biol. Chem., 268: 14065-14070; Kreitman and Pastan (1993) Bioconjug. Chem., 4: 581-585; and Buchner, et al., (1992) Anal. Biochem., 205: 263-270). Debinski, et al., For example, describe denaturation and reduction of guanidine-DTE inclusion body proteins. The proteins can be re-folded into a buffer containing the redox pH, which includes, but is not limited to, oxidized glutathione and L-arginine. The re-folding reagents can be made to flow or otherwise be made to move in contact with one or more polypeptides or other expression product or vice versa. In the case of prokaryotic production of a non-natural amino acid polypeptide, the polypeptide produced herein can be misfolded and thus lacks or has reduced biological activity. The bioactivity of the protein can be restored by "refolding". In one embodiment, a misfolded polypeptide is re-folded upon solubilization (wherein the polypeptide is also insoluble), de-folding and reducing the polypeptide chain using, by way of example, one or more chaotropic agents (in which are included but not limited to urea and / or guanidine) and a reducing agent capable of reducing disulfide bonds (in which are included but not limited to dithiothreitol, DTT or 2-mercaptoethanol, 2-ME). At a moderate concentration of the chaotrope, an oxidant is then added (for example oxygen, cystine or cystamine), which allows the re-formation of disulfide bonds. A polypeptide Unfolded or misfolded can be re-folded using standard methods known in the art, such as those described in U.S. Patent Nos. 4,511,502, 4,511,503 and 4,512,922, each of which is incorporated herein by reference in its entirety. The polypeptide can also be co-folded with other proteins to form heterodimers or heteromultimers. After re-folding or co-folding, the polypeptide can be further purified. The purification of non-natural amino acid polypeptides can be carried out using a variety of techniques, including but not limited to those described herein, by way of example hydrophobic interaction chromatography, size exclusion chromatography, ion exchange, reverse phase high performance liquid chromatography, affinity chromatography and the like or any combination thereof. The additional purification may also include a step of drying or precipitation of the purified protein. After purification, the non-natural amino acid polypeptides can be exchanged in different buffer solutions and / or concentrates by any of a variety of methods known in the art, including, but not limited to, diafiltration and dialysis. hGH that is provided as a single purified protein may be subjected to aggregation and precipitation. In certain embodiments, the purified non-natural amino acid polypeptides can be at least 90% pure (as measured by reverse phase high performance liquid chromatography, RP-HPLC or sodium dodecyl sulfate-polyacrylamide gel electrophoresis, SDS- PAGE). In certain other embodiments the purified non-natural amino acid polypeptides may be at least 95% pure or at least 98% pure or at least 99% or greater in purity. Regardless of the exact numerical value of the purity of the non-natural amino acid polypeptides, the non-natural amino acid polypeptides are sufficiently pure for use as a pharmaceutical or for further processing, in which, but not limited to, conjugation with a water soluble polymer such as PEG. In certain embodiments, molecules of non-natural amino acid polypeptides can be used as therapeutic agents in the absence of other active ingredients or proteins (other than excipient, carriers and stabilizers, serum albumin and the like) and in certain embodiments polypeptide molecules. of non-natural amino acids can be complexed with another polypeptide or a polymer. 2. Purification of Non-Amino Acid Polypeptides General purification methods. The techniques disclosed in this section can be applied to the general purification of the non-natural amino acid polypeptides described herein. Any of a variety of isolation steps can be carried out on the dialyzed cell extract, culture medium, inclusion body, periplasmic space of the host cells, cytoplasm of the host cells or other material comprising the desired polypeptide or on any mixtures of polypeptides resulting from any isolation steps including but not limited to affinity chromatography, ion exchange chromatography, hydrophobic interaction chromatography, gel filtration chromatography, high performance liquid chromatography ("HPLC"), phase HPLC inverse ("RP-HPLC"), expanded bed adsorption or any combination and / or repetition thereof and in any appropriate order. The equipment and other necessary materials used to perform the techniques described herein are commercially available. Pumps, fraction collectors, monitors, recorders and complete systems are available from, for example, Applied Biosystems (Foster City, CA), BioRad Laboratories, Inc. (Hercules, CA) and Amersham Biosciences, Inc. (Piscataway, NJ). Chromatographic materials which include, but are not limited to, exchange matrix materials, media and pH buffer solutions are also available from such companies.

The equilibrium and other steps in the column chromatography processes described herein such as washing and elution, can be carried out more quickly using specialized equipment such as a pump. Commercially available pumps include but are not limited to, HILOAD® P-50 pump, P-I peristaltic pump, P-901 pump and P-903 pump (Amersham Biosciences, Piscataway, NJ). Examples of fraction collectors include the RediFrac fraction collector, fraction collectors FRAC-100 and FRAC-200, and the fraction collector SUPERFRAC® (Amersham Eiosciences, Piscataway, NJ). Mixers are also available to form pH gradients and linear concentration gradients. Commercially available mixers include the GM-I gradient mixer and in-line mixers (Amersham Biosciences, Piscataway, NJ). The chromatographic process can be monitored using any commercially available monitor. Such monitors can be used to collect information such as UV, fluorescence, pH and conductivity. Examples of detectors include UV-I Monitor, UVICORD® S II, UV-M II Monitor, UV-900 Monitor, UPC-900 Monitor, pH / C-900 Monitor and Conductivity Monitor (Amersham Biosciences, Piscataway, NJ). Of course, whole systems are commercially available which include the various AKTA® systems from Amersham Biosciences (Piscataway, NJ).

In one embodiment of the methods and compositions described herein, for example, the polypeptide can be reduced and denatured by first denaturing the resulting purified polypeptide in urea, followed by dilution in TRIS buffer containing a reducing agent (such as as DTT) at an appropriate pH. In another embodiment, the polypeptide is denatured in urea in a concentration range of between about 2 M to about 9 M, followed by dilution in pH TRIS buffer at a pH in the range of about 5.0 to about 8.Ü. The re-folding mixture of this modality can then be incubated. In one embodiment, the re-folding mixture is incubated at room temperature for four to twenty-four hours. Then the reduced and denatured polypeptide mixture can be further isolated or purified. As stated herein, the pH of the first polypeptide mixture can be adjusted before carrying out any subsequent isolation steps. In addition, the first polypeptide mixture or any subsequent mixture thereof can be concentrated using techniques known in the art. In addition, the elution pH buffer solution comprising the first polypeptide mixture or any subsequent mixture thereof can be exchanged for an appropriate pH buffer for the next isolation step using well-known techniques for those of ordinary skill in art. Ion exchange chromatography. The techniques disclosed in this section can be applied to the ion chromatography of the non-natural amino acid polypeptides described herein. In one embodiment and as an optional additional step, ion exchange chromatography can be performed in the first polypeptide mixture, See generally ION EXCHANGE CHROMATOGRAPHY: PRINCIPLES AND METHODS (Cat. No. 18-11 14-21, Amersham Biosciences (Cat. Piscataway, NJ)). Commercially available ion exchange columns include the columns HITRAP®, HIPREP®, and HILOAD® (Amersham Biosciences, Piscataway, NJ). Such columns use strong anion exchangers such as Q SEPHAROSE® Fast Flow, Q SEPHAROSE® High Performance and Q SEPHAROSE® XL; strong cation exchangers such as SP SEPHAROSE® High Performance, SP SEPHAROSE® Fast Flow and SP SEPHAROSE® XL; weak anion exchangers such as DEAE SEPHAROSE® Fast Flow; and weak cation exchangers such as CM SEPHAROSE® Fast Flow (Amersham Biosciences, Piscataway, NJ). Anionic or cationic exchange column chromatography can be performed on the polypeptide at any stage of the purification process to substantially isolate the purified polypeptide. The cation exchange chromatography step can be carried out using any suitable cation exchange matrix.

Cationic exchange matrices include but are not limited to fibrous, porous, non-porous, microgranular, pearlized or crosslinked cation exchange matrix materials. Such cation exchange matrix materials include but are not limited to cellulose, agarose, dextran, polyacrylate, polyvinyl, polystyrene, silica, polyether or compounds of any of the foregoing. Following adsorption of the polypeptide to the cation exchange matrix, the substantially purified polypeptide can be eluted by contacting the matrix with a pH buffer solution having a sufficiently high pH or high ionic strength to displace the polypeptide from the matrix. Suitable pH regulating solutions for use in the high pH elution of the substantially purified polypeptide include but are not limited to pH regulating solutions of citrate, phosphate, formate, acetate, HEPES and MES that fluctuate in concentration of at least about 5 mM to at least about 100 mM. Reverse phase chromatography. The techniques disclosed in this section can be applied to reverse phase chromatography of the non-natural amino acid polypeptides described herein. RP-HPLC can be effected to purify proteins following suitable protocols that are known to those of ordinary skill in the art. See, for example, Pearson et al., ANAL. BIOCHEM. (1982) 124: 217-230; Rivier et al., J. CHROM. (1983) 268: 112-119; Kunitam et al., J. CHROM. (1986) 359: 391-402. RP-HPLC can be performed on the polypeptide to isolate the substantially purified polypeptide. In this regard, resins derived from silica with alkyl functionalities with a wide variety of lengths, which include but are not limited to, reams from about C3 to at least about C3o, at least C3 to at least about C20 , or at least C to at least about Cis- Alternatively, a polymeca resin can be used. For example, the resin CGlOOOsd TosoHaas Amberchrome, which is a styrene polymer resin. Also cyano or polymeric with a wide variety of alkyl chain lengths can be used. In addition, the RP-HPLC column can be washed with a solvent such as ethanol. An appropriate elution pH buffer solution containing an ionic coupling agent and an organic modifier such as methanol, isopropanol, tetrahydrofuran, acetonitrile or ethanol, can be used to elute the polypeptide from the RP-HPLC column. Commonly used ionic pairing agents include but are not limited to acetic acid, formic acid, perchloric acid, phosphoric acid, trifluoroacetic acid, heptafluorobutyric acid, triethylamine, tetramethylammonium, tetrabutylammonium, triethylammonium acetate. Elution can be carried out using one or more gradients or isocratic conditions, gradient conditions are preferred to reduce the separation time and to decrease the peak width. Another method involves the use of two gradients with different solvent concentration ranges. Examples of elution pH buffer solutions suitable for use herein may be but are not limited to solutions of ammonium acetate and acetonitrile.

Communication techniques of hydrophobic interaction chromatography. The techniques discussed in this section can be applied to the purification of hydrophobic interaction chromatography of the non-natural amino acid polypeptides described herein. Hydrophobic interaction chromatography (HIC) can be performed on the polypeptide. See generally HYDROPHOBIC INTERACTION CHROMATOGRAPHY HANDBOOK: PRINCIPLES AND METHODS (Cat. No. 18-1020-90, Amersham Biosciences (Piscataway, NJ) which is incorporated by reference herein) Appropriate HIC matrices may include but are not limited to matrices alkyl- or aryl-substituted, salts as butyl-, hexyl-, octyl- or phenyl-substituted matrices, which include matrices of agarose, cross-linked agarose, sepharose, cellulose, silica, dextran, polystyrene, poly (methacrylate) and resins in mixed mode, in which are included but not limited to a ethyleneamine resin or a butyl- or phenyl-substituted poly (methacrylate) matrix. Commercially available sources of hydrophobic interaction column chromatography include but are not limited to HITRAP®, HIPREP® and HILOAD® columns (Amersham Biosciences, Piscataway, NJ). Briefly, prior to loading, the HIC column can be equilibrated using pH regulatory elutions known to those of ordinary skill in the art, such as an acetic acid / sodium chloride solution or HEPES containing ammonium sulfate. Ammonium sulfate can be used as the pH buffer for loading the HIC column. After loading the polypeptide, the column can then be washed using pH buffer solutions under standard conditions to remove initial conditions but retaining the polypeptide on the HIC column. The polypeptide can be eluted with about 3 to about 10 column volumes of a standard pH buffer solution, such as a buffer solution of pH HEPES containing EDTA and a concentration of ammonium sulfate lower than the equilibrium pH buffer solution. or a solution regulating the pH of acetic acid / sodium chloride, among others. A decreasing linear salt gradient using for example a gradient of potassium phosphate can be used to elute the polypeptide molecules. Then the eluent can be concentrated, for example, by filtration such as diafiltration or ultrafiltration The diafiltration can be used to remove the salt used to elute the polypeptide. Other purification techniques. The techniques disclosed in this section can be applied to other purification techniques of the non-natural amino acid polypeptides described herein. Still another isolation step, for example, gel filtration (GEL FILTRATION: PRINCIPLES AND METHODS (Cat. No. 18-1022-18, Amersham Biosciences, Piscataway, NJ, which is incorporated herein by reference in its entirety), Hydroxyapatite chromatography (suitable matrices include but are not limited to HA-Ultrogel, High Resolution (Calbiochem), CHX Ceramic Hydroxyapatite (BioRad), Bio-Gel HTP-Hydroxyapatite (BioRad)), HPLC, expanded bed adsorption, Ultrafiltration, diafiltration, lyophilization and the like can be performed on the first polypeptide mixture or any subsequent mixture thereof to remove any excess salt to replace the pH buffer solution with an appropriate pH buffer for the next step of isolation or even final drug product formulation The performance of the polypeptide, in which the substantially purified polypeptide is included, can be moni performed in each step described herein using various techniques, including, but not limited to, those described herein. Such techniques can also be used to determining the yield of the substantially purified polypeptide following the last isolation step. By way of example, the performance of the polypeptide can be monitored using any of several reverse phase high pressure liquid chromatography columns, having a plurality of alkyl chain lengths, such as Cyan RP-HPLC, C? 8RP- HPLC, also as HPLC cation exchange and HPLC gel filtration. In certain embodiments, the yield of the pclipeptide after each purification step may be at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50 %, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85% , at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.9% or at least about 99.99% of the polypeptide in the starting material for each step of purifying tion.

Purity can be determined using standard techniques, such as SDS-PAGE or by measuring the polypeptide using Western blot and ELISA. For example, polyclonal antibodies can be generated against proteins isolated from yeast fermentation of negative control and recovery of cation exchange. The antibodies can also be used to test for the presence of contaminating host cell proteins. The Vydac C4 RP-HPLC material (Vydac) consists of silica gel particles, the surface of which carry C4-alkyl chains. The separation of the polypeptide from the proteinaceous impurities is based on differences in the strength of hydrophobic interactions. The elution is carried out with a gradient of acetonitrile in dilute trifluoroacetic acid. Preparative HPLC is performed using a stainless steel column (filled with 2.8 to 3.2 liters of Vydac C4 silica gel). The Ultrogel Hydroxyapatite eluate is acidified by adding trifluoroacetic acid and loaded onto the Vydac C4 column. A gradient of acetonitrile in dilute trifluoroacetic acid is used for washing and elution. The fractions are collected and immediately neutralized with phosphate buffer. Fractions of the polypeptide that are within the limits of IPC are accumulated. The DEAE Sepharose material (Pharmacia) consists of diethylammoethyl (DEAE) groups that are linked covalently to the surface of Sepharose cells. The binding of the polypeptide to the DEAE groups is moderated by ionic interactions. The acetonitrile and trifluoroacetic acid pass through the column without being retained. After these substances have been washed, the trace impurities are removed by washing the column with pH buffer solution of acetate at a low pH. The column is then washed with pH neutral phosphate buffer solution and the polypeptide is eluted with a pH buffer solution with increased ionic strength. The column is packed with fast flow Sepharose DEAE. The volume of the column is adjusted to ensure a loading of polypeptide in the range of 3-10 mg of polypeptide / ml of gel. The column is washed with water and equilibrium pH buffer solution (sodium / potassium phosphate). The accumulated fractions of the HPLC eluate are charged and the column is washed with equilibrium pH buffer. The column is then washed with washing buffer (pH buffer solution of sodium acetate) followed by washing with buffer solution of equilibrium pH. Subsequently, the polypeptide is eluted from the column with elution pH buffer (sodium chloride / sodium / potassium phosphate) and collected in a single fraction according to the main elution profile. The eluate of the DEAE Sepharose column is adjusted to the specific conductivity. The drug substance The resulting product is sterile registered in Teflon bottles stored at -70 ° C. Additional methods include but are not limited to, steps to remove endotoxins. The endotoxins are lipopolysaccharides (LPS) which are located in the outer membrane of Gram-negative host cells, such as, for example, Escherichia coli. Methods to reduce levels of endotoxin include but are not limited to, purification techniques using silica supports, glass powder or hydroxyapatite reverse phase, affinity chromatcgraphy, size exclusion, anion exchange chromatography, hydrophobic interaction chromatography or a combination of these methods and the like. Modifications or additional methods may be required to remove contaminants such as co-migrating proteins of the polypeptide of interest.

Methods for measuring endotoxin levels are known to those of ordinary skill in the art and include but are not limited to analysis of Lysate of A ebocyte Linnulus (LAL) Additional methods and procedures include but are not limited to, SDS-PAGE coupled with protein-holding methods, immunoabsorption, mass spectrometry attrition / ionization assisted by matrix laser (MALDI-MS), liquid chromatography / mass spectrometry, isoelectric, analytical anion exchange, chromatofocusing and circular dichroism. In certain embodiments, the non-natural amino acids described herein can be biosynthetically incorporated into polypeptides, thereby making non-natural amino acid polypeptides. In other embodiments, such amino acids are incorporated at a specific site within the polypeptide. In other embodiments, such amino acids incorporated into the polypeptide using a translation system. In other embodiments, such translation systems comprise: (i) an oligonucleotide encoding a polypeptide, wherein the polynucleotide comprises a selector codon corresponding to the pre-designated incorporation site of the above amino acids and (ii) a tRNA comprising the amino acid , where the tRNA is specific to the selector codon. In other embodiments of such translation systems, the polynucleotide is mRNA produced in the translation system. In other embodiments of such translation systems, the translation system comprises a plasmid or a phage comprising the polynucleotide. In other embodiments of such translation systems, the translation system comprises genomic DNA comprising the polynucleotide. In other embodiments of such translation systems, the polynucleotide is stably integrated into the genomic DNA. In other embodiments of such translation systems, the translation system comprises specific tRNA by a selector codon selected from the group that It consists of an amber codon, ocher codon, opal codon, a single codon, a rare codon, an unnatural codon, a five-base codon and a four-base codon. In other embodiments of such translation systems, tRNA is a suppressor tRNA. In other embodiments of such translation systems, the translation system comprises a tRNA that is ammoacylated to the above amino acids. In other embodiments of such translation systems, the translation system comprises a specific ammoacyl synthetase for the tRNA. In other embodiments of such translation scans, the translation system comprises an orthogonal tRNA and an orthogonal ammoacyl tRNA smtetase. In other embodiments of such translation systems, the polypeptide is synthesized by a ribosome and in additional embodiments, the translation system is a translimation system comprising a cell selected from the group consisting of a bacterial cell, archaeobacteric cell and cell. eukaryotic In other embodiments, the cell is an Escherichia coli cell, yeast cells, a cell of a Pseudomonas species, mammalian cell, plant cell or an insect cell. In other embodiments of such translation systems, the translation system is a translating system comprising cell extract from a bacterial cell, archaebacteria cell or eukaryotic cell. In other embodiments, the cell extract is from an Escherichia coli cell, a cell of a Pseudomonas species, yeast cells, mammalian cell, plant cells or an insect cell. In other embodiments, at least a portion of the polypeptide is synthesized by solid phase or solution phase peptide synthesis or a combination thereof, while in other embodiments it further comprises ligating the polypeptide to another polypeptide. In other embodiments, the non-natural amino acids described herein can be biosynthetically incorporated into a polypeptide, wherein the polypeptide is a protein homologue to a therapeutic protein selected from the group consisting of: alpha-1, antitrypsin, angiostatin, antihemolytic factor, antibody, apolipoprotein, apoprotein, atrial natri-retic factor, atrial natriuretic polypeptide, atrial peptide, chemokine CXC, T39765, NAP-2, ENA-78, gro-a, gro-b, gro-c, IP-10, GCP-2 , NAP-4, SDF-1, PF4, M1G, calcitonin, ligand c-kit, cytosine, chemokine CC, protein-1 alpha chemoattractant monocyte, protein-2 alpha chemoattractant monocyte, protein-3 alpha chemoattractant monocyte, protein -1 monocyte inflammatory alpha, monocyte inflammatory beta-1 protein, RANTES, 1309, R83915, R91733, HCC1, T58847, D31065, T64262, CD40, CD40 ligand, c-kit ligand, collagen, colony stimulating factor (CSF) , complement factor 5a inhibitor, complement receptor 1, cytosine, peptide-78 epithelial neutrophil activator, MIP-16, MCP-1, epithelial growth factor (EGF), activating peptide of epithelial neutrophil, erythropoietin (EPO), exfoliating toxin, Factor IX, Factor VII, Factor VIII, Factor X, fibroblast growth factor (FGF), fibrinogen, fibronectin, four-helix bundle protein, G-CSF, glp- 1, GM-CSF, glucocerebrosidase, gonadotropin, growth factor, growth factor receptor, grf, hedgehog protein, hemoglobin, hepatocyte growth factor (hGF), hirudin, human growth hormone (hGH), serum albumin human, ICAM-1, ICAM-1 receptor, LFA-1, LFA-1 receptor, insulin, insulin-like growth factor (IGF), IGF-1, IGF-II, interferon (IFN), IFN-alpha , IFN-beta, IFN-gamma, any interferon-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, keratinocyte growth factor (KGF), lactoferrin, leukemia inhibitory factor, luciferase, neurturin, neutrophil inhibiting factor ( NIF), onc Ostatin M, osteogenic protein, oncogene product, paracytonine, parathyroid hormone, PD-ECSF, PDGF, peptide hormone, pleiotropin, protein A; G protein, pth, pyrogenic exotoxin A, pyrogenic exotoxin B, pyrogenic exotoxin C, pyy, relaxin, renin, SCF, small biosynthetic protein, soluble complement receptor I, soluble ICAM-1, soluble interleukin receptor, soluble TNF receptor, somatomedin , somatostatin, somatotropin, streptokinase, superantigens, staphylococcal enterotoxin, SEA, SEB, SEC1, SEC2, SEC3, SED, SEE, steroid hormone receptor, superoxide dismutase, toxic shock syndrome toxin, alpha limosine 1, tissue plasminogen activator, tumor growth factor (TGF), tumor necrosis factor, tumor necrosis alpha factor, tumor necrosis beta factor, tumor necrosis factor receptor (TNRF), VLA protein -4, VCAM-1 protein, vascular endothelial growth factor (VEGF), urokinase, mos, ras, raf, met, p53, tat, fos, myc, jun, myb, rei, estrogen receptor, progesterone receptor, testosterone receptor, aldosterone receptor, receptor LDL and corticosterone.

B. In vivo post-translational modifications In producing proteins or polypeptides of interest with at least one non-natural amino acid in eukaryotic cells, such polypeptides can include eukaryotic post-translational modifications. In certain embodiments, a protein includes at least one unnatural amino acid and at least one post-translational modification that is made in vivo by a eukaryotic cell, wherein the post-translational modification is not made by a prokaryotic cell. By way of example, post-translational modification includes but is not limited to acetylation, acylation, lithium modification, palmitoinination, palmitate addition, phosphorylation, glycolipid linkage modification, glycosylation and the like. In one aspect, the post-translational modification includes annexation of an oligosaccharide (in which but not limited to (GlcNAc-Man) 2-Man-GlcNAc-GlcNAc)) to an asparagine by a GlcNAc-asparagine binding. See Table 1 listing some examples of N-linked oligosaccharides of eukaryotic proteins (additional residues may also be present, which are not shown). In another aspect, post-translational modification includes annexation of an oligosaccharide (in which but not limited to Gal-GalNAc, Gal-GlcNAc, etc.) to a serine or threonine via a link of GalNAc-serine or GalNAc- threonine or GlcNAc-threonine linkage or a GlcNAc-sepna link.

Table 1: Examples of oligosaccharides by means of GLCNAC-links In yet another aspect, the post-translational modification includes the proteolytic processing of precursors (in which they include but are not limited to, calcitonin precursor, peptide precursor, calcitonin-regulated gene, pre-proparatiroid hormone, preproinsulin, proinsulin, pre -pro-opiomelanocortin, pro-opiomelanocortin and the like), assemble a multisubunit protein or macromolecular assembly, translation to another site in the cell (in which they include but not limited to organelles, such as the endoplasmic reticulum, the Colgi apparatus, the nucleus, lusosomes, peroxisomes, mitochondria, chloroplasts, vacuoles, etc., or by means of the secretory route). In certain embodiments, the protein comprises a secretion or localization sequence, an epitope tag, a FLAC tag, a polyhistidine tag, a GST fusion or the like. An advantage of an unnatural amino acid is that it has additional chemical portions that can be used to add additional molecules. These modifications can be made in vivo in a eukaryotic or non-eukaryotic or in vitro cell. Thus, in certain modalities, post-translation modification is by means of the non-natural amino acid. For example, post-translational modification can be by means of a nucleophilic-electrophilic reaction. Most of the reactions currently used for the Selective modification of proteins involve the formation of covalent bonds between nucleophilic and electrophilic reaction partners, which include but are not limited to the reaction of α-haloketones with histidine or cysteine side chains. The selectivity in these cases is determined by the number and access of the nucleophilic residues in the protein. In the polypeptides described herein or produced using the methods described herein, other more selective reactions may be used, in which they include but are not limited to, the reaction of a non-natural keto-amino acid with hydrazine or aminooxy compounds, in vitro and in vivo. See, for example, Cornish, et al., (1996) J. Am. Chem. Soc. , 118: 8150-8151; Mahal, et al., (1997) Science, 276: 1125-1128; Wang, et al., (2001) Science, 292: 498-500; Chin, et al., (2002) JX Am. Chem. Soc, 124: 9026-9027; Chin, et al., (2002) Proc.

Nati Acad. Sci., 99: 11020-11024; Wang, et al., (2003) Proc. Nati Acad. Sci., 100: 56-61; Zhang, et al., (2003) Biochemistry, 42: 6735-6746 and Chin, et al., (2003) Science, 301: 964-7. This allows for the selective labeling of virtually any protein with a reagent host in which are included fluorophores, crosslinking agents, saccharide derivatives and cytotoxic molecules. See also, U.S. Patent No. 6,927,042 entitled "Glycoprotein synthesis" filed January 16, 2003, which is incorporated by reference herein. Post modifications Translation, in which but not limited to, by means of an azido amino acid, may also be performed by means of Staudinger ligation (in which are included but not limited to triarylphosphine reagents). See, for example, Kiick et al., (2002) Incorporation of azides in to recombine t proteins for chemoselective modi fi cation by the Sta udinger lígta tíon, PNAS 99: 19-24.

IX. Alternative systems for producing non-natural amino acid polypeptides Various strategies have been employed for introducing non-natural amino acids into proteins in non-recombinant host cells, mutagenized host cells or in cell-free systems. The alternative systems revealed in this section can be applied to the production of the non-natural amino acid. By way of example, derivatization of amino acids with reactive side chains such as Lys, Cys and Tyr resulted in the conversion of lysine to N 2 -acetyl-lysine. Chemical synthesis also provides a method of this to incorporate non-natural amino acids. With the recent development of enzymatic ligation and natural chemical ligation of peptide fragments, it is possible to manufacture larger proteins. See, for example, P. E. Dawson and S. B. H. Kent, Annu. Rev. Biochem. , 69: 923 (2000). The ligation of chemical peptide and Natural chemical ligation is described in U.S. Patent 6,184,344, U.S. Patent Publication No. 2004/0138412, U.S. Patent Publication No. 2003/0208046, WO 02/098902 and WO 03/042235, which are incorporated herein by reference in its entirety A biosynthetic general Vac method in which a suppressor tRNA chemically acylated with the desired non-natural amino acid is added to a m Vitro extract capable of supporting protein biosynthesis, has been used to specifically incorporate more than 100 non-natural amino acids into the site. a variety of proceinas of virtually any size. See, for example, V. W. Cornish, D. Mendel and P. G. Schultz, Angew. Chem. Int. Ed. Engl. , 1995, 34: 621 (1995); CJ. Noren, S.J. Anthony-Cahill, M.C. Griffith, P.G. Schultz, A generates the method for if you specify the incorporation of the amino acid to proteins, Science, 244: 182-188 (1989) and J.D. Bain, CG. Glabe, T.A. Dix, A.R. Chamberlm, E.S. Diala, Bi osyn the ti c si te-speci fi c mcorpora ti on of a non-na ture amino a cid m to polypeptide, J. Am. Chem. Soc, 111: 8013-8014 (1989). A wide range of functional groups have been introduced to proteins for studies of protein stability, protein folding, enzymatic mechanism and signal transduction. A live m method, called selective pressure incorporation, was developed to take advantage of the promiscuity of wild-type synthetases. See, N. Budisa, C. Mmks, S.

Alefelder, W. Wenqer, F. M. Dong, L. Moroder and R. Huber, FASEB J., 13: 41 (1999). An aucotropic strain, in which the relevant metabolic pathway that supplies the cell with a particular natural amino acid is quenched, is cultured in minimal media containing limited concentrations of the natural amino acid, while the transcription of the target gene is repressed. At the start of a stationary growth phase, the natural amino acid is depleted and replaced with the unnatural amino acid analogue. The induction of expression of the recombinant protein results in the accumulation of a protein containing the unnatural analogue. For example, using this strategy om and p-fluorophenylalanines have been incorporated into proteins and exhibit two distinctive highlights in the UV spectrum that can be easily identified, see for example, C. Minks, R. Huber, L. 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 cyto-oligosaccharide ligands by 19 F NMR, see for example H. Duewel, E. Daub, V. Robinson and JF Honek, Biochemistry, 36: 3404 ( 1997) and trifluoroleucine has been used in place of leucine, resulting in increased thermal and chemical stability of a leucine-zipper protein. See, for example, Y. Tang, G. Ghirlanda, W. A. Petka, T. Nakajima, W. F. DeGrado and D. A. Tirrell, Angew. Chem. Int. Ed. Engl., 40: 1494 (2001). Further, selenomethionine and teluromethion are incorporated into various cobinders to facilitate phase resolution in X-ray crystallography. See for example, W. A. Hendrickson, J. R. Horton and D. M. Lemaster, EMBO J., 9: 1665 (1990); J. O. Boles, K. Lewmski, M. Kunkle, J. D. Odom, B. Dunlap, L. Lebioda and M. Hatada, Nat. Struct. Biol. , 1: 283 (1994); N. Budisa, B. Steipe, P. Demapge, C. Eckerskorn, J. Kellermann and R. Huber, Eur. J. Biochem., 230: 788 (1995) and N. Budisa, W. Karnbrock, S. Stembacher, A. Humm, L. Prade, T. Neuefe d, L. Moroder and R. Huber, J. Mol. Biol., 270: 616 (1997). Analogs of metionma with alquene or alquene runcionaliaades have also been incorporated efficiently, allowing the additional modification of proteins by chemical means. See for example, J. C. M. van Hest and D. A. Tirrell, FEBS Lett., 428: 68 (1998); J. C. M. van Hest, K. L. Knck and D. A. Tirrell, J. Am. Chem. Soc, 122: 1282 (2000) and K. L. Knck and D. A. Tirrell, Tetrahedron, 56: 9487 (2000); U.S. Patent 6,586,207; US Patent Publication 2002/0042097, which are incorporated herein by reference in their entirety The success of this method depends on the recognition of non-natural amino acid analogs by ammoacyl-tRNA synthetases, which generally require high selectivity to ensure fidelity of protein translation. One way to expand the scope of this method is to relax the specificity of substrate in the aminoacyl-tRNA synthetases, which has been obtained in a limited number of cases. By way of example only, the replacement of Ala294 by Gly in phenylalanyl-tRNA synthetase (PheRS) of Escherichia coli increases the size of the substrate binding cavity and results in the clearance of p-Cl-phenylalanine (p-Cl-). Phe). See, M. Ibba, P. Kast and H. Hennecke, Biochemistry, 33: 7107 (1994). A strain of Escherichia coli harboring this mutant PheRS allows the incorporation of p-CI-phenylalanine or p-Br-phenylalanine instead of phenylalanine. See for example, M. Ibba and H. Hennecke, FEBS Lett. , 364: 272 (1995) and N. Sharma, R. Furter, P. Kast and D. A. Tirrell, FEBS Lett. , 467: 37 (2000). Similarly, a Phel30Ser point mutation near the tyrosyl-tRNA synthetase amino acid binding site of Escherichia coli was shown to allow azathosine to be incorporated more efficiently than tyrosine. See, F. Hamano-Takaku, T. Iwama, S. Saito-Yano, K. Takaku, Y. Monden, M. Kitabatake, D. Soil and S. Nishimura, J. Biol. Chem., 275: 40324 • (2000). Another strategy to incorporate non-natural amino acids into proteins in vivo is to modify the synthetases that have reading-proof mechanisms. These synthetases can not discriminate and therefore activate amino acids that are structurally similar to the natural cognates amino acids. This error is corrected in a separate site, which deacylates the most charged amino acid of the tRNA to maintain the fidelity of protein translation. If the smtetase correction activity is enabled, structural analogs that are poorly acylated can escape the editing function and be incorporated. This procedure has been recently demonstrated with the valil-tRNA smtetase (ValRS), See, V. Doring, HD Mootz, LA Nangle, TL Hendrickson, V. de Crecy-Lagard, P. Schimmel and P. Marliere, Science, 292: 501 (2001). ValRS may mis-acilate Val tRNA with Cys, Thr, or ammobutyrate (Abu); these non-cognate amino acids are subsequently hydrolyzed by the domain d edition. After the random mutagenesis of the Eschep chia coli chromosome a strain of Escheri chia col i mutant was selected to have a mutation in the ValRS editing site. This editing ValRS incorrectly loads ARNtVal with Cys. Because Abu roughly resembles Cys (the -SH group of Cys is replaced with -CH3 in Abu), the ValRS mutant when this strain of Eschep chia coli mutant is cultured in the presence of Abu. The mass spectrometric analysis shows that approximately 24% of the values are replaced by Abu in each valma position in the natural protein. Solid phase synthesis methods and semi-synthetic methods have also allowed the synthesis of a variety of proteins containing new amino acids. For example, see the following publications and references cited therein, which are as follows: Crick, F.H.C., Barrett, L. Brenner, S. Watts-Tobm, R. Genera l na ture of the genetic code for proteins. Nature, 192: 1227-1232 (1961); Kaiser, E.T. Syn thetic approaches to biologically active peptides and proteins incl udmg enyzmes, Acc Chem Res. , 22: 47-54 (1989); Nakatsuka, T-, Sasaki, T., Kaiser, E.T. Peptide segmen t coupling ca talyzed by the semisyn thetic enzyme thiosubtil ism, J Am Chem Soc, 109: 3808-3810 (1987); Schnolzer, M., Kent, S B H. Constructing proteins by dovetailmg unprotected synthetic peptides: backbone-5 engmeered HIV protease, Science, 256 (5054): 221-225 (1992); ohaiken, i.M. Semisynthetic peptides and proteins, CRC "rit Rev Biochem 11 (3): 255-301 (1981); Offbrd, RE Protein engmeepng by chemi cal means Protein Eng., 1 (3): 151-157 (1987); and Jackson , DY, Burnier, J., Quan, C, Stanley, M., Tom, J., Wells, JA A Designed Peptide Ligasefor Total Syn thesis of Ribonuclease A wi th Unna tural Ca talytic Residues, Science, 266 (5183): 243 (1994) Chemical modification has been used by introducing a variety of unnatural side chains, in which co-factors, spin markers and oligonucleotides are included in vitro proteins See, for example, Corey, DR, Schultz, PG Generation of a hybrid sequence-specific smgle-stranded deoxypbonuclease, Science, 238 (4832): 1401-1403 (1987); Kaiser, ET, Lawrence DS, Rokita, SE The chemica l modifi cation of enzyma tic speci fi ci ty , Annu Rev. Biochem., 54: 565-595 (1985); Kaiser, E.T., Lawrence, D.S. Chemical mutation of enzyz active sites, Science, 226 (4674): 505-511 (1984); Neet, K.E., Nancí A, Koshland, D.E. Properties of thiol-subtilism, J Biol. Chem., 243 (24): 6392-6401 (1968); Polgar, L. (ed.)., M.L. Bender, A new enzyme contaming a synthetically formed active site. Thiol-subtilism. J. Am. Chem Soc. , 88: 3153-3154 (1966) and Pollack, SJ. , Nakayama, G. Schultz, P.G. Introduction of nucleophiles and spectroscopic probes into antibody combimng sites, Science, 224 (4881): 1038-1040 (1988).

Alternatively, biosynthetic mixtures employing chemically modified aminoacyl-tRNA have been used to incorporate several biophysical probes into proteins synthesized in Vitro. See the following publications and references cited therein: Brunner, J. New Photolabelmg and crosslmkmg methods, Annu. Rev Biochem., 483-514 (1993) and Kpeg, Xi. C, Walter, P., Hohnson, A.E. Photocrosslinkmg of the signal sequence of nascent preprolactm of the 54-kilodalton polypeptide of the signal recognition partiole, Proc. Nati Acad. Sci., 8604-8608 (1986) Previously, it has been shown that non-natural amino acids can be specifically incorporated into the site to m Vitro proteins by the addition of the chemically aminoacylated suppressor tRNA to programmed protein synthesis reactions with a gene containing a mutation in the sense of desired amber. Using these methods, a number of the 20 common amino acids with narrow structural homologs, for example fluorophenylalanine by phenylalanine, can be substituted using autotrophic strains for a particular amino acid. See for example, Noren, C.J., Anthony-Cahill, Gnffith, M.C., Schultz, P.G. A general method for site-specific mcorporation of unnatural ammo acids into proteins, Science, 244: 182-188 (1989); M.W. Nowak, et al., Science, 268: 439-42 (1995); Bam, J.D., Glabe, C.G., Dix, T.A., Chamberlm, A.R., Diala, E.S. Biosynthetic s1 t ^ -npecific Incorporation of a non-natural ammo acid into a polypeptide, J. Am Chem Soc. , 111: 8013-8014 (1989); N. Budisa et al., FASEB J. 13: 41-51 (1999); Ellman, J.A., Mendel, D., Anthony-Cahill, S., Noren, C.J., Schultz, P.G. Biosynthetic method for introducing unnatural ammo acids site-specifically mto proteins. Methods in Enz., Vol. 202, 301-06 (199) and Mendel, D., Cornish, V.W. and Schultz, P.G. Site-Directed Mutagenesis with an Expanded Genetic Code, Annu Rev Biophys. Biomol Struct. 24, 435-62 (1995). For example, a suppressor tRNA was repaired that recognized the UAG retention codon and was chemically ammoacylated with an unnatural amino acid. Conventional site-directed mutagenesis was used to introduce the TAG retention codon, at the site of interest in the protein gene. See, for example, Sayers, J.R., Schmidt, W. Eckstem, F. 5 ', 3 'Exonuclease in phosphorothioa te-based olignoucleotide-directed mutagensis, Nucleic Acids Res., 791-802 (1988). When the acylated suppressor tRNA and the mutant gene were combined in an in vitro transcription / translation system, the unnatural amino acid was incorporated in response to the UAG codon that gave a protein containing the amino acid at the specified position. Experiments using [3 H] -Phe and experiments with a-hydroxy acids showed that only the desired amino acid is incorporated in the position specified by the UAG codon and that this amino acid is not incorporated elsewhere in the protein. See, for example, Noren, et al, supra; Kobayashi et al., (2003) Nature Structural Biology 10 (6): 425-432 and Ellmap, J.A., Mendel, D., Schultz, P.G. If te-specifi c incorporation of navel backbone structures into proteins, Science, 197-200 (1992). Microinjection techniques have also been used to incorporate non-natural amino acids into proteins. See for example, MW Nowak, PC Kearney, JR Sampson, ME Sales, CG Labarca, SK Silverman, WG Zhong, J. Thorson, JN Aon, N. Davidson, PG Schultz, DA Dougherty and HA Lester, Science, 268: 439 (1995) and DA Dougherty, Curr. Opin. Chem. Biol. , 4: 645 (2000). A Xenopus oocyte was coinjected with two RNA species manufactured in vitro: an mRNA that encodes the target protein with a UAG retention codon at the amino acid position of interest and a suppressor tRNA aminoacylated amber with the desired non-natural amino acid. The oocyte production machinery then inserts the unnatural amino acid in the position specified by UAG. This method has allowed in vivo structure-function studies of integrated membrane proteins, which are generally not prone to in vitro expression systems. Examples include the incorporation of a fluorescent amino acid to the tachiquinin neurokinin-2 receptor to measure distances by fluorescence resonance energy transfer, see for example, G. Turcatti, K. Nemeth, MD Edgcrton, 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 residues exposed on the surface in ion channels,, see for example, 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 a real-time ion channel, see for example, 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 the fundamental ion channel chains to test for their gate mechanisms. See for example, PM England, Y. Zhang, DA Dougherty and HA Lester, Cell, 96: 89 (1999) and T. Lu, AY Ting, J. Mainland, LY Jan, PG Schultz and J. Yang, Nat. Neurosci . , 4: 239 (2001).

The ability to incorporate non-natural amino acids directly into proteins in vivo offers a wide variety of advantages including, but not limited to, high-throughput mutant proteins, technical ease, the potential to study mutant proteins in cells or possibly in living organisms and the use of these mutant proteins in therapeutic treatments. The ability to include unnatural amino acids with various sizes, acidities, nucleophilicities, hydrophobicities, and other protein properties can greatly expand the ability to rationally and systematically regulate protein structures, both to test protein function and to create new proteins or organisms with new properties In an attempt to incorporate specifically into the para-F-Phe site, a pair of tARNPheCUA / pheilalanyl-tRNA synthetase amber suppressor was used in a layer of Escheri chia coli Phe auxotropic, p-F-Phe resistant. See, for example, R. Furter, Protein Sci .. 7: 419 (1998). It may also be possible to obtain the expression of a desired polynucleotide using a cell-free (in-vitro) translation system. The translation systems can be cellular or cell-free and can be prokaryotic or eukaryotic. Cell translation systems include, but are not limited to, whole cell preparations such as permeabilized cells or cell cultures wherein a desired nucleic acid sequence can be transcribed to mRNA and the translated mRNA. Cell-free translation systems are crcially available and many different types and systems are well known. Examples of cell-free systems include, but are not limited to, used prokaryotes such as used from Escheri chia coli and used eukaryotes such as wheat germ extracts, used insect cell, used rabbit reticulocyte, used oocyte rabbit and Used of human cells. Extracts or used eukaryotic may be preferred when the resulting protein is glycosylated, phosphorylated or otherwise modified because many such modifications are only possible in eukaryotic systems. Some of these extracts and used are crcially available (Promega, Madison, Wis., Stratagene, La Jolla, California, Amersham, Arlington Heights, III, GIBCO / BRL, Grand Island, N.Y.). Membranous extracts, such as canine pancreatic extracts containing microsomal membranes, are also available that are useful for translating secretory proteins. In these systems, the can include either mRNA as a template (in-vitro translation) or DNA as a template (combined in-vitro transcription and translation), in vi tro synthesis is directed by the ribosomes. Considerable effort has been applied to the development of expression systems for cell-free proteins. See, for example, Kim, D.-M. and J.R. Swartz, Biotechnology and Bioengmeerm, 74: 309-316 (2001); Kim, D.-M. and J.R. Swartz, Biotechnol ogy Let ters, 22, 1537-1542, (2000); Kim, D.-M. and J.R. Swartz, Biotechnology Progress, 16, 385-390, (2000); Kim, D.-M. and J.R. Swartz, Bi otechnology and Bioengmeerm, 66, 130-188, (1999); and Patnaik, R. and J.R. Swartz, Biotechniques 24, 862-868, (1998); U.S. Patent No. 6,337,191; U.S. Patent Publication No. 2002/0081660; WO 00/55353; WO 90/05785, which are incorporated DOT reference herein. Another method that can be applied to the expression of polypeptides comprising an unnatural amino acid includes the fusion technique of mRNA-peptide. See, for example, R. Roberts and J. Szostak, Proc. Na ti Acad. Sci. (USA) 94 12297-12302 (1997); A. Frankel et al., Chemis try & Biology 10, 1043-1050 (2003). In this procedure, a mRNA template linked to puromicma is translated into a peptide on the ribosome. If one or more tRNA molecules have been modified, non-natural amino acids can be incorporated into the peptide as well. After the last codon of mRNA has been read, the puromicma captures the C-terminus of the peptide. If it is found that the resulting mRNA-peptide conjugate has interesting properties in an in vi tro analysis, its identity can be easily revealed from the mRNA sequence. In this way, polypeptide libraries comprising one or more can be selected non-natural amino acids to identify polypeptides having desired properties. More recently, translations of ribosome m vi tro with purified components have been reported that allow the synthesis of peptides substituted with non-natural amino acids. See, for example, A. Forster et al., Proc Nati Acad. Sc. (USA) 100 6353 (2003). It is also possible to use reconstituted translation systems. Amounts of purified translation factors have also been used successfully to translate mRNA to protein as well as combinations of lysates or lysates supplemented with purified translation factors such as start factor-1 (IF-1), IF-2, IF-3, T factor of elongation (EF-Tu) or termination factors. Cell-free systems can also be coupled transcription / translation systems where DNA is introduced into the system, transcribed to mRNA and translated mRNA as described in Current Protocols in Molecular Biology (F. M. Ausubel et al., Willey Interscience, 1993), which is specifically incorporated herein by reference. The RNA transcribed in the eucaponic transcription system can be in the form of heteronuclear RNA (hnRNA) or mature mRNA of poly A tail of 5 'end of crowns (7-met? L guanos a) and 3' end which can be a advantage in certain translation systems. For example, crowned mRNAs are translated with great efficiency into the lysate system of reticulocyte tRNA can be aminoacylated with a desired amino acid by any term or technique, in which are included but not limited to chemical or enzymatic aminoacylation. The aminoacylation can be effected by aminoacyl tRNA syntheses or by other enzymatic molecules, which include but not be limited to ribozyme. The term "ribozyme" is interchangeable with "catalytic RNA". Cech et al. (Cech, 1987 Science, 236: 1532-1539, MacCorkle et al., 1987, Concepts Biochem, 64: 221-226) demonstrated that the presence of RNAs that are stable in nature can act as catalysts (ribozymes). However, although it has been shown that these natural RNA catalysts only act on ribonucleic acid substrates for splicing and splicing, the recent development of artificial evolution of ribozymes has expanded the catalysis repertoire to various chemical reactions. Studies have identified RNA molecules that catalyze the aminoacyl-RNA bonds in their own (2 ') 3' terminology (Illangakekare et al., 1995 Science 267: 643-647), and an RNA molecule that can transfer an amino acid from one RNA molecule to another (Lohse et al., 1996 Nature 381: 442-44). U.S. Patent Application Publication 2003/0228593, which is incorporated by reference herein, discloses methods for constructing ribozymes and their use in the tRNA aminoacylation with naturally encoded and non-naturally encoded amino acids. Immobilized substrates of enzymatic molecules that can aminoacylate tRNA, which include but not be limited to ribozymes, can allow the efficient agility purification of aminoacylated products. Examples of suitable substrates include agorose, sepharose and magnetic beads. The production and use of a ribozyme-immobilized substrate form for aminoacylation is described in Chemistry and Biology 2003, 10: 1077-1084 and patent application publication 2003/0228593, which are incorporated by reference herein. Chemical aminoacylation methods include, but are not limited to, those presented by Hecht and coworkers (Hecht, SM Acc. Chem. Res. 1992, 25, 545; Heckler, TG; Roesser, JR; Xu, C; Chang, P.; Hecht, SM Biochemistry 1988, 27, 7254; Hecht, SM; Alford, BL; Kuroda, Y .; Kitano, SJ Biol. Chem. 1978, 253, 4517) and by Schultz, Chamberlin, Dougherty and others (Cornish, VW; Mendel, D. Schultz, PG Angew, Chem. Int. Ed. Engl. 1995, 34, 621; Robertson, SA; Ellman, JA; Schultz, PGJ Am. Chem. Soc. 1991, 113, 2722; Noren, CJ; Anthony-Cahill, SJ; Griffith, M. C; Schultz, PG Science 1989, 244, 182; Bain, JD; Glabe, CG; Dix, TA; Chamberlin, ARJ Am.

Chem. Soc. 1989, 111, 8013; Bain, J. D. et al. Nature 1992, 356, 537; Gallivan, J. P.; Lester, H. A .; Dougherty, D. A: Chem. Biol. 1997, 4, 740; Turcatti, et al. J. Biol. Chem. 1996, 271, 19991; Nowak, M. W. et al. Science, 1995, 268, 439; Saks, M. E. et al. J. Biol. Chem. 1996, 271, 23169; Hohsaka, T. et al. J. Am. Chem. Soc. 1999, 121, 34), which are incorporated by reference herein, to avoid the use of synthetases in ammoacylation. Such methods or other chemical aminoacylation methods can be used to ammoacrylate tRNA molecules. Methods to generate catalytic RNA may involve, generate separate accumulations of random sequences, perform direct evolution in the accumulations, select the accumulations as desirable aminoacylation activity and select frequencies of those ribozymes that exhibit ammoacylation activity. desired. Ribozymes may comprise portions and / or regions that facilitate acylation activity, such as a portion of GGU and a U-rich reqion. For example, it has been reported that U-rich regions may facilitate recognition of an amino acid substrate. , and a GGU portion can form base pairs with the terms 3 'of a tRNA. In combination, the GGU and U-rich region facilitate the simultaneous recognition of both the amino acid and tRNA simultaneously, and by this they facilitate the ammoacylation of the 3'-tRNA term.

Ribozymes can be generated by in vitro selection using a conjugated r24mini partially randomized with tRNAAsnCCCG, followed by systematic designs of a consensus sequence found in the active clones. An exemplary ribozyme obtained by this method is termed "riboxime Fx3" and is described in U.S. Patent Application Publication No. 2003/0228593, the content of which is incorporated in the patent by reference, acts as a versatile catalyst for the synthesis of several aminoacyls tRNA loaded with unnatural amino acids cognates. Immobilization on a substrate can be used to enable purification by sufficient affinity of aminoacylated tRNAs. Examples of suitable substrates include but are not filed with agarose, sepharose and magnified beads. Ribozymes can be immobilized on resins by taking advantage of the chemical structure of RNA, such as 3'-cis-diol on RNA ribose can be oxidized with thiodate to produce the corresponding dialdehyde to facilitate the immobilization of RNA on the resin . Various types of resins can be used in which non-expensive hydrazide resins are included where the reductive amino acid makes the interaction between the resin and the ribozyme an irreversible bond. Synthesis of aminoacyl-tRNA can be significantly facilitated by this column aminoacylation technique. Kourouklis et al. Methods 2005; 36: 239-4 describe a column-based aminoacylation system. The isolation of the aminoacylated tRNAs can be effected in a variety of ways. An appropriate method is to elute the aminoacylated tRNAs from a column with a pH regulating solution such as a buffer solution of sodium acetate with 10 nN EDTA, a pH regulating solution containing N- (2-hydroxyethyl) piperazine-N '- (3-propanesulfonic acid) 50 nN, 12.5 nN KCl, pH 7.0, 10 nN EDNA, or simply EDTA with pH-regulated water (pH 7.0). The aminoacylated tRNAs can be added to translation reductions in order to incorporate the amino acid with which the tRNA was aminoacylated at a position of choice into a polypeptide manufactured by the translation reaction. Examples of translation systems in which the aminoacylated tRNAs of the present invention can be used, include but are not limited to cellular ligands. The cell ligands provide necessary reaction components for the in vitro translation of a polypeptide from an input mRNA. Examples of such reaction components include but are not limited to ribosomal proteins, rRNA, amino acids, tRNA, GTP, ATP, translation start and delivery factors, and additional factors associated with translation. Additionally, the translation systems can be batch translations or translation by compartments. The Batch translation systems combine reaction components in a single compartment, while translation systems per compartment separate the reaction reaction components from the reaction products that can inhibit translation efficiency. Such translation systems are commercially available. In addition, a coupled transcription / translation system can be used. The coupled transcription / translation systems allow both the transcription of an input DNA to a corresponding mRNA, which in turn is translated by the reaction components. An example of a commercially available coupled transcription / translation system is the Rapid Translation System (RTS, Roche, Inc.). The system includes a mixture containing an E. coli lysate to provide translation components such as ribosomes and translation factors. Additionally, an RNA polymerase is included for the transcription of input DNA to a mRNA template for use in translation. The RTS may use division by compartments of the reaction components by means of a membrane interposed between the reaction components, in which a supply / waste compartment and a transcription / translation compartment are included. The amino acylation of tRNA can be effected by other agents, which include but are not limited to transferases, polymerases, catalytic antibodies, multifunctional proteins and the like. Stephan in Scientist 2005 Octubre10; pages 30-33 describe additional methods for incorporating non-naturally encoded amino acids into proteins. Lu et al. In Mol Cell. 2001 Oct; 8 (4): 759-89 describes a method in which a protein is chemically linked to a synthetic peptide containing unnatural amino acids (expressed protein ligation).

X. Modifications post-translation of non-natural amino acid components of a polypeptide Methods, compositions, techniques and strategies for specifically incorporating non-natural amino acids into the site during the in vivo translation of proteins have been developed. By incorporating a non-natural amino acid with a side chain chemistry that is orthogonal to that of the amino acids that occur stably in nature, this technology makes possible site-specific derivation of recombinant proteins. As a result, a major advantage of the methods, compositions, techniques and strategies described herein is that derived proteins can now be prepared as defined homogeneous products. However, the methods, compositions, The reaction mixtures, techniques and strategies described herein are not limited to unnatural amino acid polypeptides formed by live protein m translation techniques, in which it includes unnatural amino acid polypeptides formed by any technique, which include example only techniques m Vi tro, expressed protein ligation, chemical synthesis, techniques based on ribosazymes, (see for example, section in the present entitled "Expression in Alternative Systems"). The ability to incorporate non-natural amino acids into recombinant proteins greatly expands the chemistries that can be implemented for post-translational derivation, where such derivation occurs either m vi or m Vi tro. More specifically, the derivatization of proteins using the reductive reaction or reductive tuning reactions between a carbonyl compound, such as, by way of example, aldehydes and an aromatic amine to form an alkylated amine, including a secondary amine or a tertiary amine, which linkage to a non-natural amino acid portion of a polypeptide offers several advantages. First, amino acids that occur stably in nature (a) do not contain aromatic amine groups that can react with carbonyl groups to form alkylated aromatic amines, thereby creating secondary amine or tertiary amine bonds and (b) aromatic amine groups can reacting with carbonyl-containing groups to form alkylated amines, in which secondary amines or tertiary amines are included and thus reagents designed to form such alkylated amines will react specifically at the site 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 alkylated amines and the corresponding amine linkage). Such a site-specific derivation is illustrated in Figures 19-34, wherein the side chain containing an aromatic amine is preferably seduced alkylated on side chains containing protonated amines or portions of imidazole. Second, such alkylated aromatic amines have amine linkages that are stable under physiological conditions, suggesting that proteins derived by such linkages are valid candidates for therapeutic applications. Third, the stability of such an amine linkage can be manipulated based on the identity (i.e., functional groups and / or structures) of the non-natural amino acid to which the link has been formed. In some embodiments, the alkylated amine, which includes a secondary amine or a tertiary amine, linkage to the non-natural amino acid polypeptide has a half-life of decomposition of less than one hour, in other modalities less than one day, in other embodiments less than two days, in other modalities less than a week and in other modalities more than a week. In still other embodiments, the resulting alkylated amine includes a secondary amine or a tertiary amine, is stable for at least four weeks under biological conditions. In still other embodiments, the resulting alkylated amine that includes a secondary amine or a tertiary amine is stable for at least three weeks under biological conditions. In still other embodiments, the resulting alkylated amine, which includes a secondary amine or a tertiary amine, is stable for at least two weeks under biological conditions. In still other embodiments, the resulting alkylated amine, which includes a secondary amine or a tertiary amine, is stable for at least one week under biological conditions. In other embodiments, the resulting alkylated amine includes a secondary amine or a tertiary amine, the bond is stable for at least six days under biological conditions. In other embodiments, the resulting alkylated amine, which includes a secondary amine or a tertiary amine, the bond is stable for at least five days under biological conditions. In other embodiments, the resulting alkylated amine, which includes a secondary amine or a tertiary amine, the bond is stable for at least four days under biological conditions. In other embodiments, the resulting alkylated amine, which includes a secondary amine or a tertiary amine, is stable for at least three days under biological conditions. In other embodiments, the amine alkylated, which includes a secondary amine or a tertiary amine the bond is stable for at least two days under biological conditions. In other embodiments, the resulting alkylated amine, which includes a secondary amine or a tertiary amine, is stable for at least one day under biological conditions. In other embodiments, the resulting alkylated amine, which includes a secondary amine or a tertiary amine, is stable for up to 24 hours under biological conditions. In other embodiments, the resulting alkylated amine, which includes a secondary amine or a tertiary amine, is stable for up to 12 hours under biological conditions. In other embodiments, the resulting alkylated amine, which includes a secondary amine or a tertiary amine, is stable for up to 6 hours under biological conditions. In other embodiments, the resulting alkylated amine, which includes a secondary amine or a tertiary amine, is stable for up to 3 hours under biological conditions. In other embodiments, the resulting alkylated amine, which includes a secondary amine or a tertiary amine, is stable for up to 2 hours under biological conditions. In other embodiments, the resulting alkylated amine, which includes a secondary amine or a tertiary amine, is stable for up to one hour under biological conditions. In still other embodiments, the resulting alkylated amine, which includes a secondary amine or a tertiary amine the bond is stable for at least two weeks under moderately acidic conditions; in other embodiments, the resulting alkylated amine, which includes a secondary amine or a tertiary amine, the bond is stable for at least five days under moderately acidic conditions. In other embodiments, the unnatural amino acid polypeptide is stable for at least one day at a pH between about 2 to about 8; in other embodiments, at a pH of from about 2 to about 6; in another embodiment, at a pH of about 2 to about 4. In other embodiments, the unnatural amino acid polypeptide is stable for at least one day at a pH between about 6 and about 10; in other embodiments, from a pH of from about 4 to about 8; in another embodiment, at a pH of about 4 to about 10. In other embodiments, the unnatural amino acid polypeptide is stable for at least one day at a pH of about 2 and about 15; in other embodiments, from a pH of from about 3 to about 7; in another embodiment, at a pH of from about 3 to about 10. In other embodiments, using the strategies, methods, compositions and techniques described herein, the synthesis of an alkylated amine, including secondary amine or tertiary amine bonds to a non-natural amino acid polypeptide can vary to alter the average amine composition at required needs (for example, for a therapeutic use such as sustained release or diagnostic use, commercial products or industrial use or military use). 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 (in which they include but are not limited to antibodies and fragments of antibodies) and in those that include but are not limited to, the study of protein structure and function > . See for example, Dougherty, (2000) Unnatura l 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, uses based on analysis, cosmetics, plant biology, environmental, energy production and / or military uses. However, the non-natural amino acid polypeptides described above may undergo additional modifications to incorporate new functionalities or modified functionalities, in which they include manipulating the therapeutic effectiveness of the polypeptide, improving the safety profile of the polypeptide, adjusting the pharmacokinetics, pharmacology and / or or pharmacodynamics of the polypeptide (eg, increase water solubility, bioavailability, increase serum half-life, increase life therapeutic means, modulate the immunopenicity, modulate the biological activity or prolong the circulation time), provide additional functionality to the polypeptide, incorporate a label, marker or detectable signal to the polypeptide, facilitate the isolation properties of the polypeptide and any combination of the aforementioned modifications previously . The methods, compositions, strategies and techniques described herein are not limited to a particular type, class or family of polypeptides or proteins. 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, apolipoprotein, apoprotein, atpal natriyrético factor, atrial natriuretic polypeptide, atrial peptide, chemokine CXC, T39765, NAP-2, ENA-78, gro-a, gro-b, gro-c, IP-10, GCP-2, NAP-4, SDF-1, PF4, M1G, calciton, ligand c kit, cytosma, chemokine CC, protein-1 alpha chemoattractant monocyte, protein-2 alpha chemoattractant monocyte, protein-3 alpha chemoattractant monocyte, protein-1 alpha-inflammatory rhonocyte, protein-1 beta-inflammatory monocyte, RANTES, 1309 , R83915, R91733, HCC1, T58847, D31065, T64262, CD40, CD40 ligand, c-kit ligand, collagen, colony stimulating factor (CSF), complement factor 5a inhibitor, complement receptor 1, cytosm, peptide -78 epithelial neutrophil activator, MIP-16, MCP-1, epithelial growth factor (EGF), epithelial neutrophil activating peptide, erythropoietin (EPO), exfoliating toxin, Factor IX, Factor VII, Factor VIII, Factor X, growth factor of fibroblast (FGF), fibrinogen, fibronectin, four-helix 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 (hCH), human serum albumin, ICAM-1, ICAM-1 receptor, LFA-1, LFA-1 receptor, insulin, growth factor insulin-like (IGF), IGF-1, IGF-II, interferon (IFN), IFN-alpha, IFN-beta, IFN-gamma, any interferon-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, factor Keratinocyte growth (KGF) , lactoferrin, leukemia inhibitory factor, luciferase, neurturin, neutrophil inhibiting factor (NIF), oncostatin M, osteogenic protein, oncogene product, paracytonine, parathyroid hormone, PD-ECSF, PDGF, peptide hormone, pleiotropin, protein A; G protein, pth, pyrogenic exotoxin A, pyrogenic exotoxin B, pyrogenic exotoxin C, pyy, relaxin, renin, SCF, small biosynthetic protein, soluble complement receptor I, soluble ICAM-1, soluble interleukin receptor, soluble TNF receptor, somatomedin, somatostatin, somatoin, streptokinase, superantigens, staphylococcal enterotoxin, SEA, SEB, SEC1, SEC2, SEC3, SED, SEE, steroid hormone receptor, superoxide dismutase, toxin of toxic shock syndrome, limosine alpha 1, plasminogen activator of tissue, tumor growth factor (TGF), tumor necrosis factor, tumor necrosis factor alpha, tumor necrosis factor beta, tumor necrosis factor receptor (TNRF), VLA-4 protein, VCAM protein- 1, vascular endothelial growth factor (VEGF), urokinase, mos, ras, raf, met, p53, tat, fos, myc, jun, myb, rei, esen receptor, progesterone receptor, testosterone receptor, aldosterone receptor , LDL receptor and corticosterone. The non-natural amino acid polypeptide can be homologous to any polypeptide member of the growth hormone gene superfamily. Such modifications include the incorporation of additional functionality wherein the non-natural amino acid component of the polypeptide in which they are included but not limited to a label; a dye; a polymer; a water soluble polymer; a polyethylene glycol derivative; a photocrosslinker; a cytotoxic compound; a drug; an affinity marker; a photoaffinity marker; a reactive compound; a resin; a second protein or polypeptide or polypeptide analogue; an antibody or antibody fragment; a metal chelator; a co-factor; a fatty acid; a carbohydrate; a polynucleotide; a DNA; an RNA; an antisense polynucleotide; a saccharide, a water-soluble dendrimer, a cyclodextrin, a biomaterial; a nanoparticle; a spin marker; a fluorophore, a portion containing metal; a radioactive portion; a new functional group; a group that interacts covalently or non-covalently with other molecules; a photo-charged portion; an excitable portion by actinic radiation; a ligand; a photoisomerizable portion; biotin; a biotin analogue; a portion that incorporates an atom. heavy; a group chemically cleaved it; a photocleavable group, an elongated side chain; a carbon-bonded sugar; a redox-active agent; an amino thioacid; a toxic portion; an isotopically labeled portion; a biophysical probe; a phosphorescent group; a chemiluminescent group; a dense group of elecs; a magnetic group; an intercalary group; a chromophore; an energy transfer agent; a biologically active agent; a detectable marker; a small molecule; an inhibitory ribonucleic acid; a radionucleotide; a neu capture agent; a biotin derivative; quantum dot (s); a nanotransmitter; a radio transmitter; an abzyme, an activated complex activator, a virus, an adjuvant, an aglycan, an allergen, an angiostatin, an antihormone, an antioxidant, an aptamer, a guide RNA, a saponin, a shuttle vector, a macromolecule, an mimotope, a receptor, a reverse micelle and any combination thereof. Thus, by way of example only, an unnatural amino acid polypeptide containing any of the following amino acids may be further modified using the methods and compositions described herein: is selected from the group consisting of a monocyclic aryl ring, a bicyclic aryl ring, a multicyclic aryl ring, a monocyclic heteroaryl ring, a bicyclic heteroaryl ring and a multicyclic heteroaryl ring; A is independently CRa or N; B is independently CRa, N, 0 or S; each Ra is independently selected from the group consisting of H, halogen, alkyl, - (NR ') 2, -C (0) R', C (0) N (R ') 2, -OR and -S (0) 2R', wherein k is 1, 2 or 3 and n is 0, 1, 2, 3, 4, 5, or 6; Ri is H, an amino protecting group, resin, amino acid, polypeptide or polynucleotide and R2 is OH, an ester, resin, amino acid, polypeptide or polynucleotide protecting group; each R3 and R4 is independently H, halogen, lower alkyl or substituted lower alkyl or R3 and R4 or two R3 groups optionally form a cycloalkyl or heterocycloalkyl; M is H or -CH2R5; or the portion M-N-C (R5) can form a ring structure of 4 to 7 members; R5 is alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, substituted alkoxy, alkylalkoxy, substituted alkylalkoxy, polyalkylene oxide, substituted polyalkylene oxide, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocycle , substituted heterocycle, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl,, -C (0) R ", -C (0) OR", -C (0) N (R ") 2, -C (O) NHCH ( R ") 2, - (alkylene or alkylene) -N (R") 2 '- (alkenylene or substituted alkenylene) -N (R ") 2, - (alkylene or substituted alkylene) - (aryl or substituted aryl), - (alkenylene or substituted alkenylene) - (aryl or substituted aryl), - (alkylene or substituted alkylene) -ON (R ") 2, - (alkylene or substituted alkylene) -C (O) SR", - (alkylene or substituted alkylene) -SS- (aryl or substituted aryl), wherein each R "is independently hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocycle, heterocycle substituted, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl or C (O) OR '; or R5 is LX, wherein X is selected from the group consisting of a label, a dye, a polymer "; a water soluble polymer; a derivative of polyethylene glycol; a photocrosslinker; a cytotoxic compound; a drug; an affinity marker; a photoaffinity marker; a reactive compound; a resin; a second protein or polypeptide or polypeptide analogue; an antibody or antibody fragment; a metal chelator; a co-factor; a fatty acid; a carbohydrate; a polynucleotide; a DNA; an RNA; an antisense polynucleotide; a saccharide, a water-soluble dendrimer, a cyclodextrin, a biomaterial; a nanoparticle; a spin marker; a fluorophore, a portion containing metal; a radioactive portion; a new functional group; a group that interacts covalently or non-covalently with other molecules; a photoenaged portion; an excitable portion by actinic radiation; a ligand; a photoisomerizable portion; biotin; a biotin analogue; a portion that incorporates a heavy atom; a group chemically cleaved it; a group photocleavable; an elongated side chain; a carbon-bonded sugar; a redox-active agent; an amino thioacid; a toxic portion; an isotopically labeled portion; a biophysical probe; a phosphorescent group; a chemiluminescent group; a dense group of electrons; a magnetic group; an intercalary group; a chromophore; an energy transfer agent; a biologically active agent; a detectable marker; a small molecule; an inhibitory ribonucleic acid; a raclionucleotide; an electron capture agent; a biotin derivative; quantum dot (s); a nanotransmitter; a radio transmitter; an abzyme, an activated complex activator, a virus, an adjuvant, an aglycan, an allergen, an angiostatin, an antihormone, an antioxidant, an aptamer, a guide RNA, a saponin, a shuttle vector, a macromolecule, an mimotope, a receptor, a reverse micelle and any combination thereof and L is optional and when present is a linker selected from the group consisting of alkylene, substituted alkylene, alkenylene, substituted alkenylene, -0-, -0- (alkylene) or substituted alkylene), - (alkylene or substituted alkylene) -0-, -C (0) -, -C (0) - (alkylene or substituted alkylene), (alkylene or substituted alkylene) -C (0) -, - C (0) N (R ') -, -C (0) N (R') - (alkylene or substituted alkylene) -, - (alkylene or substituted alkylene) -0C (0) N (R ') -, - N (R ') C (0) -, -N (R') C (0) - (alkylene or substituted alkylene) -, (alkylene or substituted alkylene) -NR 'C (0) -, -S-, -S- (alkylene or substituted alkylene) -, -S (0) k- wherein k is 1, 2 or 3, -S (0) k- (alkylene or substituted alkylene) -, -C (S) -, -C (S) - (alkylene or substituted alkylene) -, CSN (R ') -, CSN (R') - (alkylene or substituted alkylene) -, -N (R ') CO- (alkylene or substituted alkylene) - , -N (R ') C (0) 0-, - (alkylene or substituted alkylene) -0-N = CR' -, - (alkylene or substituted alkylene) -C (O) NR '- (alkylene or substituted alkylene) ) -, (alkylene or substituted alkylene) -S (0) y- (alkylene or substituted alkylene) -S-, - (alkylene or substituted alkylene) -SS-, S (0) kN (R ') -, N ( R ') C (0) N (R') -, - N (R ') C (S) N (R') -, - N (R ') S (0) kN (R') -, -M (R ') - N =, -C (R') = N-, -C (R ') = NN (R') -, -C (R ') = NN =, -C (R') 2- N = N and -C (R ') 2-N (R') -N (R ') -; or R5 and any Ra optionally form a cycloalkyl or a heterocycloalkyl and each R 'is independently H, alkyl or substituted alkyl. Such non-natural amino acids include but are not limited to, amino acids with the following structures: wherein each A 'is independently selected from < "K" N XI or - M And more than two A1 can be { - ÑH with the condition that A1 is selected from ("k ,, or \ In addition, such non-natural amino acids include but are not limited to amino acids with the following structures: wherein G is an amine protecting group, in which, but not limited to, A ° r ° -cc'3 A oA? °° J NO. where: is selected from the group consisting of a monocyclic aryl ring, a bicyclic aryl ring, a multicyclic aryl ring, a monocyclic heteroaryl ring, a bicyclic heteroaryl ring and a multicyclic heteroaryl ring; A is independently CRa or N; B is independently CRa, N, O or S; each Ra is independently selected from the group consisting of H, halogen, alkyl, -N02, -CN, substituted alkyl, -N (R ') 2, -C (0) kR', -C (0) N (R ' ) 2, -OR 'and -S (0) kR', wherein k is 1, 2 or 3 and n is 0, 1, 2, 3, 4, 5, or 6; Ri is H, an amino protecting group, resin, amino acid, polypeptide or polynucleotide and R2 is OH, an ester, resin, amino acid, polypeptide or polynucleotide protecting group; each R3 and R4 is independently H, halogen, lower alkyl or substituted lower alkyl or R3 and R4 or two R3 groups optionally form a cycloalkyl or a heterocycloalkyl; Y is -NH-NH2, -NH-NHR ', -CR' = NR ', -N02 or -N3 and each R' is independently H, alkyl or substituted alkyl. In addition, such non-natural amino acids include but are not limited to the following amino acids with the following structures: where: is selected from the group consisting of a monocyclic aryl ring, a bicyclic aryl ring, a multicyclic aryl ring, a monocyclic heteroaryl ring, a bicyclic heteroaryl ring and a multicyclic heteroaryl ring; A is independently CRa or N; B is independently CRa, N, O or S; each Ra cs independently selected from the group consisting of H, halogen, alkyl, -N02, -CN, substituted alkyl, -N (R ') 2, -C (0) kR', -C (0) N (R ' ) 2, -OR 'and -S (0) kR', wherein k is l, 2 or 3 and n is 0, 1, 2, 3, 4, 5, or 6; Ri is H, an amino protecting group, resin, amino acid, polypeptide or polynucleotide and R2 is OH, an ester, resin, amino acid, polypeptide or polynucleotide protecting group; each R3 and R4 is independently H, halogen, lower alkyl or substituted lower alkyl or R3 and R4 or two R3 groups optionally form a cycloalkyl or a heterocycloalkyl; M is H or -CH2R5; or the portion M-N-C (Rs) can form a ring structure of 4 to 7 members; R5 is alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy substituted, alkylalkoxy, substituted alkylalkoxy, polyalkylene oxide, substituted polyalkylene oxide, cycloalkyl, substituted cycloalkyl, aplo, substituted substituted, heteroaryl, substituted heteroaryl, heterocycle, substituted heterocycle, alkalip, substituted alkaryl, aralkyl, substituted aralkyl,, -C (0) R ", -C (0) OR", -C (0) N (R) 2, -C (0) NHCH (R ") 2, - (alkylene or alkylene) -N (R") 2 »- (alkenylene or substituted alkenylene) - N (R") 2, - (alkylene or substituted alkylene) - (substituted aryl or aryl), - (substituted alkenylene or alkenylene) - (substituted or substituted aryl), - substituted alkylene or alkylene) -ON (R ") 2, - (alkylene or substituted alkylene) -C (0) SR", - (alkylene or substituted alkylene) -SS- (substituted aryl or aryl), wherein each R "is independently hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, alkoxy Substituted, aplo, substituted substituted, heteroaryl, substituted heteroaryl, heterocycle, substituted heterocycle, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl or C (0) 0R '; or R5 is L-X, where X is selected from the group that consists of a marker; a dye; a polymer ", a water-soluble polymer, a polyethylene glycol derivative, a photocrosslinker, a cytotoxic compound, a drug, an affinity tag, a photo-affinity tag, a reactive compound, a resin, a second protein or polypeptide, polypeptide analogue; an antibody or fragment of antibodies; a metal chelator; a co-factor; a fatty acid; a carbohydrate; a polynucleotide; a DNA; an RNA; an antisense polynucleotide; a saccharide, a water-soluble dendrimer, a cyclodextrin, a biomaterial; a nanoparticle; a spin marker; a fluorophore, a portion containing metal; a radioactive portion; a new functional group; a group that interacts covalently or non-covalently with other molecules; a photoenaged portion; an excitable portion by actinic radiation; a ligand; a photoisomerizable portion; bic? a biotin analogue; a portion that incorporates a heavy atom; a group chemically cleaved it; a photocleavable group; an elongated side chain; a carbon-bonded sugar; a redox-active agent; an amino thioacid; a toxic portion; an isotopically labeled portion; a biophysical probe; a phosphorescent group; a chemiluminescent group; a dense group of electrons; a magnetic group; an intercalary group; a chromophore; an energy transfer agent; a biologically active agent; a detectable marker; a small molecule; an inhibitory ribonucleic acid; a radionucleotide; an electron capture agent; a biotin derivative; quantum dot (s); a nanotransmitter; a radio transmitter; an abzyme, an activated complex activator, a virus, an adjuvant, an aglycan, an allergan, an angiostatin, an antihormone, an antioxidant, an aptamer, a guide RNA, a saponin, a vector of shuttle, a macromolecule, a mimotope, a receptor, a reverse micelle and any combination thereof and L is optional and when present is a linker selected from the group consisting of alkylene, substituted alkylene, alkenylene, substituted alkenylene, -0- , -0- (alkylene or substituted alkylene), - (alkylene or substituted alkylene) -0-, -C (0) -, -C (0) - (alkylene or substituted alkylene), (alkylene or substituted alkylene) -C (0) -, -C (0) N (R ') -, -C (0) N (R') - (alkylene or substituted alkylene) -, - (alkylene or substituted alkylene) -0C (0'N ( R ') -, -N (R') C (0) -, -N (D ') C (0) - (alkylene or substituted alkylene) -, (alkylene or substituted alkylene) -NR' C (0) - , -S-, -S- (alkylene or substituted alkylene) -, -S (0) k- wherein k is 1, 2 or 3, -S (0) - (alkylene or substituted alkylene) -, -C ( S) -, -C (S) - (alkylene or substituted alkylene) -, CSN (R ') -, CSN (R') - (alkylene or substituted alkylene) -, -N (R ') C0- (alkylene or substituted alkylene) -, -N (R ') C (0) 0-, - (a alkylene or substituted alkylene) -0-N = CR '-, - (alkylene or substituted alkylene) -C (0) NR' - (alkylene or substituted alkylene) -, (alkylene or substituted alkylene) -S (0) - ( alkylene or substituted alkylene) -S-, - (alkylene or substituted alkylene) -SS-, S (0) kN (R ') -, N (R ') C (0) N (R') -, - N (R ') C (S) N (R') -, - N (R ') S (0) kN (R') -, -N (R ') - N =, -C (R') = N-, -C (R ') = NN (R') -, -C (R ') = NN =, -C (R') 2-N = N and -C (R ') 2-N (R') -N (R ') -; or R5 and any Ra optionally form a cycloalkyl or a heterocycloalkyl and each R 'is independently H, alkyl or substituted alkyl. Such non-natural amino acids include, but are not limited to the following structures: H, (I ....... or (? Viien where, each A1 is independently selected from C'R "N. with the remaining A1 selected from Ra or s; wherein: L 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 lower heteroalkylene, alkarylene, substituted alkarylene, aralkylene or substituted aralkylene; Q 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, -0- (alkylene or substituted alkylene), -S- (alkylene or substituted alkylene), wherein k is 1, 2 0 3, -S (0) k (alkylene or substituted alkylene), -C (0) - (alkylene or substituted alkylene), -C (S) - (alkylene or substituted alkylene), -NR '- (alkylene or substituted alkylene) -, -CON (R ") - (substituted alkylene or alkylene) ) -, -CSN (R ') - (alkylene or substituted alkylene) -, -N (R') CO- (alkylene or substituted alkylene) - and wherein each R 'is independently H, alkyl or substituted alkyl; Ri is H, an amino protecting group, resin, amino acid, polypeptide or polynucleotide and R2 is OH, an ester, resin, amino acid, polypeptide or polynucleotide protecting group; each R3 and R4 is independently H, halogen, lower alkyl or substituted lower alkyl or R3 and R or two R3 groups optionally form a cycloalkyl or heterocycloalkyl; each Ra is independently selected from the group consisting of H, halogen, alkyl, -N02, -CN, substituted alkyl, -N (R ') 2, -C (0) k (R') 2, -C (0) N (R ') 2, -OR and -S (0) 2R', wherein k is 1, 2 or 3; Rβ is a protected aldehyde or a masked aldehyde, wherein the protective group includes but is not limited to, ) < - > A * 3. wherein each X is independently selected from the group consisting of -o, -S-, -N (i¡) -, -N (R.}. -, -N (?) - and -N (OML ') .: X, is OK, - < J? C. -SR, -N () :. -N (R) (Ac), -N (R;? (ONI! :) 0 N. and wherein each R 'and R is independently H, alkyl or substituted alkyl; (ID wherein: L 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 lower heteroalkylene, alkarylene, substituted alkarylene, aralkylene or substituted aralkylene; Q 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, -0- -0- (alkylene or substituted alkylene) -, -S-, -S- (alkylene or substituted alkylene) -, -S (0) k-, -S (0) k- (alkylene or substituted alkylene) -, wherein k is 1, 2 or 3, -S (0) k (alkylene or substituted alkylene) -, -C (0) -, -C (0) - (alkylene or substituted alkylene) ) -, -C (S) -, -C (S) - (alkylene or substituted alkylene) -, -N (R ') -, -N (R') - (alkylene or substituted alkylene) -, C (0) ) N (R ') -, -C (0) n (R') - (alkylene or substituted alkylene) -, -CSN (R ") -, -CSN (R'J - (alkylene or substituted alkylene) -, N (R ') C0-, -N (R') C0- (aluuylene or substituted alkylene) -, N (R ') C (0) 0-, -S (0) kN (R') -, -N (R ') C (0) N (R') -, -N (R ') C (S) ) N (R ') -, -N (R'J S (0) kN (R') -, -N (R ') - N =, -C (R ") = N-, -C (R') ) = NN (R ') -, C (R') = NN =, -C (R ') 2 = NN = and -C (R') 2-N (R ') -N (R') -, wherein each R 'is independently H, alkyl or substituted alkyl, Ri is H, an amino protecting group, resin, amino acid, polypeptide or polynucleotide and R2 is OH, an ester, resin, amino acid, polypeptide or polynucleotide protecting group; and R4 is independently H, halogen, lower alkyl or substituted lower alkyl or R3 and R4 or two R3 groups optionally form a cycloalkyl or heterocycloalkyl; each R a is independently selected from the group consisting of H, halogen, alkyl, -N02, -CN , substituted alkyl, -N (R ') 2, -C (0) kR', -C (0) N (R ') 2-0R' and -S (0) kR ', in where k is l, 2 or 3; n is 0, 1, 2, 3, 4, 5 or 6; M is H or -CH2R5; Rs is alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, substituted alkoxy, alkylalkoxy, substituted alkylalkoxy, polyalkylene oxide, substituted polyalkylene oxide, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocycle. , substituted heterocycle, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl,, -C (0) R ", -C (0) OR", -C (0) N (R ") 2, -C (0) HCH ( R ") 2, - (alkylene or alkylene) -N (R") 2 '- (alkenylene or substituted alkenylene) -N (R ") 2, - (alkylene or substituted alkylene) - (aryl or substituted aryl), - (alkenylene or substituted alkenylene) - (aryl or substituted aryl), - (alkylene or substituted alkylene) -ON (R'J 2, - (alkylene or substituted alkylene) -C (0) SR ", - (alkylene or substituted alkylene) ) -SS- (aryl or substituted aryl), wherein each R "is independently hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, alkoxy sust ituido, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocycle, substituted heterocycle, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl or C (0) 0R '; or R5 is L-X, wherein X is selected from the group consisting of a label; a dye; a "polymer"; a water soluble polymer; a polyethylene glycol derivative; a photoreticulator; a cytotoxic compound; a drug; an affinity marker; a photoaffinity marker; a reactive compound; a resin; a second protein or polypeptide or polypeptide analogue; an antibody or antibody fragment; a metal chelator; a co-factor; a fatty acid; a carbohydrate; a polynucleotide; a DNA; an RNA; an antisense polynucleotide; a saccharide, a water-soluble dendrimer, a cyclodextrin, a biomaterial; a nanoparticle; a spin marker; a fluorophore, a portion containing metal; a radioactive portion; a new functional group; vn group that interacts covalently or non-covalently with other molecules; a photoenaged portion; an excitable portion by actinic radiation; a ligand; a photoisomerizable portion; biotin; a biotin analogue; a portion that incorporates a heavy atom; a chemically cleavable group; a photocleavable group; an elongated side chain; a carbon-bonded sugar; a redox-active agent; an amino thioacid; a toxic portion; an isotopically labeled portion; a biophysical probe; a phosphorescent group; a chemiluminescent group; a dense group of electrons; a magnetic group; an intercalary group; a chromophore; an energy transfer agent; a biologically active agent; a detectable marker; a small molecule; an inhibitory ribonucleic acid; a radionucleotide; an electron capture agent; a biotin derivative; quantum dot (s); a nanotransmitter; a radio transmitter; an abzyme, an activated complex activator, a virus, an adjuvant, an aglycan, an allergan, an angiostatma, an antihormone, an antioxidant, an aptamer, a guide RNA, a saponin, a shuttle vector, a macromolecule, an mimotope, a receptor, a reverse micelle and any combination thereof and L is optional and when present is a linker selected from the group consisting of alkylene, substituted alkylene, alkenylene, substituted alkenylene, -0-, -0- (alkylene) or substituted alkylene), - (alkylene or substituted alkylene) -0-, -C (0) -, -C (0) - (alkylene or substituted alkylene), (alkylene or substituted alkylene) -C (0) -, - C (0) N (R ') -, -C (0) N (R') - (alkylene or substituted alkylene) -, - (alkylene or substituted alkylene) -0C (0) N (R ') -, - N (R ') C (0) -, -N (R') C (0) - (alkylene or substituted alkylene) -, (alkylene or substituted alkylene) -NR 'C (0) -, -S-, - S- (alkylene or substituted alkylene) -, -S (0) k- where k is 1, 2 or 3, -S (0) k- (alkylene) or substituted alkylene) -, -C (S) -, -C (S) - (alkylene or substituted alkylene) -, CSN (R ') -, CSN (R') - (alkylene or substituted alkylene) -, -N (R ') C0- (alkylene or substituted alkylene) -, -N (R') C (0) 0-, - (alkylene or substituted alkylene) -0-N = CR '-, - (alkylene or substituted alkylene) -C (0) NR '- (alkylene or substituted alkylene) -, (alkylene or substituted alkylene) -S (0) k- (alkylene or substituted alkylene) -S-, - (alkylene or substituted alkylene) -SS-, S (0) kN (R ') -, N (R') C (0) N (R ') -, - N (R') C (S) N (R ') -, - N (R') S (0) kN (R ') -, -N (R') - N =, -C (R ') = N-, -C (R') = NN (R ') -, -C (R') = NN =, -C (R ') 2 -N = N and - C (R ') 2-N (R') -N (R ') -; or R5 and any Ra optionally form a cycloaikyl or a heterocycloalkyl and each R 'is independently H, alkyl or substituted alkyl; is selected from the group consisting of a monocyclic aryl ring, a cyclic aryl ring, a multicyclic aryl ring, a hecarcaryl ring mcno < A cycloalkyl, a bicyclic heteroaryl ring and a multicyclic heteroaryl ring; A is independently CRa or N and B is independently CRa, N, O or S. In one aspect of the methods and compositions described herein are compositions that include at least one polypeptide with at least one, which include 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 modified post-translation. The post-translational modified non-natural amino acids may be the same or different, including, but not limited to, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 , 13, 14, 15, 16, 17, 18, 19, 20 or more different sites in the protein comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more non-natural amino acids modified post-translation. In another aspect, a composition includes a polypeptide with at least one, but less than the total of a particular amino acid present in the polypeptide is substituted with the post-translational modified non-natural amino acid. For a given polypeptide with more than one post-translationally modified non-natural amino acid, the unnatural amino acids modified post-translational aimepte may be identical or different (in which, but not limited to, to the polypeptide which may include two or more different types) of non-natural amino acids modified post-translation, or it may include two unmodified modified non-natural amino acids post-translation). For a given polypeptide with more than two post-translational modified non-natural amino acids, the non-natural post-translational modified amino acids may be the same, different or a multiple combination of non-natural modified post-translational amino acids from the same group with at least one non-natural amino acid. natural modified post-translation.

A. Methods for post-translationally modifying unnatural amino acid polypeptides using a single step of post-translational modification: Reducing alkylation reactions of unnatural amino acids containing aromatic amine with carbonyl-containing reagents The side chains of the amino acids occurring from Stable manner in nature lack highly nucleophilic sites. Accordingly, the incorporation of a non-natural amino acid with a side chain containing nucleophil: co, which includes, by way of example only, an amino acid containing an aromatic amine group or a substituted aromatic amine group, makes possible the site-specific alkylation of this side chain via nucleophilic addition to a carbonyl-containing reagent, in which an aldehyde-containing reagent is included, followed by a reduction reaction. This reductive alkylation reaction generates an amine bond, which includes secondary and tertiary amines. The methods for derivatizing and / or further modifying can be carried out with naturally-synthesized polypeptides or chemically synthesized polypeptides that have been purified prior to the reductive alkylation step or after the reductive alkylation step. In addition, the methods for further derivatizing and / or purifying can be carried out with synthetic polymers, polysaccharides or polynucleotides that have been purified before or after such modifications. The post-translational modification of polypeptides based on the reductive alkylation of an aromatic amine-containing polypeptide with an aldehyde-containing reagent has distinct advantages. First, the aromatic amines can be seduced alkylated with carbonyl-containing compounds, in which aldehydes and ketones are included, in a pH range of about 4 to about 10 (and in additional embodiments, in a pH range of about 4 to about 7) with a reducing agent such as NabCNH to generate secondary or tertiary amine bonds. Other reducing agents that can be used include but are not limited to Na2S, Na2S204, LYH4, B2H6 and NaBH4. Second, under these reaction conditions the chemistry is selective for non-natural amino acids since the side chains of the amino acids that occur stably in nature are not reactive. This allows site-specific derivation of polypeptides that have incorporated non-natural amino acids containing portions of aromatic amine or protected portions of aldehyde, in which recombinant proteins are included by way of example. Such protein-derived polypeptides can thereby be prepared as defined homogeneous products. Third, the gentle conditions necessary to perform a reaction of one a portion of the aromatic amine on an amino acid, that the acid incorporated into a polypeptide, with an aldehyde-containing reagent in general does not irreversibly destroy the tertiary structure of the polypeptide (except, of course, where the purpose of the reaction is to destroy such a tertiary structure ). Similarly, the mild conditions necessary to effect the reaction of an aldehyde moiety on an amino acid, that acid incorporated into a polypeptide and deprotected, with an aromatic amine-containing reagent generally does not irreversibly destroy the tertiary structure of the polypeptide (except, of course, where the purpose of the reaction is to destroy such a tertiary structure). Fourth, the reaction occurs rapidly at room temperature, which allows the use of many types of polypeptides or reagents that would otherwise be unstable at higher temperatures. Fifth, the reaction occurs easily in aqueous conditions, allowing through the use of incompatible polypeptides and reagents (to any extent) with non-aqueous solutions. Sixth, the reaction occurs rapidly even when the ratio of polypeptide or amino acid to reagent is stoichiometric, stoichiometric or quasi stoichiometric, such that it is not necessary to add excess reagent or polypeptide to obtain a useful amount of reaction product . Seventh, the resulting amine can be produced selectively and / or regiospecifically, depending on the design of the amine and carbonyl portions of the reagents. Similarly, reductive alkylation of aromatic amines with aldehyde-containing reagents and reductive tuning of aldehydes with aromatic amine-containing reagents generates amine, in which secondary and tertiary amines are included, bonds that are stable under biological conditions. By way of example only, the following non-natural amino acids can be alkylated seductively with aldehyde-containing reactants described herein, in of the group that consists of a monocyclic aryl ring, a bicyclic aryl ring, a multicyclic aryl ring, a monocyclic heteroaryl ring, a bicyclic heteroaryl ring and a multicyclic heteroaryl ring; A is independently CRa or N; B is independently CRa, N, 0 or S; each Ra is independently selected from the group consisting of H, halogen, alkyl, -N02, -CN, substituted alkyl, -N (R ') 2-, -C (0) kR', -C (0) N (R ') 2, -OR' and -S (0) kR ', in where k is 1, 2 or 3 and n is 0, 1, 2, 3, 4, 5, or 6; Ri is H, an amino protecting group, resin, amino acid, polypeptide or polynucleotide and R2 is OH, an ester, resin, amino acid, polypeptide or polynucleotide protecting group; each of R and R4 is independently H, halogen, lower alkyl, or substituted lower alkyl or R3 and R4 or two R groups optionally form a cycloalkyl or a heterocycloalkyl; M is H or -CH2R5; wherein the portion M-N-C (R5) can form a ring structure of 4 to 7 members; R5 is alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, substituted alkoxy, alkylalkoxy, substituted alkylalkoxy, polyalkylene oxide, substituted polyalkylene oxide, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocycle , substituted heterocycle, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl,, -C (0) R ", -C (0) OR", -C (0) N (R ") 2, -C (0) NHCH ( R ") 2, - (alkylene or alkylene) -N (R") 2 '- (alkenylene or substituted alkenylene) -N (R ") 2, - (alkylene or substituted alkylene) - (aryl or substituted aryl), - (alkenylene or substituted alkenylene) - (aryl or substituted aryl), - (alkylene or substituted alkylene) -ON (R ") 2, - (alkylene or substituted alkylene) -C (O) SR", - (alkylene or substituted alkylene) ) -SS- (aryl or substituted aryl), where each R "is independently hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocycle, substituted heterocycle, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl or C (0) ) OR '; or R5 is LX, wherein X is selected from the group consisting of a label, a dye, a polymer "; a water soluble polymer; a polyethylene glycol derivative; a photocrosslinker; a cytotoxic compound; a drug; an affinity marker; a photoaffinity marker; a reactive compound; a resin; a second protein or polypeptide or polypeptide analogue; an antibody or antibody fragment; a metal chelator; a cc-factor; a fatty acid; a carbohydrate; a polynucleotide; a DNA; an RNA; an antisense polynucleotide; a saccharide, a water-soluble dendrimer, a cyclodextrin, a biomaterial; a nanoparticle; a spin marker; a fluorophore, a portion containing metal; a radioactive portion; a new functional group; a group that interacts covalently or non-covalently with other molecules; a photoenaged portion; an excitable portion by actinic radiation; a ligand; a photoisomerizable portion; biotin; a biotin analogue; a portion that incorporates a heavy atom; a chemically cleavable group; a photocleavable group; an elongated side chain; a carbon sugar linked a redox-active agent; an amino thioacid; a toxic portion; an isotopically labeled portion; a biophysical probe; a phosphorescent group; a chemiluminescent group; a dense group of electrons; a magnetic group; an intercalary group; a chromophore; an energy transfer agent; a biologically active agent; a detectable marker; a small molecule; an inhibitory ribonucleic acid; a radionucleotide; an electron capture agent; a biotin derivative; quantum dot (s); a nanotransmitter; a radio transmitter; an abzyme, an activated complex activator, a virus, an adjuvant, an aglycan, an allergen, an angiostatin, an antihormone, an antioxidant, an aptamer, a guide RNA, a saponin, a shuttle vector, a macromolecule, an mimotope, a receptor, a reverse micelle and any combination thereof and L is optional and when present is a linker selected from the group consisting of alkylene, substituted alkylene, alkenylene, substituted alkenylene, -O-, -O- (alkylene) or substituted alkylene), - (alkylene or substituted alkylene) -0-, -C (0) -, -C (0) - (alkylene or substituted alkylene), (alkylene or substituted alkylene) -C (0) -, - C (0) N (R ') -, -C (0) N (R') - (alkylene or substituted alkylene) -, - (alkylene or substituted alkylene) -0C (0) N (R ') -, - N (R ') C (0) -, -N (R') C (0) - (alkylene or substituted alkylene) -, (alkylene or substituted alkylene) -NR 'C (0) -, -S-, - S- (alkylene or substituted alkylene) -, -S (0) - where k is 1, 2 or 3, -S (O) k- (alkylene or substituted alkylene) -, -C (S) -, -C (S) - (alkylene or substituted alkylene) -, CSN (R ') -, CSN (R ') - (alkylene or substituted alkylene) -, -N (R') CO- (alkylene or substituted alkylene) -, -N (R ') C (O) O-, - (alkylene or substituted alkylene) - 0-N = CR '-, - (alkylene or substituted alkylene) -C (O) NR' - (alkylene or substituted alkylene) -, (alkylene or substituted alkylene) -S (O) - (alkylene or substituted alkylene) - S-, - (alkylene or substituted alkylene) -SS-, S (0) kN (R ') -, N (R') C (0) N (R ') -, - N (R') C (S) ) N (R ') -, -N (R') S (O) kN (R ') -, -N (R') - N =, -C (R ') = N-, -C (R') ) = NN (R ') -, -C (R') = NN =, -C (R'i 2 -N = N and -C (R ') 2- (R') -N (R ') - or R5 and any Ra optionally form a cycloalkyl or a heterocycloalkyl and each R 'is independently H, alkyl or substituted alkyl Such non-natural amino acids include but are not limited to, amino acids with the following structures: M 1 or 1 -Mi and more than two A 'can be. { -Nll with the condition that A 'be selected from < K, o \ In addition, such non-natural amino acids include but are not limited to, amino acids with the following structures: where G is an amine protecting group in which it is included but not limited to wherein (S, ', ..-. ^' 4'- -N-R5? r; is selected from the group consisting of a monocyclic aryl ring, a bicyclic aryl ring, a multicyclic aryl ring, a ring of monocyclic heteroaryl, a bicyclic heteroaryl ring and a multicyclic heteroaryl ring: A is independently CRa or N; B is independently CRa, N, O or S, each Ra is independently selected from the group consisting of H, halogen, alkyl, - NO2, -CN, alkyl substituted, -N (R ') 2, -C (0) kR', -C (0) N (R ') 2, -OR' and -S (0) kR ', where k is l, 2 or 3 and n is 0, 1, 2, 3, 4, 5, or 6; Ri is H, an amino protecting group, resin, amino acid, polypeptide or polynucleotide and R2 is OH, an ester, resin, amino acid, polypeptide or polynucleotide protecting group; each R and R is independently H, halogen, lower alkyl or substituted lower alkyl or R and R4 or two R groups optionally form a cycloalkyl or a heterocycloalkyl; M is H or -CH2Rs; or the portion M-N-C (R5) can form a ring structure of 4 to 7 members; R5 is alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, substituted alkoxy, alkylalkoxy, substituted alkylalkoxy, polyalkylene oxide, substituted polyalkylene oxide, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocycle , substituted heterocycle, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl,, -C (0) R ", -C (0) OR", -C (0) N (R ") 2, -C (0) NHCH ( R'J 2, - (alkylene or alkylene) -N (R ") 2 '- (alkenylene or substituted alkenylene) -N (R") 2, - (alkylene or substituted alkylene) - (aryl or substituted aryl), - (alkenylene or substituted alkenylene) - (aryl or substituted aryl), - (alkylene or substituted alkylene) -O (R ") 2 - (alkylene or substituted alkylene) -C (0) SR", - (alkylene or substituted alkylene) -SS- (aryl or substituted aryl), wherein each R "is independently hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocycle, heterocycle substituted, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl or C (O) OR '; or R5 is LX, wherein X is selected from the group consisting of a label, a dye, a polymer "; a water soluble polymer; a polyethylene glycol derivative; a photocrosslinker; a cytotoxic compound; a drug; an affinity marker; a photoaffinity marker; a reactive compound; a resin; a second protein or polypeptide or polypeptide analogue; an antibody or antibody fragment; a metal chelator; a co-factor; a fatty acid; a carbohydrate; a polynucleotide; a DNA; an RNA; an antisense polynucleotide; a saccharide, a water-soluble dendrimer, a cyclodextrin, a biomaterial; a nanoparticle; a spin marker; a fluorophore, a portion containing metal; a radioactive portion; a new functional group; a group that interacts covalently or non-covalently with other molecules; a photographed portion; an excitable portion by actinic radiation; a ligand; a photoisomerizable portion; biotin; a biotin analogue; a portion that incorporates a heavy atom; a group chemically cleaved it; a group photocleavable; an elongated side chain; a carbon-bonded sugar; a redox-active agent; an amino thioacid; a toxic portion; an isotopically labeled portion; a biophysical probe; a phosphorescent group; a chemiluminescent group; a dense group of electrons; a magnetic group; an intercalary group; a chromophore; an energy transfer agent; a biologically active agent; a detectable marker; a small molecule; an inhibitory ribonucleic acid; a radionucleotide; an electron capture agent; a biotin derivative; quantum dot (s); a nanotransmitter; a radio transmitter; an abzyme, an activated complex activator, a virus, an adjuvant, an aglycan, an allergen, an angiostatin, an antihormone, an antioxidant, an aptamer, a guide RNA, a saponin, a shuttle vector, a macromolecule, an mimotope, a receptor, a reverse micelle and any combination thereof and L is optional and when present is a linker selected from the group consisting of alkylene, substituted alkylene, alkenylene, substituted alkenylene, -0-, -0- (alkylene) or substituted alkylene), - (alkylene or substituted alkylene) -0-, -C (0) -, -C (0) - (alkylene or substituted alkylene), (alkylene or substituted alkylene) -C (0) -, - C (0) N (R ') -, -C (0) N (R') - (alkylene or substituted alkylene) -, - (alkylene or substituted alkylene) -0C (0) N (R ') -, - N (R ') C (0) -, -N (R') C (0) - (alkylene or substituted alkylene) -, (alkylene or substituted alkylene) -NR 'C (0) -, -S-, -S- (alkylene or substituted alkylene) -, -S (0) k- wherein k is 1, 2 or 3, -S (0) k- (alkylene or substituted alkylene) -, -C (S) -, -C (S) - (alkylene or substituted alkylene) -, CSN (R ') -, CSN (R') - (alkylene or substituted alkylene) -, -N (R ') CO- (alkylene or substituted alkylene) - , -N (R ') C (0) 0-, - (alkylene or substituted alkylene) -0-N = CR' -, - (alkylene or substituted alkylene) -C (O) NR '- (alkylene or substituted alkylene) ) -, (alkylene or substituted alkylene) -S (0) k- (alkylene or substituted alkylene) -S-, - (alkylene or substituted alkylene) -SS-, S (0) kN (R ') -, N ( R ') C (0) N (R') -, - R RJ f (S) N (RJ -, - N (R ') S (0) and (P') -, - N (R ') - N =, -C (R ') = N-, -C (R') = NN (R ') -, -C (R') = NN =, -C (R ') 2 -N = N and - C (R ') 2-N (R') -N (R ') - or R5 and any Ra optionally form a cycloalkyl or a heterocycloalkyl and each R' is independently H, alkyl or substituted alkyl. but are not limited to, amino acids with the following structures: where, each 1 is independently selected from C N, or c - ^. ^ X'- and up to two A 'can be - A ^ J *; with the remaining A1 selected from CR, or Ni.

Such reductive alkylation reactions post-translationally modify unnatural amino acid polypeptides containing aromatic amine to non-natural amino acid polypeptides containing unnatural amino acids containing mono-alkylated or di-alkylated aromatic amine. The types of polypeptides comprising such non-natural amino acids containing aromatic amine are practically unlimited insofar as the unnatural amino acid containing aromatic amine are located on the polypeptide, such that the aldehyde-containing reagent can react with an aromatic amine group and not to create a resulting modified non-natural amino acid that destroys the tertiary structure of the polypeptide (except, of course, if such is the purpose of the reaction). By way of example only, the carbonyl containing reagents that are reactive with the non-natural aromatic amine-containing amino acids described herein and which may also be used to optionally modify the non-natural amino acid polypeptides containing aromatic amine include aldehyde-containing compounds with the following structure, where: R? is alkyl, substituted alkyl, alkynyl, substituted alkenyl, alkynyl, substituted alkynyl, substituted alkoxy, alkylalkoxy, substituted alkylalkoxy, polyalkylene oxide, substituted polyalkylene oxide, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocycle, substituted heterocycle, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl, - C (0) R ", -C (0) OR", -C (0) N (R ") 2, -C (0) NHCH (R") 2, - (alkylene or alkylene) -N (R " ) 2 - (alkenylene or substituted alkenylene) -N (R ") 2, - (alkylene or substituted alkylene) - (aryl or substituted aryl), - (substituted alkenylene or alkenylene) - (substituted aryl or aryl), - ( alkylene or substituted alkylene) -ON (R ") 2, - (alkylene or substituted alkylene) -C (O) SR", - (alkylene or substituted alkylene) -SS- (aryl or substituted aryl), wherein each R " is independently hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, alkoxy substituted, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocycle, substituted heterocycle, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl or C (0) OR '; or Rs is L-X, wherein X is selected from the group consisting of a marker; a dye; a polymer ", a water-soluble polymer, a polyethylene glycol derivative, a photocrosslinker, a cytotoxic compound, a drug, an affinity tag, a photoaffinity tag, a reactive compound, a resin, a second protein or polypeptide or analogue polypeptide, an antibody or antibody fragment, a metal chelator, a co-factor, a fatty acid, a carbohydrate, a polynucleotide, a DNA, an RNA, an antisense polynucleotide, a saccharide, a water-soluble dendrimer, a cyclodextrin a biomaterial, a nanoparticle, a spin marker, a fluorophore, a metal-containing portion, a radioactive portion, a new functional group, a group that interacts covalently or non-covalently with other molecules, a photoenaged portion, a portion excitable by actinic radiation, a ligand, a photoisomerizable portion, biotin, a biotin analogue, a portion that incorporates a heavy atom, a chemical cleavable group mind, a photocleavable group; an elongated side chain; a carbon-bonded sugar; a redox-active agent; an amino thioacid; a toxic portion; an isotopically labeled portion; a probe biophysics; a phosphorescent group; a chemiluminescent group; a dense group of electrons; a magnetic group; an intercalary group; a chromophore; an energy transfer agent; a biologically active agent; a detectable marker; a small molecule; an inhibitory ribonucleic acid; a radionucleotide; an electron capture agent; a biotin derivative; quantum dot (s); a nanotransmitter; a radio transmitter; an abzyme, an activated complex activator, a virus, an adjuvant, an aglycan, an allergen, an angiostatin, an antihormone, an antioxidant, an aptamer, a guide RNA, a saponin, a shuttle vector, a macromolecule, an mimotope, a receptor, a reverse micelle and any combination thereof and L is optional and when present is a linker selected from the group consisting of alkylene, substituted alkylene, alkenylene, substituted alkenylene, -O-, -O- (alkylene) or substituted alkylene), - (alkylene or substituted alkylene) -0-, -C (0) -, -C (0) - (alkylene or substituted alkylene), (alkylene or substituted alkylene) -C (0) -, - C (0) N (R ') -, -C (0) N (R') - (alkylene or substituted alkylene) -, - (alkylene or substituted alkylene) -0C (0) N (R ') -, - N (R ') C (0) -, -N (R') C (0) - (alkylene or substituted alkylene) -, (alkylene or substituted alkylene) -NR 'C (0) -, -S-, - S- (alkylene or substituted alkylene) -, -S (0) k- where k is 1, 2 or 3, -S (0) y- (alkyl) ene or substituted alkylene) -, -C (S) -, -C (S) - (alkylene or substituted alkylene) -, CSN (R ') -, CSN (R') - (alkylene or substituted alkylene) -, -N (R ') CO- (alkylene or substituted alkylene) -, -N (R') C (0) 0-, - (alkylene or substituted alkylene) -0-N = CR '-, - (alkylene or substituted alkylene) -C (0) NR' - (alkylene or substituted alkylene) -, (alkylene or substituted alkylene) -S (0) k- (alkylene or substituted alkylene) -S-, - (alkylene or substituted alkylene) -SS-, S (0) kN (R ') -, N (R') C (0) N (R ') -, - N (R') C (S) N (R ') -, -N (R') S (O) kN (R ') -, -N (R') - N =, -C (R ') = N-, -C (R') = NN ( R ') -, -C (R') = NN =, -C (R ') 2-N = N and -C (R') 2-N (R ') -N (R') -; each R 'is independently H, alkyl or substituted alkyl. In addition, by way of example only, the carbonyl-containing reagents that are reactive with the non-natural aromatic amine-containing amino acids described herein and that can be used to further modify non-natural amino acid polypeptides containing aromatic amine are compounds that contain carbonyl in which are included diketones, ketoaldehydes and dialdehydes with the following structures wherein each Ri is independently selected from H, optionally substituted alkyl, optionally substituted alkene, optionally substituted alkyne, optionally substituted cycloalkyl, optionally substituted heterocycle, optionally substituted aryl or optionally substituted heteroaryl; R5 is alkylene, substituted alkylene, alkenylene, substituted alkenylene, alkynylene, substituted alkynylene, polyalkylene oxide, substituted polyalkylene oxide, cycloalkylene, substituted cycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, heterocyclealkylene, substituted heterocyclealkylene, -C (0) ) R ", -C (0) OR", -C (0) N (R'J -, - (alkylene or substituted alkylene) -N (R ") -. - (substituted alkylene-alkylene) -N ( R'J -, (alkylene or substituted alkylene) -ON (R'J-, (alkylene or substituted alkylene) -C (O) SR "-, wherein each R" - is independently hydrogen, alkyl, substituted alkyl, alkenyl , substituted alkenyl, alkoxy, substituted alkoxy, alkylene, substituted alkylene, alkenylene, substituted alkenylene, alkynylene, substituted alkynylene, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocycle, substituted heterocycle, alkaryl, substituted alkaryl, aralkyl or substituted aralkyl; R5 is LX, in do nde X is selected from the group consisting of a marker; a dye; a "polymer"; a water soluble polymer; a polyethylene glycol derivative; a photocrosslinker; a cytotoxic compound; a drug; an affinity marker; a photoaffinity marker; a reactive compound; a resin; a second protein or polypeptide or polypeptide analogue; an antibody or antibody fragment; a metal chelator; a co-factor; a fatty acid; a carbohydrate; a polynucleotide; a DNA; an RNA; an antisense polynucleotide; a saccharide, a water-soluble dendrimer, a cyclodextrin, a biomaterial; a nanoparticle; a spin marker; a fluorophore, a portion containing metal; a radioactive portion; a new functional group; a group that interacts covalently or non-covalently with other molecules; a photoenaged portion; an excitable portion by actinic radiation; a ligand; a photcisomerizable portion; biotin; a biotin analogue; a portion that incorporates a heavy atom; a group chemically cleaved it; a photocleavable group; an elongated side chain; a carbon-bonded sugar; a redox-active agent; an amino thioacid; a toxic portion; an isotopically labeled portion; a biophysical probe; a phosphorescent group; a chemiluminescent group; a dense group of electrons; a magnetic group; an intercalary group; a chromophore; an energy transfer agent; a biologically active agent; a detectable marker; a small molecule; an inhibitory ribonucleic acid; a radionucleotide; an electron capture factor; a biotin derivative; quantum dot (s); a nanotransmitter; a radio transmitter; an abzyme, an activated complex activator, a virus, an adjuvant, an aglycan, an allergana, an angiostatin, an antihormone, an antioxidant, an aptamer, a guide RNA, a saponin, a shuttle vector, a macromolecule, a mimotope, a receptor, a reverse micelle and any combination thereof and L is optional and when present is a linker selected from the group consisting of of alkylene, substituted alkylene, alkenylene, substituted alkenylene, -0-, -0- (alkylene or substituted alkylene), - (alkylene or substituted alkylene) -O-, -C (0) -, -C (0) - ( alkylene or substituted alkylene), (alkylene or substituted alkylene) -C (O) -, -C (0) N (R ') -, -C (0) N (R') - (alkylene or substituted alkylene) -, - (substituted alkylene or alkyl) -0C (0) N (R ') -, -N (R') C (0) -, -N (R ') C (0) - (alkylene or substituted alkylene) - , (alkylene or substituted alkylene) -NR 'C (0) -, -S-, -S- (alkylene or substituted alkylene) -, -S (0) k- where k is 1, 2 or 3, -S (0) k- (alkylene or substituted alkylene) -, -C (S) -, -C (S) - (alkylene or substituted alkylene) -, CSN (R ') -, CSN (R') - (alkylene or substituted alkylene) -, -N (R ') CO - (alkylene or substituted alkylene) -, -N (R ') C (0) 0-, - (alkylene or substituted alkylene) -0-N = CR' -, - (alkylene or substituted alkylene) -C (O) NR '- (alkylene or substituted alkylene) -, (alkylene or substituted alkylene) -S (0) k- (alkylene or substituted alkylene) -S-, - (alkylene or substituted alkylene) -SS-, S (0) kN (R ') -, N (R') C (0) N (R ') -, - N (R') C (S) N (R ') -, - N (R') S (0) kN (R ') -, -N (R') - N =, -C (R ') = N-, -C (R') = NN (R ') -, -C (R') = NN =, -C (R ') 2-N = N and -C (R') 2-N (R ') -N (R') -; each R 'is independently H, alkyl or substituted alkyl; n is 1, 2 or 3.

The types of carbonyl-containing reagents that can react with unnatural amino acids containing aromatic amine are practically unlimited, while the unnatural amino acid containing aromatic amine is located on the polypeptide, such that the carbonyl-containing reagent can react with the aromatic amine group and not creating a resulting modified non-natural amino acid that destroys the tertiary structure of the polypeptide (except, of course, if such destruction is the purpose of the reaction). Such carbonyl-containing reagents include but are not limited to reagents that contain at least a portion of aldehyde and reagents that contain at least two aldehyde portions. Additionally, the reaction between an aldehyde moiety and an aromatic amine moiety is easy and such reductive alkylation reactions have at least one of the following characteristics: (i) it occurs in a pH range of about 4 to about 7, ( ii) generate an amine bond that is stable under biological conditions; (iii) is site specific; (iv) does not irreversibly destroy the tertiary structure of a polypeptide; (v) occurs rapidly at room temperature; (vi) occurs easily in aqueous conditions; (vii) occurs easily when the proportion of the non-natural amino acid comprising the aromatic amine portion to the aldehyde-containing reactant is stoichiometric, stoichiometric or almost stoichiometric and (vm) is regioceptive and / or regiospecific. The orthogonal nature of the reductive alkylation reactions results in regioceptivity and / or regiospecificity, thereby allowing post-site-specific modification of unnatural amino acid polypeptides without affecting other amino acids in the polypeptide containing the amino acid (s). s) unnatural (is). Illustrative embodiments of methods for reductively alkylating an unnatural amino acid containing aromatic amine in a polypeptide are illustrated in Figures 20-34. Certain embodiments include a single reductive alkylation of an unnatural amino acid containing aromatic amine on a polypeptide with a carbonyl-containing reagent, including an aldehyde-containing reagent, which produces a secondary amine moiety, while other embodiments include two reductive alkylations of an unnatural amino acid containing aromatic amine on a polypeptide with a carbonyl-containing reagent, including an aldehyde-containing reagent, which produces an amine that produces a tertiary amine moiety. Additionally, certain embodiments include a single reductive alkylation of an unnatural amino acid containing aromatic amine on a polypeptide with a reagent containing at least two carbonyl groups, producing by this a portion of secondary amine. Still other embodiments include double reductive alkylations of an unnatural amino acid containing aromatic amine on a polypeptide with a reagent containing at least two carbonyl groups thereby producing a tertiary amine moiety. In these illustrative embodiments, an aldehyde-containing reagent is added to a pH regulated solution (pH of about 4 to about 7) of an aromatic amine-containing amino acid polypeptide and a reducing oil such as by way of example only, sodium cyanobide. The reaction proceeds at room temperature and the resulting non-natural amino acid polypeptide containing alkylated aromatic amine can be purified by HPLC, FPLC or size exclusion chromatography. In other embodiments, chemistry of multiple linkers can specifically react at the site with an unnatural amino acid polypeptide containing aromatic amine. In one embodiment, the linker methods described in the patent use linkers that contain the carbonyl functionality, include an aldehyde functionality, over at least one linker term (mono, bi or multifunctional). Reductive alkylation of a polypeptide containing aromatic amine with an aldehyde-derivative linker generates a stable amine bond. Bi- and / or multi-functional linkers, also known as linkers Heterofunctionals (eg, carbonyl, in which an aldehyde is included with one or more other chemical linkers) allow specific site-specific connection of different molecules (eg, other polypeptides, polynucleic acids, polymers or small molecules) to the polypeptide of non-natural amino acid, whereas mono-functional linkers, also known as homofunctional linkers (carbonyl-substituted, in which substituted aldehydes are included, in all terms) facilitate the specific dimer- or oligomerization of the amino acid polypeptide site non-natural aromatic amine. By combining this linker strategy with the in vivo translation technology described herein, it becomes possible to specify the three-dimensional structure of the chemically made proteins.

B. Methods for post-translationally modifying unnatural amino acid polypeptides using two stages of post-translational modification: Formation of unnatural amino acids containing aromatic amine followed by reductive alkylation reactions of unnatural amino acids containing aromatic amine with carbonyl-containing reagents . Amino acids containing aromatic amine can be incorporated translationally into polypeptides before the reductive alkylation with carbonyl-containing reagents, including aldehyde-containing reagents. Alternatively, amino acid precursors containing aromatic amine, such as amino acids containing substituted aromatic moieties can be translationally incorporated into polypeptides and subsequently transformed to amino acids containing aromatic amine prior to reductive alkylation with carbonyl-containing reagents. The latter method involves two post-translation modifications, while the first involves a single post-translation modification. The methods for derivatizing and / or further modifying can be effected with naturally generated polypeptides or chemically synthesized polypeptides that can be purified before or after these modification methods. In addition, the methods for derivatizing and / or modifying further may be carried out on synthetic polymers, polysaccharides or polynucleotides which may be modified before or after these modification methods. By way of example only, the following non-natural amino acids can be reduced to generate amino acids containing aromatic amine, where: is selected from the competing group of a monocyclic aryl ring, a bicyclic aryl ring, a multicicyl aryl ring, a monocyclic ring heroaryl, a bicyclic heteroaryl ring, and a multiciclic heteroaryl ring; A is independently CRa or N; B is independently CRa, N; O, or S; each Ra is independently selected from groups consisting of H, halogen, alkyl, -NO2, -CN, substituted alkyl, -N (R ') 2, -C (0)? PJ, -C (0) N (R') ) 2, -OR ', and -S (0)? R', where k is 1,2, or 3; and n is 0, 1, 2, 3, 4, 5, or 6; Ri is H, an amino protecting group, resin, amino acid, polypeptide or polynucleotide; and R2 is OH, an ester, resin, amino acid, polypeptide or polynucleotide protecting group; each of R3 and R4 is independently H, halogen, lower alkyl or substituted higher alkyl, or R and R4 or two R3 groups optionally form a cycloalkyl or a heterocycloalkyl; Y is -NH-NH2, -NH-NHR ', CR' = NR ', -N02, or -N3, and each R' is independently H, alkyl or substituted alkyl. Such non-natural amino acids include but are not limited to amino acids with the following structure: Illustrative embodiments for reductively generating an unnatural amino acid containing aromatic amine on a natural polypeptide, synthetic polymer, polysaccharide, polynucleotide or chemically synthesized polypeptide are presented in Figures 14 and 15. Certain embodiments include the reduction of imine substituents to unnatural amino acids containing aromatic primary amine, imine substituents to non-natural amino acids containing aromatic secondary amine, reduction of hydraziamiana substituents to non-natural amino acids containing aromatic primary amine, reduction of hydraziamiana substituents to non-natural amino acids containing aromatic secondary amine, reduction of nitro substituents to amino acids natural containing aromatic primary amine and reduction of azido substituents to non-natural amino acids containing aromatic primary amine. In these illustrative embodiments, the substituted aromatic portions are reduced in a pH-regulated solution (pH about 4 to about 7) and the reducing agents used include but are not limited to TCEP, Na2S20, LiAlH4, B2H6, NaBH4 or NaBCNH3. The reaction proceeds at room temperature and the resulting non-natural amino acid polypeptide containing aromatic amine, synthetic polymer, polysaccharide, polynucleotide or chemically synthesized polypeptide can be purified by HPLC, FPLC or size exclusion chromatography. The reductive alkylation of such an aromatic amine containing amino acid generated from the reduction of amino acids containing substituted aromatic moiety is understandable in section A: Method for modifying post-transducer polypeptides that have non-naural amino acids using a single step of post-translational modifi cation: Reductive alkylation reactions of non-na tural amino acids that have aromatic amine with reactants that have aldehyde. Illustrative embodiments of the reductive alkylation of the aromatic amine containing amino acid prepared by reductions of a protected (or masked) amine are presented in Figures 15 and 34. The reductive alkylation step follows the first post-translation step that seductively generates the non-natural amino acids containing aromatic amine on the polypeptides. The reductive alkylations are the second post-translational reaction, which by this modifies the unnatural amino acid polypeptides containing aromatic amine to non-natural amino acid polypeptides with non-natural amino acids containing alkylated aromatic amine. Such post-translation modifications or postincorporation modifications can also be applied to aromatic amine-containing amino acids incorporated into synthetic polymers, polysaccharides, polynucleotides or chemically synthesized polypeptides. The types of polypeptides comprising such non-natural amino acids containing aromatic amine are practically unlimited, while the non-natural amino acid contains aromatic amine located on the polypeptide, such that the reagent containing carbonyl, in which reagents are included containing aldehyde, can react with the aromatic amine group and not create a resulting modified non-natural amino acid that destroys the tertiary structure of the polypeptide (except, of course, if such destruction is the purpose of the reaction). By way of example only, the aldehyde-containing reagents which are reactive with the non-natural amino acids containing aromatic amine described in present, and can also be used to further modify non-natural amino acid polypeptides containing aromatic amine are compounds with the following structure Q where; R5 is alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, substituted alkoxy, alkylalkoxy, substituted alkylalkoxy, polyalkylene oxide, substituted polyalkylene oxide, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocycle , substituted heterocycle, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl,, -C (0) R ", -C (0) OR", -C (0) N (R ") 2, -C (O) NHCH ( R ") 2, - (alkylene or alkylene) -N (R") 2 '- (alkenylene or substituted alkenylene) -N (R ") 2, - (alkylene or substituted alkylene) - (aryl or substituted aryl), - (alkenylene or substituted alkenylene) - (aryl or substituted aryl), - (alkylene or substituted alkylene) -ON (R ") 2, - (alkylene or substituted alkylene) -C (0) SR", - (alkylene or substituted alkylene) ) -SS- (aryl or substituted aryl), wherein each R "is independently hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, tuido, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocycle, substituted heterocycle, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl or -C (0) OR '; or R5 is L-X, wherein X is selected from the group consisting of a label; a dye; a polymer ", a water-soluble polymer, a polyethylene glycol derivative, a photocrosslinker, a cytotoxic compound, a drug, an affinity tag, a photoaffinity tag, a reactive compound, a resin, a second protein or polypeptide or analogue polypeptide, an antibody or antibody fragment, a metal chelator, a co-factor, a fatty acid, a carbohydrate, a polynucleotide, a DNA, an RNA, an antisense polynucleotide, a saccharide, a water-soluble dendrimer, a cyclodextrin a biomaterial, a nanoparticle, a spin marker, a fluorophore, a metal-containing portion, a radioactive portion, a new functional group, a group that interacts covalently or non-covalently with other molecules, a photoenaged portion, a portion excitable by actinic radiation, a ligand, a photoisomerizable portion, biotin, a biotin analogue, a portion that incorporates a heavy atom, a chemical cleavage group, amente, a photocleavable group; an elongated side chain; a carbon-bonded sugar; a redox-active agent; an amino thioacid; a toxic portion; an isotopically labeled portion; a biophysical probe; a phosphorescent group; a chemiluminescent group; a dense group of electrons; a magnetic group; an intercalary group; a chromophore; a transfer agent Energy; a biologically active agent; a detectable marker; a small molecule; an inhibitory ribonucleic acid; a radionucleotide; an electron capture agent; a biotin derivative; quantum dot (s); a nanotransmitter; a radio transmitter; an abzyme, an activated complex activator, a virus, an adjuvant, an aglycan, an allergen, an angiostatin, an antihormone, an antioxidant, an aptamer, a guide RNA, a saponin, a shuttle vector, a macromolecule, an mimotope, a receptor, a reverse micelle and any combination thereof and L is optional and when present is a linker selected from the group consisting of alkylene, substituted alkylene, alkenylene, substituted alkenylene, -0-, -0- (alkylene) or substituted alkylene), - (alkylene or substituted alkylene) -0-, -C (0) -, -C (0) - (alkylene or substituted alkylene), (alkylene or substituted alkylene) -C (0) -, - C (0) N (R ') -, -C (0) N (R') - (alkylene or substituted alkylene) -, - (alkylene or substituted alkylene) -0C (0) N (R ') -, - N (R ') C (0) -, -N (R') C (0) - (alkylene or substituted alkylene) -, (alkylene or substituted alkylene) -NR 'C (0) -, -S-, - S- (alkylene or substituted alkylene) -, -S (0) k- where k is 1, 2 or 3, -S (0) k- (alkylene) or substituted alkylene) -, -C (S) -, -C (S) - (alkylene or substituted alkylene) -, CSN (R ') -, CSN (R') - (alkylene or substituted alkylene) -, -N (R ') C0- (alkylene or substituted alkylene) -, -N (R') C (0) 0-, - (alkylene or substituted alkylene) -0-N = CR '-, - (alkylene or substituted alkylene) - C (O) NR '- (alkylene or substituted alkylene) -, (alkylene or substituted alkylene) -S (0) k- (alkylene or substituted alkylene) -S-, - (alkylene or substituted alkylene) -SS-, S (0) kN (R ') -, N (R') C (0) N (R ') -, - N (R') C (S) N (R ') -, - N (R') S (0) kN (R ') -, -N (R') - N =, -C (R ') = N-, -C (R') = NN (R ') -, -C (R') = NN =, -C (R ') 2-N = N and -C (R') 2-N (R ') -N (R') -; each R 'is independently H, alkyl or substituted alkyl. In addition, by way of example only, the carbonyl-containing reagents are reactive with the non-natural aromatic amine-containing amino acids described herein and which can be used to further modify the non-natural amino acid polypeptides containing aromatic amine are compounds containing dicarbonyl, in which diketones, ketoaldehydes and dialdehydes are included with the following structures, i? H ° o where; each Ri is independently selected from H, optionally substituted alkyl, optionally substituted alkene, optionally substituted alkyne, optionally substituted cycloalkyl, optionally substituted heterocycle, optionally substituted aryl or optionally substituted heteroaryl; Rs is alkylene, substituted alkylene, alkenylene, substituted alkenylene, alkynylene, substituted alkynylene, polyalkylene oxide, substituted polyalkylene oxide, cycloalkylene, substituted cycloalkylene, substituted arylene arylene, heteroarylene, substituted heteroarylene, heterocycloalkylene, substituted heterocycloalkylene, -C (0) R "-, - C (0) OR "-, - C (0) N (R") -, - (substituted alkylene or alkylene) -ON (R ") -, - (substituted alkenylene or alkenylene) -N (R'J -, - (alkylene or substituted alkylene) R ", - (substituted alkylene or alkylene) -C (0) SR" -, wherein each R "is independently of hydrogen, alkyl, substituted alkyl, substituted alkenylene, alkenii, 1-xhoxyl alcohol, substituted alkylene, alkenylene substituted, alkynylene, substituted alkynylene, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocycle, substituted heterocycle, alkaryl, substituted alkaryl, aralkyl, or substituted aralkyl or R5 is LX, wherein X is selected from the group consisting of a labeled r; a dye; a polymer ", a water-soluble polymer, a polyethylene glycol derivative, a photocrosslinker, a cytotoxic compound, a drug, an affinity tag, a photoaffinity tag, a reactive compound, a resin, a second protein or polypeptide or analogue polypeptide, an antibody or fragment of antibodies, a chelator of metals, a co-factor, a fatty acid, a carbohydrate, a polynucleotide, a DNA, an RNA, a antisense polynucleotide; a saccharide, an aqua-soluble dendrimer, a cyclodextrin, a biomaterial; a nanoparticle; a spin marker; a fluorophore, a portion containing metal; a radioactive portion; a new functional group; a group that interacts covalently or non-covalently with other molecules; a photoenaged portion; an excitable portion by actinic radiation; a liqando; a photoisomerizable portion; biotin; a biotin analogue; a portion that incorporates a heavy atom; a chemically cleavable group; a photocleavable group; an elongated side chain; a carbon-bonded sugar; a redox-active agent; an amino thioacid; a toxic portion; an isotopically labeled portion; a biophysical probe; a phosphorescent group; a chemiluminescent group; a dense group of electrons; a magnetic group; an intercalary group; a chromophore; an energy transfer agent; a biologically active agent; a detectable marker; a small molecule; an inhibitory ribonucleic acid; a radionucleotide; an electron capture agent; a biotin derivative; quantum dot (s); a nanotransmitter; a radio transmitter; an abzyme, an activated complex activator, a virus, an adjuvant, an aglycan, an allergen, an angiostatin, an antihormone, an antioxidant, an aptamer, a guide RNA, a saponin, a shuttle vector, a macromolecule, an mimotope, a receptor, a reverse micelle and any combination thereof and L is optional and when present is a linker selected from the group consisting of alkylene, substituted alkylene, alkenylene, substituted alkenylene, -0-, -0- (alkylene or substituted alkylene), - (alkylene or substituted alkylene) -0-, - C (0) -, -C (0) - (alkylene or substituted alkylene), (alkylene or substituted alkylene) -C (0) -, -C (0) N (R ') -, -C (0) N (R ') - (alkylene or substituted alkylene) -, - (alkylene or substituted alkylene) -0C (0) N (R') -, -N (R ') C (0) -, -N (R') C (0) - (alkylene or substituted alkylene) -, (alkylene or substituted alkylene) -NR 'C (0) -, -S-, -S- (alkylene or substituted alkylene) -, -S (0) k- wherein k is 1, 2 or 3, -S (0) k- (substituted alkylene or alkylene) -, -CJ S) -, -C (S) - (alkylene or substituted alkylene) -, CSN (R ') -, CSN (R ') - (alkylene or substituted alkylene) -, -N (R') CO- (alkylene or substituted alkylene) -, -N (R ') C (0) 0-, - (alkylene or alkylene substituted) -0-N = CR '-, - (alkylene or substituted alkylene) -C (0) NR' - (alkylene or alkylene) used) -, (alkylene or substituted alkylene) -S (0) k- (alkylene or substituted alkylene) -S-, - (alkylene or substituted alkylene) -SS-, S (0) kN (R ') -, - N (R ') C (0) N (R') -, - N (R ') C (S) N (R') -, - N (R ') S (0) kN (R') -, -N (R ') - N =, -C (R') = N-, -C (R ') = NN (R') -, -C (R ') = NN =, -C (R') 2-N = N and -C (R ') 2-N (R') -N (R ') -; each R 'is independently H, alkyl or substituted alkyl; n is 1, 2 or 3. The types of carbonyl-containing reagents that can react with non-natural amino acids containing aromatic amine are practically unlimited, while the unnatural amino acid containing aromatic amine is located on the polypeptide, such that the carbonyl-containing reagent, in which aldehyde-containing reagents are included, can react with the aromatic amine group and not create a modified unnatural amino acid, resulting that destroys the tertiary structure of the polypeptide (except, of course, if such destruction is the purpose of the reaction). Such carbonyl-containing reagents include but are not limited to, reagents containing at least one carbonyl moiety and / or reagents containing at least two carbonyl moieties. Additionally, the reaction between a carbonyl moiety, by way of example only an aldehyde portion and an aromatic amine moiety is easy and such reductive alkylation reactions have at least one of the following characteristics: (i) it occurs in a pH range from about 4 to about 7, (n) generates an amine bond that is stable to or biological conditions; (m) is site specific; (iv) does not irreversibly destroy the tertiary structure of a polypeptide; (v) occurs rapidly at room temperature; (vi) occurs easily in aqueous conditions; (vn) occurs easily when the ratio of the non-natural amino acid comprising the aromatic amine portion to the aldehyde-containing reagent is stoichiometric, stoichiometric or quasi-stoichiometric; and (vm) is selective regio and / or specific regio. The orthogonal nature of the reductive alkylation reactions results in regioselectivity and / or regiospecifity, thereby allowing site-specific modification of unnatural amino acid polypeptides without affecting other amino acids in the non-natural amino acid polypeptide. Certain embodiments include a single reductive alkylation of the aromatic amine portion with a carbonyl-containing reagent yielding a secondary amine portion, while other embodiments include two reductive alkylanons of the aromatic amine portion with two carbonyl-containing reagents that produce a tertiary amine portion. Reagents containing carbonyl in this embodiment can be identical or different. Additionally, certain embodiments include a single reductive alkylation of the aromatic amine portion with a reagent containing at least two carbonyl groups, thereby producing a secondary amine moiety. Still other embodiments include two reductive alkylations, also referred to as reductive double alkylation, of the aromatic amine moiety with a reagent containing at least two carbonyl groups, thereby producing a cyclic tertiary amine moiety. In these illustrative embodiments, the carbonyl-containing reagent is added to a pH-regulated solution (pH about 4 to about 7) of the non-natural amino acid containing aromatic amine on a polymer for example a natural polypeptide, synthetic polymer, polysaccharide, polynucleotide or chemically synthesized polypeptide and a reducing agent, such as by way of example only, NaBCNH3 , TCEP, Na2S20, LiAlH4, B2H6 and NaBH4. The reaction proceeds at room temperature and the resulting non-natural amino acid polypeptide containing alkylated aromatic amine can be purified by HPLC, FPLC or size exclusion chromatography. In other embodiments, multiple linker chemistry can specifically react at the site with an unnatural amino acid polypeptide containing aromatic amine. In one embodiment, the linker methods described in the patent use linkers containing the carbonyl functionality, in which the aldehyde functionality is included, over at least one linker term (mono, bi or multi-functional). Reductive alkylation of a carbonyl-derivative linker with an aromatic amine-containing polypeptide generates a stable amine bond. Bi- and / or multi-functional linkers, also known as heterofunctional linkers (for example, aldehyde with one, plus, or other binding tape) allow specific site-specific connection of different molecules (eg, other polypeptides, polynucleic acids , polymers or molecules small) to the unnatural amino acid polypeptide, whereas the mono-functional linkers, also known as homofunctional linkers (eg, completely substituted by aldehyde in all terms) facilitate the specific dimer- or oligomerization of the amino acid polypeptide site non-natural aromatic amine. By combining this linker strategy with the m vivo translation technology described herein, it becomes possible to specify the three-dimensional structures of the chemically processed polypeptides.

C. Methods for post-translationally modifying unnatural amino acid polypeptides using two stages of post-translational modification: Protection of unnatural amino acids containing aldehyde protected by reductive amination of unnatural amino acids containing aldehyde followed by reagents containing aromatic amine . The incorporation of non-natural amino acids with side chain containing masked or protected electropile to polypeptides allows site-specific reduction of the side chains. Such non-natural amino acids include masked or protected carbonyl groups, such as, by way of example only, amino acids containing masked aldehyde groups or protected aldehyde group, and wherein the site-specific reducing animation is carried out after unmasking or deprotection of the aldehyde, via nucleopilic addition of an aromatic amine to the side chain containing available aldehyde. This reductive amination reaction generates an amine bond, in which secondary and tertiary amines are included. Methods for derivatizing and / or further modifying can be carried out with naturally generated polypeptides or chemically synthesized polypeptides that have been purified prior to the reductive amination step or after the step of reductive animation. In addition, methods for derivatizing and / or further modifying can be carried out with synthetic polymers, polysaccharides or polynucleotides which can be purified before or after these modification methods. By way of example only, the following non-natural amino acids are the type of amino acids that contain masked / protected aldehyde portion that can be incorporated into polypeptides, then unmasked / unprotected before reductively aminating the side chain containing available aldehyde to generate a link of amine. where : L is optional and when present is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylnene, substituted lower alkenylene, alkynene, lower heteroalkylene, substituted heteroalkylene, lower heterocyclearkylene, substituted lower heterocyclealkylene, arylene, substituted arylene, heteroarylene , substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene or substituted aralkylene; Q is optional and when present is a linker selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O- (alkylene or substituted alkylene) -, -S - (substituted alkylene or alkylene) -, wherein k is 1,2, or 3, -S- (0) k (substituted alkylene or alkylene) -, -C (0) - (alkylene or substituted alkylene) -, - C (S) - (substituted alkylene or alkylene) -, -NR '- (substituted alkylene or alkylene) -, -CON (R ") - (substituted alkylene or alkylene) -, -CSN (R") -, -CSN (R ') - (substituted alkylene or alkylene) -, -N (R') CO- (substituted alkylene or alkylene) -, and wherein each R 'is independently H, alkyl or substituted alkyl, Ri is H a amino, amino acid, polypeptide or polynucleotide group and R2 is OH, is a protective group ester, resin, amino acid, polypeptide or polynucleotide; each R3 and R4 is independently H, halogen, lower alkyl, substituted lower alkyl, or R3? R4, or two groups of R3 # optionally form a cycloalkyl or a heterocycloalkyl; each Ra is independently selected from the group consisting of H, halogen, alkyl, -N02, -CN, substituted alkyl, -N (R ') 2, -C (0) kR', -C (0) N (R ') 2, -0R', and -S (0) kR ', where k is 1, 2, or 3; Re is an aldehyde, a protected aldehyde or a masked aldehyde, wherein the protective group includes but is not limited to: wherein each Xi is independently selected from a group consisting of -0-, -S-, -N (H) -, -N (Ac) -, and -N (OMe) -; X2 is -OR, -OAc, -SR, -N (R) 2, -N (R) (Ac), -N (R) (0Me), or N3, Y where each R 'and R is independently H, alkyl or substituted alkyl. Such non-natural amino acids can also be in the form of a salt or can be incorporated into a non-natural amino acid polypeptide, polymer or a polynucleotide. Such non-natural amino acids can also be incorporated into a non-natural amino acid polypeptide and then modified post-translationally by deprotection to form an aldehyde group "in situ" followed by reductive amidation of aldehyde with an aromatic amine-containing reagent, thereby forming non-natural amino acids, which have the structure of formula (D) as part of the non-natural amino acid polypeptide. In addition, non-natural amino acids having the formula structure of (E) can also be incorporated into a polymer or a polynucleotide and are deprotected to form an "in-situ" dldehyde group followed by reductive amidation of the aldehyde with a reagent containing aromatic amine. By way of example only, aromatic amine-containing reagents that are reactive with the unnatural, unmasked / deprotected aldehyde-containing amino acids described herein are compounds with the following structure: R, where is selected from the group consisting of a monocyclic aryl ring and a bicyclic aryl ring, a multicyclic aryl ring, a monocyclic heteroaryl ring, a bicyclic heteroaryl ring and a monocyclic heteroaryl ring. A is independently CRZ or N; and B is independently CRa, N, 0 or S; each Ra is independently selected from the group consisting of H, halogen, alkyl, -N02, -CN, substituted alkyl, -N (R ') 2, -C (0)? R', -C (0) N (R ') ) 2, -0R ', and -S (0)? R', where k is 1,2, or 3, n is 0, 1, 2, 3, 4, 5 or 6; M is H or CH2R5; Rs is alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alcoholyl, substituted alkoxy, alkylalcohoxy, substituted alkylalkoxy, polyalkylene oxide, substituted polyalkylene oxide, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl , heterocycle, substituted heterocycle, substituted alkaryl alkaryl, aralkyl, substituted aralkyl, -C (0) R ", -C (0) OR", -C (0) N (R'J 2, -C (0) NHCH ( R'J 2, - (substituted alkylene or alkylene) -N (R ") 2 r ~ (substituted alkenylene or alkenylene) -N (R") 2, - (substituted alkylene or alkylene) - (substituted aryl or aryl), - (alkenylene or substituted alkenylene) - (substituted aryl or aryl), - (substituted alkylene or alkylene) -ON (R ") 2, - (substituted alkylene or alkylene) - C (O) SR ", - (substituted alkylene or alkylene) -SS- (substituted aryl or aryl), wherein each R" is independently hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alcohol, substituted alkoxy, aryl, Substituted aryl, heteroaryl, substituted heteroaryl, heterocycle, substituted heterocycle, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl, or -C (0) OR '; or R5 is L-X, wherein X is selected from the group consisting of a label; a dye; a polymer ", a water-soluble polymer, a polyethylene glycol derivative, a photocrosslinker, a cyto-chemical compound, a drug, an affinity tag, a photoaffinity tag, a reactive compound, a resin, a second protein or polypeptide or analogue polypeptide, an antibody or antibody fragment, a metal chelator, a co-factor, a fatty acid, a carbohydrate, a polynucleotide, a DNA, an RNA, an antisense polynucleotide, a saccharide, a water-soluble dendrimer, a cyclodextrin a biomaterial, a nanoparticle, a spin marker, a fluorophore, a metal-containing portion, a radioactive portion, a new functional group, a group that interacts covalently or non-covalently with other molecules, a photoenaged portion, a portion excitable by actinic radiation, a ligand, a photoisomerizable portion, biotin, a biotin analogue, a portion that incorporates a heavy atom, a chemical cleavage group, amente a group photocleavable; an elongated side chain; a carbon-bonded sugar; a redox-active agent; an amino thioacid; a toxic portion; an isotopically labeled portion; a biophysical probe; a phosphorescent group; a chemiluminescent group; a dense group of electrons; a magnetic group; an intercalary group; a chromophore; an energy transfer agent; a biologically active agent; a detectable marker; a small molecule; an inhibitory ribonucleic acid; a radionucleotide; an electron capture agent; a biotin derivative; quantum dot (s); a nanotransmitter; a radio transmitter; an abzyme, an activated complex activator, a virus, an adjuvant, an aglycan, an allergen, an angiostatin, an antihormone, an antioxidant, an aptamer, a guide RNA, a saponin, a shuttle vector, a macromolecule, an mimotope, a receptor, a reverse micelle and any combination thereof and L is optional and when present is a linker selected from the group consisting of alkylene, substituted alkylene, alkenylene, substituted alkenylene, -0-, -0- (alkylene) or substituted alkylene), - (alkylene or substituted alkylene) -0-, -C (0) -, -C (0) - (alkylene or substituted alkylene), (alkylene or substituted alkylene) -C (0) -, - C (0) N (R ') -, -C (0) N (R') - (alkylene or substituted alkylene) -, - (alkylene or substituted alkylene) -0C (0) N (R ') -, - N (R ') C (0) -, -N (R') C (0) - (alkylene or substituted alkylene) -, (alkylene or substituted alkylene) -NR 'C (0) -, -S-, -S- (alkylene or substituted alkylene) -, -S (0) - wherein k is 1, 2 or 3, -S (0) k- (alkylene or substituted alkylene) -, -C (S) -, - C (S) - (alkylene or substituted alkylene) -, CSN (R ') -, CSN (R') - (alkylene or substituted alkylene) -, -N (R ') CO- (alkylene or substituted alkylene) -, -N (R ') C (O) 0-, - (alkylene or substituted alkylene) -0-N = CR' -, - (alkylene or substituted alkylene) -C (0) NR '- (alkylene or substituted alkylene) -, (alkylene or substituted alkylene) -S (0) k- (alkylene or substituted alkylene) -S-, - (alkylene or substituted alkylene) -SS-, S (0) N (R ') -, -N ( R ') C (0) N (R') -, - N (R ') C (S) N (R') -, - N (R ') S (0) kN (R') -, - (R ') - N =, -C (R') = N-, -C (R ') = NN (R') -, -C (R ') = NN =, -C (R') 2- N = - N and -C (R ') 2-N (R') -N (R ') -; each R 'is independently H, alkyl or substituted alkyl. Such reagents include compounds having the following structures, These compounds can also be used to further modify polypeptides of non-natural amino acid-containing amino acids (natural or chemically synthesized), synthetic polymers, polysaccharides or polynucleotides.

The reductive amination reactions post-translationally modify the unnatural amino acid polypeptides containing unmasked / unprotected aldehyde to non-natural amino acid polypeptides containing non-natural amino acid containing aromatic amine. The types of polypeptides comprising such non-natural amino acids containing aromatic amine are practically unlimited, while the unnatural amino acid containing unmasked / deprotected aldehyde is located on the polypeptide, such that the reactant containing aromatic amine can react with the available aldehyde group and not creating a resulting modified non-natural amino acid that destroys the tertiary structure of the polypeptide (except, of course, if such destruction is the purpose of the reaction). Additionally, the reaction between an aldehyde portion and an aromatic amine moiety is easy and such reductive amination reactions have at least one of the following characteristics: (i) it occurs in a pH range of about 4 to about 7, (ii) ) generates an amine bond that is stable under biological conditions; (iii) is site specific; (iv) does not irreversibly destroy the tertiary structure of a polypeptide; (v) occurs rapidly at room temperature; (vi) occurs easily in aqueous conditions; (vii) occurs easily when the proportion of the non-natural amino acid comprising the Aromatic amine portion to the aldehyde-containing reagent is stoichiometric, stoichiometric or quasi-stoichiometric; and (viii) it is selective regio and / or specific regio. The orthogonal nature of the reductive alkylation reactions results in the regio selectivity and / or specific regio, thereby allowing site-specific post-translational modification of unnatural amino acid polypeptides without affecting other amino acids in the unnatural amino acid polypeptide . An illustrative embodiment for seductively amending an unnatural amino acid containing unmasked or deprotected aldehyde on a polypeptide is illustrated in Figure 18. In addition, certain embodiments include a single reductive amination of an unnatural amino acid containing unmasked or deprotected aldehyde on a polypeptide with a reagent containing aromatic amine, thereby producing a secondary amine moiety. Additionally, certain embodiments include a reductive amination of a non-natural amino acid containing carbonyl on a polypeptide, in which the carbonyl portion contains at least two unmasked or protected aldehyde groups, with an aromatic amine-containing reagent, thereby producing a of cyclic tertiary amine. Still other embodiments include two reductive aminations of an unnatural amino acid containing unmasked or deprotected aldehyde on the basis of a polypeptide containing at least two aldehyde groups, with reagents containing identical or different aromatic amine-containing reagents, thereby producing two portions of secondary amine. In these illustrative embodiments, an aromatic amine-containing reagent is attached to a pH-regulated solution (pH about 4 to about 7) of the unnatural amino acid polypeptide containing unmasked or deprotected aldehyde and a reducing agent, such as example only, TCEP, Na2S20, L? AlH4, B2H5; NaBH4 or NaBCNH3. The reaction proceeds at room temperature and the resulting non-natural amino acid polypeptide containing aminated aldehyde can be purified by HPLC, FPLC or size exclusion chromatography. In other embodiments, multi-linker chemistry can specifically react at the site with an unnatural amino acid polypeptide containing unmasked or deprotected aldehyde. In one embodiment, the linker methods described in the patent use linkers containing the aromatic amine functionality, on at least one linker term (mono, bi or multi-functional). The reductive animation of an aromatic amine linker-depurated with an unmasked or deprotected aldehyde-containing polypeptide generates a stable amine bond. Bi-and / or multi-functional linkers, also known as heterofunctional linkers (eg, aromatic amine with one or more, Binding ribbons) allow site-specific connection of different molecules (eg, other polypeptides, polynucleic acids, polymers or small molecules) to the non-natural amino acid polypeptide, as mono-functional linkers, also known as homofunctional linkers (all amine -aromatics substituted in all terms) facilitate the dimer- or site-specific oligomerization of the non-natural amino acid polypeptide containing unmasked or deprotected aldehyde. By combining this linker strategy with the in vivo translation technology described in the patent, it becomes possible to specify the three-dimensional structures of the chemically processed proteins.

D. Example of Aggregate Functionality: Macromolecular Polymers Coupled to Non-Natural Amino Acid Polypeptides Various modifications to the non-natural amino acid polypeptides described herein may be effected using the compositions, methods, techniques and strategies described herein. These modifications include the incorporation of additional functionality on the non-natural amino acid component of the polypeptide, which include but are not limited to: a marker; a dye; a polymer; a water soluble polymer; a polyethylene glycol derivative; a photocrosslinker; a cytotoxic compound; a drug; an affinity marker; a photoaffinity marker; a reactive compound; a resin; a second protein or polypeptide or polypeptide analogue; an antibody or antibody fragment; a metal chelator; a co-factor; a fatty acid; a carbohydrate; a polynucleotide; a DNA; an RNA; an antisense polynucleotide; a saccharide, a water-soluble dendrimer, a cyclodextrin, a biomaterial; a nanoparticle; a spin marker; a fluorophore, a portion containing metal; a radioactive portion; a new functional group; a group that interacts covalently or non-covalently with other molecules; a photoenaged portion; an excitable portion by actinic radiation; a ligand; a photoisomerizable portion; biotin; a biotome analogue; a portion that incorporates a heavy atom; a chemically cleavable group; a photocleavable group; an elongated side chain; a carbon-bonded sugar; a redox-active agent; an amino thioacid; a toxic portion; an isotopically labeled portion; a biophysical probe; a phosphorescent group; a chemiluminescent group; a dense group of electrons; a magnetic group; an intercalary group; a chromophore; an energy transfer agent; a biologically active agent; a detectable marker; a small molecule; an inhibitory ribonucleic acid; a radionucleotide; an electron capture agent; a biotin derivative; quantum dot (s); a nanotransmitter; a radio transmitter; an abzima, an activated complex activator, a virus, an adjuvant, an aglycan, an allergen, an angiostatin, an antihormone, an antioxidant, an aptamer, a guide RNA, a saponin, a shuttle vector, a macromolecule, a mimotope, a receptor, a reverse micelle and any combination thereof. As a non-limiting illustrative 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 in the present are also applicable (with appropriate modifications, if necessary for which that of skill in the art could be made with the disclosures herein) to add other functionalities, which include but are not limited to those listed above. A wide variety of macromolecular polymers and other molecules can be linked to the non-natural amino acid polypeptides described herein to modulate biological properties of the unnatural 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 linked to the non-natural amino acid polypeptide via the non-natural amino acid or any functional substituent of the non-natural amino acid or any substituent or functional group added to the non-natural amino acid. Water-soluble polymers can be coupled to non-natural amino acids incorporated into polypeptides (natural or synthetic), polynucleotides, polysaccharides or synthetic polymers. The water soluble polymers can be coupled via a non-natural amino acid incorporated in the polypeptide or any functional group or substituent of an unnatural amino acid or any functional group or substituent added to the non-natural amino acid, zn some cases, the unnatural amino acid ponpeptides described herein comprise one or more non-natural amino acid (s) linked to water-soluble polymers and one or more amino acids that are stably present in nature coupled to water-soluble polymers. The covalent annexation of hydrophilic polymers to a biologically active molecule represents a method to increase the solubility in water (such as in a physiological environment), bioavailability, increase the half-life in the serum, increase the therapeutic half-life, modulate the immunogenicity, modulate the biological activity or prolong the circulation time of the biologically active molecule, in which it includes proteins, peptides and particularly hydrophobic molecules. Additional important aspects of such hydrophilic polymers can include biocompatibility, lack of toxicity and lack of immunogenicity. For therapeutic use, the final product preparation, a pharmaceutically acceptable polymer can be used. Examples of hydrophilic polymers include, but are not limited to, polyalkyl ethers and alkoxy-capped analogs thereof (eg, polyoxyethylene glycol, polyoxyethylene / propylene glycol and methoxy or ethoxy-crown analogs thereof, especially polyoxyethylene glycol, the latter is also known as polyethylene glycol or PEG); polyvinyl pyrrolidones; polyvinyl alkyl ethers; polyoxazolines, polyalkyl oxazolines and polyhydroxyalkyl oxazolines; polyacrylamides, polyalkyl acrylamides and polyhydroxyalkyl acrylamides (for example, polyhydroxypropylmethacrylamide and derivatives thereof); polyhydroxyalkyl acrylates; polysialic acids and analogue thereof; hydrophilic peptide sequences; polysaccharides and their derivatives, wherein dextran and dextran derivatives are included, for example, carboxymethyldextran, dextran sulfate, aminodextran; cellulose and its derivatives, for example, carboxymethyl cellulose, hydroxyalkyl celluloses; chitin and its derivatives, for example, chitosan, succinyl chitosan, carboxymethylchitin, carboxymethylchitosan; hyaluronic acid and its derivatives; starches; Alkinates; chondroitin sulfate; albumin; pullulana and carboxymethyl pullulana; polyamino acids and derivatives thereof, for example, polyglutamic acids, polysilvia, polyaspartic acid, polyaspartamides; copolymers of maleic anhydride such as: maleic anhydride styrene copolymer, maleic anhydride divmileter copolymer; polyvinyl alcohols; copolymers thereof; terpolymers thereof; mixtures thereof; and derivatives of the above. The water soluble polymer can be in any structural form in which they are included but not limited to linear, hairpin or branched. In some embodiments, the fundamental polymer chains that are soluble in water, with from about 2 to about 300 terms, are particularly useful. Multifunctional polymeric derivatives include but are not limited to, linear polymers having two terms, each term being linked to a functional group which may be the same or different. In some embodiments, the water-soluble polymer comprises a portion of poly (ethylene glycol). The molecular weight of the polymer can be of a wide range, in which but not limited, between about 100 Da and about 100,000 Da or more. The molecular weight of the polymer can be between about 100 Da and about 100,000 Da, which includes but is 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 poly (ethylene glycol) molecule is a branched polymer. The molecular weight of branched chain PEG may be between about 1,000 Da and about 100,000 Da, which includes but is 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 modalities, the weight 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 above list for substantially water soluble base chains is not intended to be in any way exhaustive and is only illustrative and that all polymeric materials having the qualities described above are contemplated as being suitable for use. in the methods and compositions described herein. As described above, one example of a hydrophilic polymer is poly (ethylene glycol), abbreviated PEG, which has been used extensively in pharmaceuticals, in artificial implants and in other applications where biocompatibility, lack of toxicity and lack of immunogenicity are of importance . The polymer moieties: described herein will use PEG as an exemplary hydrophilic polymer with the understanding that other hydrophilic polymers can similarly be used in such embodiments. PEG is a well-known water-soluble polymer, which is commercially available or can be prepared by ethylene glycol ring opening polymerization, according to methods well known in the art (Sandler and Karo, Polymer Synthesis, Academic Press, New York, Vol. 3, pages 138-161). PEG is commonly clear, colorless, odorless, soluble in water, heat stable, inert to many chemical agents, does not hydrolyze or deteriorate and is generally non-toxic. It is considered that poly (ethylene glycol) is biocompatible, meaning that PEG is capable of coexisting with living tissues or organisms without causing harm. More specifically, PEG is substantially non-immunogenic, ie, that PEG does not tend to produce an immune response in the body. When it is attached to a molecule that has some desirable function in the body, such as a biologically active agent, PEG tends to mask the agent and can reduce or eliminate any immune response, such that an organism can tolerate the presence of the agent . PEG conjugates do not have to produce a substantial immune response or cause coagulation or other undesirable effects. The term "PEG" is widely used to encompass any polyethylene glycol molecule, regardless of size or modification at one end of the PEG and can be represented as linked to an unnatural amino acid polypeptide by the formula: XO- (CH2CH20) n- CH2CH2-Y wherein n is 2 to 10,000 and X is H or a terminal modification, wherein, but not limited to, a C? -4 alkyl, a protective group or a terminal functional group. The term PEG includes, but is not limited to, poly (ethylene glycol) in any of its forms, in which are included bifunctional PEG, multi-arm PEG, derivatized PEG, fork PEG, branched PEG (with each chain having a weight molecular from approximately 1 kDa to approximately 100 kDa, from approximately 1 kDa to approximately 50 kDa or from approximately 1 kDa to approximately 20 kDa), PEG pending (ie, PEG or related polymers having one or more functional groups pending to the fundamental chain of the polymer) or PEG with degradable linkages therein. In one embodiment, the 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 soluble polymer comprises a portion of poly (ethylene glycol). The molecular weight of the polymer can be of a wide range, in which but not limited to, between about 100 Da and about 100,000 Da or more. The molecular weight of the polymer can be between about 100 Da and about 100,000 Da, which includes but is 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 0 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 poly (ethylene glycol) molecule is a branched polymer. The molecular weight of the branched chain PEG can be between about 1,000 Da and about 100,000 Da, which includes but is 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 0 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 approximately 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. A wide range of PEG molecules are described in, including, but not limited to, the catalog of Shearwater Polymers, Inc. catalog of Nektar Ther peutics, incorporated herein by reference. Specific examples of terminal functional groups in the literature include but are not limited to L-succinimidyl carbonate (see, for example, U.S. Patent Nos. 5,281,698, 5,468,478), amine (See for example, Buckmann et al., Makromol., Chern., 182: 1379 (1981), Zalipsky et al., Polym, J. 19: 1177 (1983)), hydrazide (See, for example, Andresz et al., Makromol, Chem. 179: 301 (1978)), succinimidyl proionate and butanoate. of succinimidyl (see for example, Olson et al., in: Poly (ethylene glycol) Chemistry &Biological Applications, pp 170-181, Harris &Zalipsky Eds., ACS, Washington, DC, 1997; See also US Patent No. 5,672,662), succinimidyl succinate (see for example, Abuchowski et al., Biochem. Biophys., 7: 175 (1984) and Joppich et al., Makromol. Chem. 180: 1381 (1979), succinimidyl (see for example, U.S. Patent No. 4,670,417), benzotriazole carbonate (see for example, U.S. Patent No. 5,650,234), glycidyl ester (see, for example, Pitha et al., Eur. J Biochem. 94: 11 (1979 ), Elling et al., Biotech, Appl. Biochem 13: 354 (1991), oxycarbonylimidazole (see for example, Beauchamp, et al., Anal. Biochem. 131: 25 (1983), Tondelli et al., J. Controlled Reasease 1: 251 (1985)), p-nitrophenyl carbonate (see for example, Veronese, et al., Appl. Biochem. Biotech., 11: 141 (1985) and Sartore et al., Appl. Biochem. Biotech. , 27: 45 (1991)), aldehyde (see for example, Harris et al., J. Pclym, Sci. Chem. Ed. 22: 341 (1984), U.S. Patent No. 5,824,784, U.S. Patent No. 5,252,714), maleimide ( see, for example, Goodson et al., Bio / Technology 8: 343 (1990), Romani et al., in: Chemistry of Peptides and Proteins 2:29 (1984)) and Kogan, Synthetic Comm. 22: 2417 (1992)) , ortopyridyl disulfide (see for example, Woghiren, et al. Bioconj. Chem. 4: 314 (1993)), acrylon (see for example, Sawhney et al., Macromolecules, 26: 581 (1993)), vinylsulfone (see for example, US Patent No. 5,900,461). All references and prior patents are incorporated herein by reference.

In some cases, a PEG ends at one end with hydroxy or methoxy, that is, X is H or CH3 ("methoxy PEG'J.

Alternatively, the PEG can terminate with a reactive group, thereby forming a bifunctional polymer. Typical reactive groups may include those reactive groups which are commonly used to react the functional groups found in the common 20 amino acids (in which but not limited to maleimide groups, activated carbonates (in which but not limited to ester are included) p-nitrophenyl), activated esters (in which but not limited to N-hydroxysuccinimide, p-nitrophenyl ester) and aldehydes) are also functional amphos that are inert to the 20 common amino acids but react specifically with functional groups complementary to the non-natural amino acids (which include, but are not limited to, atomic amine groups). It will be noted that the other end of the PEG, which is shown in the above formula by Y, will be attached either directly or indirectly to a polypeptide (synthetic or natural), polynucleotide, polysaccharide or synthetic polymer via an unnatural amino acid. When Y is an aldehyde group, then the PEG residue containing aldehyde can react with an unnatural amino acid containing aromatic amine in a polypeptide to form a PEG group linked to the polypeptide via an amine linkage. Examples of appropriate reaction conditions, methods and purification reagents known in all this specification and in the figures Attached Figure 35 presents a scheme illustrating the comparison between the N-terminal PEG coating and the PEG coating based on the reductive alkylation of the aromatic amine on amino acids that have been incorporated into peptides. The fiwra illustrates that under the reaction conditions used, the PEG coating of a natural peptide is only easily obtained in the terminal amine, whereas under the reaction conditions used for the coating with PEG of non-natural peptides incorporating non-natural amino acids. containing aromatic amine moieties can be obtained at the site of incorporation of the unnatural amino acid into the sequence, without any reaction at the protonated terminal amine. The PEG coating site depends on the site of incorporation of the non-natural amino acid containing the aromatic amine portion. Such a site may be a terminal site or it may be any site within the sequence of the peptide. By way of example only and not as limitation as to the types or classes of PEG reagents that can be used with the compositions, methods, techniques and strategies described herein, Figure 36 presents illustrative examples of PEG reagents that contain aldehyde which can react with unnatural amino acid peptides containing aromatic amine to form non-natural amino acid polypeptides linked to the PEG group via an amine bond. Also shown in Figure 36 are examples of PEG reagents containing branched aldehydes that can be reacted with amino acid polypeptides containing aromatic amine to form unnatural amino acid polypeptides linked to PEG groups. Heterobifunctional derivatives are also particularly useful when it is desired to attach different molecules to each term of the PEG polymer. In some embodiments, one PEG terminus contains a portion of aldehyde that can react with an aromatic amine group present in an unnatural amino acid to form an amine bond between the PEG and the peptide, while at another end the PEG contains another functionality that may undergo additional reaction by treatment with an appropriate agent. By way of example, such functionality can be an amine group available to act as a nucleotide on a variety of electrophiles, in which carbonyl-containing reagents are included. Thus, in some embodiments, the polypeptide comprising the non-natural amino acid is linked to a water-soluble polymer, such as polyethylene glycol (PEG), via the side chain of the non-natural amino acid. The non-natural amino acid methods and compositions described herein provide a highly efficient method for modification selective protein with PEG derivatives, which involves the selective incorporation of non-natural amino acids, which include but are not limited to those amino acids that contain functional groups or substituents not found in the 20 amino acids naturally incorporated into proteins in response to a codon selector and the subsequent modification of those amino acids with an appropriately reactive PEG derivative. Known chemistry methodologies of a wide variety are suitable for use with the non-natural amino acid methods and compositions described herein to incorporate a water-soluble polymer into the protein. The fundamental chain of the polymer can be linear or branched. Branched polymer backbones are generally known in the art. Commonly, a branched polymer has a central branch core portion and a plurality of linear polymer chains linked to the central branch core. PEG is used in branched formats which can be prepared by the addition of ethylene oxide to various forms, such as glycerol, glycerol oligomers, pentaerythritol and sorbitol. The central branch portion may also be derived from several amino acids, such as lysine. The branched poly (ethylene glycol) can be represented in general form as R (-PEG-OH) m in which R is derived from a portion of glycerol, glycerol or pentaerythritol oligomers and m represents the number of arms. PEG molecules of multiple arms, such as those described in the US Patents. Nos. 5,932,462 5,643,575; 5,229,490; 4,289,872; U.S. Patent Application 2003/0143596; WO 96/21469 and WO 93/21259, each of which is incorporated by reference herein in its entirety, can also be used as the fundamental chain of the polymer. The branched PEG may also be in the form of a hairpin PEG represented by PEG (-YCHZ2) n, wherein Y is a linking group and Z is an activated terminal group linked to CH by a chain of atoms of defined length. Still another branched form, the pending PEG, has reactive groups, such as carboxyl, along the PEG backbone, rather than at the end of the PEG chain. Figure 36 shows non-limiting examples of PEG reagents containing branched aldehyde used in the methods described herein. In addition to these forms of PEG, the polymer can also be prepared with weak or degradable bonds in the fundamental chain. For example, PEG can be prepared with ester linkages in the polymer backbone that are subjected to hydrolysis. As shown hereinafter, this hydrolysis results in the cleavage of the polymer into lower molecular weight fragments: -PEG-C02-PEG-H-H20? PEG-CO2H + HO-PEG- It will be understood by those skilled in the art that the term poly (ethylene glycol) or PEG represents or includes all forms known in the art in which they are included but not limited to those disclosed herein. The molecular weight of the polymer can be of a wide range, in which but not limited to, between about 100 Da and about 100,000 Da or more. The molecular weight of the polymer can be between about 100 Da and about 100,000 Da, which include but are not limited to, 100,000 Da, 95,000 Ca, 90,000 Da, 85,000 Da, 80,000 Da, 75,000 Da, 70,000 Da, 65,000 Da , 60,000 Da, 55,000 0 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 approximately 40,000 Da. In order to maximize the desired properties of the PEG, the molecular weight and hydration status of the PEG polymer or polymers attached to the biologically active molecule must be sufficiently high to impart the advantageous characteristics commonly associated in the annexation of the PEG polymer, such as water solubility and increased circulating half-life, as long as they do not adversely impact the bioactivity of the original molecule. The methods and compositions described herein can be used to produce substantially homogeneous preparations of polymer conjugates: "substantially homogeneous" proteins, as used herein, means that conjugated polymer polymers: protein is observed to be greater than half of the total protein. The polymer: protein conjugate has biological activity and the "substantially homogeneous" PEG-coated polypeptide preparations present provided herein are those that are sufficiently homogeneous to show the advantages of a homogeneous preparation, eg, ease in clinical application in predictability of pharmacokinetics from batch to batch. It may also be chosen to prepare a mixture of conjugated polymer molecules: protein and the advantage provided herein is that the ratio of mono-polymer conjugate: protein to be included in the mixture. Thus, if desired, a mixture of several proteins can be prepared with various numbers of attached polymer portions (ie, di-, tri-, tetra-, etc.) and combine said conjugates with the conjugate of mono-polymers: protein prepared using the methods described herein and having a mixture with a predetermined proportion of conjugates of mono-polymers: protein. The proportion of polyethylene glycol molecules to obtain proteins will vary, as with their entrances in the reaction mixture. In general, the optimum ratio (in terms of reaction efficiency in which there is a minimum excess of unreacted protein or polymer) can be determined by the molecular weight of the polyethylene glycol selected and the number of reactive groups available. Since it is concerned with molecular weight, commonly the higher the molecular weight of the polymer, the less is the number of polymer molecules that will be attached to the protein. Similarly, the branching of the polymer must be taken into account when these parameters are used. In general, the higher the molecular weight (or more branches), the higher the ratio of polymers: protein. As used herein and when conjugates of polymer: hydrophilic protein polypeptide are contemplated, the term "therapeutically effective amount" refers to a amount that gives the desired benefit to a patient. The amount will vary from one individual to another and will depend on a variety of factors, including the physical condition of the patient and the fundamental condition to be treated. A therapeutically effective amount of the present compositions can be readily determined by one skilled in the art using publicly available materials and methods. The number of water soluble polymers linked to a modified or unmodified unmodified amino acid polypeptide (i.e., the extension of the PEG coating or glycosylation) described herein may be adjusted to provide an altered pharmacological, pharmacokinetic or pharmacodynamic characteristic ( in which are included but not limited to increased or decreased) such as in vivo half-life. In some embodiments, the half-life of the polypeptide is increased by at least about 10, 20, 30, 40, 50, 60, 70, 80, 90 percent, twice, five times, 10 times, 50 times or at least approximately 100 times with respect to an unmodified polypeptide. In one embodiment, a polypeptide comprising an unnatural amino acid containing aromatic amine is modified with a PEG derivative containing a terminal aldehyde moiety that is directly linked to the PEG backbone. In another embodiment, a polypeptide that comprising an unnatural amino acid containing aromatic amine is modified with a branched PEG derivative containing a terminal aldehyde moiety, each branched PEG chain having a MW that ranges from about 10-40 kDa and in other moieties of about 5-20 kDa In some embodiments, the aldehyde-terminal PEG derivative will have the structure: RO- (CH 2 CH 20) -O- (CH 2) m-CH 2 -NH-CH 2 -C (O) H wherein R is a simple alkyl (methyl, ethyl , propyl, etc.), m is 2-10 and n is 100-1,000 (that is, the average molecular weight is between 5-40 kDa). The molecular weight of the polymer can be of a wide range, in which but not limited to between about 100 and about 100,000 Da or more. The molecular weight of the polymer can be between 100 Da and about 100,000 Da, which include but are 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 0 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 approximately 100 Da and approximately 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 another embodiment, a polypeptide comprising an aromatic amine amino acid is modified with a branched PEG derivative containing a terminal aldehyde moiety, thereby forming an amine bond with each chain of the branched PEG having a fluctuating MW of about 10. -40 kDa and in other modalities, of approximately 5-20 kDa. The molecular weight of the branched-chain PEG can be between about 1,000 Da and about 100,000 Da, which include but are 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 approximately 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. Some reviews and monographs regarding the functionalization and conjugation of PEG are available. See, for example, Harris, Ma cromol. Chem. Phys. C25: 325-373 (1985); Scouten, Methods in Enzymology 125: 30-65 (1987); Wong et al. , Enzyme My crob. Technol. 14: 866-874 (1992); Delgado et al. , Cri ti cal Reviews ín Therapeuti c Drug Carri er Sys tems 9: 249-304 (1992); Zalipsky, Bioconj uga te Chem. 6: 150-165 (1995). Methods for activating polymers can also be found in WO 94/17039, U.S. Patent No. 5,324,844, W094 / 18247, WO 94/04193, U.S. Patent No. 5,219,564, U.S. Patent No. 5,122,614, WO 90/13540, U.S. Patent No. 5,281,698 and WO 93/15189, and for conjugation between activated polymers and enzymes in which they include but are not limited to Coagulation Factor VIII (WO 94/15625), hemoglobin (WO 94/09027), oxygen carrier molecule (U.S. Patent No. 4,412,989), ribonuclease, and superoxide dismutase (Veronese et al., App. Biochem. Biotech 11: 141-52 (1985)), all of which are incorporated herein by reference in their entirety.

If necessary, the unnatural amino acid polypeptides coated with PEG described herein obtained from hydrophobic chromatography can be further encoded by one or more methods known to those skilled in the art in which they include but are not limited to affinity chromatography; anionic or cation exchange chromatography (using in those that include but are not limited to DEAE SEPHAROSE); chromatography on silica; Reverse phase HPLC; gel filtration (using, in which are included but not limited to SEPHAFEX G-75); hydrophobic interaction chromatography; size exclusion chromatography, metal-chelate chromatography; ultrafiltration / diafiltration; ethanol precipitation; precipitation of ammonium sulfate; chromathenkeque; displacement chromatography; electrophoretic procedures (in which are included but not limited to preparative isoelectric focusing), differential solubility (in which are included but not limited to precipitation of ammonium sulfate) or extraction. The apparent molecular weight can be estimated by GPC by comparison to globular protein standards (Preneta AZ, PROTEIN PURIFICATION METHODS, A PRACTICAL APPROACH (Harris and Angal, Eds.) - IRL Press 1989, 293-306). The purity of the unnatural amino acid polypeptide polypeptide: PEG can be determined by proteolytic degradation (in which are included but not limited to trypsin cleavage) followed by mass spectrometric analysis. Pepinsky RB. , et. a l. , J. Pharmcol & Exp. Ther. 297 (3): 1059-66 (2001). PEG coating of natural polypeptides by reductive alkylation of terminal amine moieties requires more time than the time required for PEG coating of an aromatic amine moiety over an unnatural amino acid. Additionally, the last PEG coating method is site specific and can occur at any site in the pclipotide where the non-natural amino acid has been incorporated. Figure 37 shows the PEG coating of a terminal amine of an MT-9 peptide using stoichiometric or stoichiometric proportions, at pH 5, requires at least 12 hours for a PEG coating of about 50% to occur. In contrast, Figure 38 shows a non-limiting example with PEG of a peptide containing an amino acid containing an aromatic amine moiety, which include but are not limited to pAF2 (p-amino-phenylalanine). In Fiqura 38, peptide MT-9 is coated with almost complete PEG with PEG 20k aldehyde or PEG 40k aldehyde in stoichiometric or quasi-stoichiometric proportions, at pH 4, after reacting for about one hour. This demonstrates the beneficial characteristics of the coating with PEG using reductive alkylation of a amino acid containing a portion of aromatic amine, such as selectivity (only the coating with PEG on the amino acid containing aromatic amine); fast reaction time (approximately one hour against 12 hours); stoichiometric or quasi-stoichiometric ratios in place of an excess of PEG aldehyde reagent (using thereby less reactive) and specificity (in contrast to only coating with N-terminal PEG, coating with PEG via reductive alkylation of an amino acid containing a portion of aromatic amine can occur anywhere in the peptide sequence). A non-limiting example of the PEG coating of hGH ("human growth hormone") is shown in Figure 39, wherein the tyrosine residue at position 35 of the hGH protein has been replaced translationally with an amino acid containing a portion of aromatic amine, which includes but is not limited to pAF2 (p-amino-phenylalanine), which is then seduced with a PEG containing an aldehyde portion. Also shown in Figure 39 is a non-limiting example of the PEG coating of IFNα, wherein an amino acid containing an aromatic amine moiety, in which but not limited to pAF2, is incorporated into the IFNα sequence and is subsequently seduced by a branched PEG containing an aldehyde moiety.

A water soluble polymer linked to an unnatural amino acid of a polypeptide described herein may be further derivatized or substituted without limitation.

E. Additional examples of modifications using site-specific derivation A non-limiting example of selective, site-specific derivation of polypeptides / proteins is shown in FIGS. 20-34, wherein the reductive alkylation of the aromatic amine portion, in which but not limited to pAF2 (p-amino-phenylalanine), occurs preferentially over other side chain groups such as, by way of example lysine, or any derivation of the end group. Such selective, site-specific derivation may allow the modification of polypeptides / proteins to design agonists and / or antagonists, the coating with specific peg of the polypeptide / protein site, prodrug design, polypeptide / protein glycosylation, polypeptide / protein dimerization , small molecule polypeptide / protein drug conjugates and small molecule drug conjugates of antibodies.

F. Enhancement of affinity for serum albumin Several molecules can also be fused to the non-natural amino acid polypeptides described in present to modulate half-life in the serum. In some embodiments, the molecules are linked or fused to the modified or unmodified unmodified amino acid polypeptides described herein to improve the affinity for endogenous serum albumin in an animal. For example, in some cases, a recombinant fusion of a polypeptide and an albumin binding sequence is made. Exemplary albumin binding sequences include, but are not limited to, the albumin binding domain of streptococcal G protein (see, eg, Makrides et al, J. Pharma col. Exp. Ther 277: 534-542 (1996 ) and Sjolander et al., J, Immunol. Methods 201: 115-123 (1997)), or albumin binding peptides such as those described in, for example, Dennos et al., J. Biol. Chem. 277: 35035-35043 (2002). In other embodiments, the modified or unmodified, non-natural amino acid polypeptides described herein are acylated with fatty acids. In some cases, fatty acids promote binding to serum albumin. See, for example, Kurtzhals et al., Biochem. J. 312: 725-731 (1995). In other embodiments, the modified or unmodified unnatural amino acid polypeptides described herein are fused directly with serum albumin (in which but not limited to, serum albumin is included) human) . Those of skill in the art will recognize that a wide variety of other molecules can also be linked to unnatural, modified or unmodified amino acid polypeptides, as described herein, to modulate the binding to serum albumin or other components of the serum .

G. Glycosylation of non-natural amino acid polypeptides described herein The methods and compositions described herein include polypeptides that incorporate one or more non-natural amino acids carrying saccharide residues. The saccharide residues may be either natural (in which, but not limited to, N-acetyl glucosamine) or unnatural (in which, but not limited to, 3-fluorogalactose). Saccharides can be bound to unnatural amino acids either through an N- or O-linked glycosidic bond (in which, but not limited to, N-acerylgalactose-L-serine) or a non-natural bond (in which include, but are not limited to, a heterocycle, in which a nitrogen-containing heterocycle, bond or the corresponding C- or S-linked glycoside is included). The saccharide portions (in which, but not limited to, glycosyl) can be added to the non-natural amino acid polypeptides either m vivo or m vi tro. In some embodiments, a polypeptide comprising a A non-natural amino acid containing aromatic amine is modified with a saccharide derived with an aldehyde group to generate the corresponding glycosylated polypeptide linked via an amine linkage. In other embodiments, a polypeptide comprising an unnatural amino acid containing protected aldehyde which, upon deprotection, is modified with a saccharide derived with an aromatic amine group to generate the corresponding glycosylated polypeptide linked via an amine linkage. Once attached to the non-natural amino acid, the saccharide can be further elaborated by treatment with glycosyltransferases and other enzymes to generate an oligosaccharide linked to the non-natural amino acid polypeptide. See, for example, H. Liu et al., J. Am. Chem. Soc. 125: 1702-1703 (2003).

H. Use of Linker Groups and Applications, Where Polypeptide Dimer and Multimer are Included In addition to adding functionality directly to the non-natural amino acid polypeptide, the unnatural amino acid portion of the polypeptide can first be modified with a multifunctional linker molecule (eg, example, bi-, tri, tetra-) which is subsequently modified further. That is, at least one end of the multifunctional linker molecule reacts with at least one non-natural amino acid in a polypeptide and the at least one other end of the Multifunctional linker is available for additional functionalization. If all the ends of the multifunctional linker are idyllic, then (depending on the stoichiometric conditions) homomultimers of the non-natural amino acid polypeptide can be formed. If the ends of the multifunctional linker have different chemical reactivities, then at least one end of the multifunctional linker group will be linked to the unnatural amino acid polypeptide and another end may subsequently react with a different functionality, in which are included by way of example only : a marker; a dye; a polymer; a water soluble polymer; a polyethylene glycol derivative; a photo-reticulator; a cytotoxic compound; a drug; a marker by affinity; a marker by photoaffinity; a reactive compound; a resin; a second protein or polypeptide or polypeptide analogue; an antibody or antibody fragment; a metal chelator; a cofactor; a fatty acid; a carbohydrate; a polynucleotide; a DNA; an RNA; an antisense polynucleotide; a saccharide, a water-soluble dendrimer, a cyclodextrin, a biomaterial; a nanoparticle; a rotating marker; a fluorophore, a portion containing metal; a radioactive portion; a novel functional group; a group that interacts covalently or non-covalently with other molecules; a photo-charged portion; an excitable portion of actinic radiation; a ligand; a portion photoisomerizable; biotin; a biotin analogue; a portion that incorporates a heavy atom; a chemically segmentable group; a photosegmentable group; an elongated side chain; a sugar linked to carbon; a redox active agent; an amino thioacid; a toxic portion; an isotopically labeled portion; a biophysical probe; a phosphorescent group; a chemo-luminescent group; a dense group of electrons; a magnetic group; an interleaving group; a chromophore; an agent that transfers energy; a biologically active agent; a detectable marker; a small molecule; an inhibitory ribonucleic acid; a radionucleotide; an agent that captures neutrons; a biotin derivative; quantum dot (s); a nanotransmitter; a radio transmitter; an abzyme, an activated complex activator, a virus, an adjuvant, an aglycan, an allergen, an angioestatin, an antihormone, an antioxidant, an aptamer, a grumed RNA, a shuttle vector, a macromolecule, a mimotope, a receptor , a reverse micelle and any combination thereof The multifunctional linker group has the general structure: wherein: each X is an aldehyde or an aromatic amine; each L is independently selected from the group consisting of alkylene, substituted alkylene, alkenylene, substituted alkenylene, -0-, -0- (alkylene or alkylene) substituted) -, -S-, -S- (alkylene or substituted alkylene) -, -S (0) k- where k is 1, 2 or 3, -S (O) k (alkylene or substituted alkylene) -, C (0) -, -C (0) - (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 (0) 0- (alkylene or alkylene Substituted) -, -0-CON (R ') - (alkylene or substituted alkylene) -, -CSN (R') -, -CSN (R ') - (alkylene or substituted alkylene) -, N (R') CO- (alkylene or substituted alkylene) -, -N (R ') C (0) 0-, -N (R') C (0) 0- (alkylene or Substituted alkylene) -, -S (0) kN (R ') -, N (R') C (0) N (R ') -, - N (R') C (0) N (R ') - (alkylene or substituted alkylene) -, -N (R ') C (S) N (R') -, - N (R ') S (O) icN (R') -, - N (R ') - N =, -C (R ') = N-, C (R') = NN (R ') -, -C (R') = NN =, -C (R ') 2-N = N- and - 25 C (R ') 2-N (R') -N (R ') -; wherein R 'is independently H, alkyl or substituted alkyl; Li is optional, and when present, is C (R ') P-NR' -C (0) 0- (alkylene or substituted alkylene) - where p is 0, 1 or 2; W is an aldehyde or an aromatic amine; and n is 1 to 3. The methods and compositions described herein also provide combinations of polypeptide, such as homodimers, heterodimers. homomultimers or heteromult (ie, trimers, tetramers, etc.). By way of example only, the following description focuses on the members of the GH supergene family, however, the methods, techniques and compositions described in this section can be applied to virtually any other polypeptide that can provide benefit in the form of dimers. and multimers, which include by way of example only: alpha-1-antitrypsin, angiostatin, antihemolytic factor, antibody, apolipoprotein, apoprotein, atrial natriuretic factor, atrial natriuretic polypeptide, atrial peptide, chemokine CXC, T39765, NAP-2 , ENA-78, qro-a, qro-b, gro-c, IP-10, GCP-2, NAP-4, SDF-I, PF4, MIG, calcitonin, c-kit ligand, cytokine, chemokine CC, protein -1 monocyte chemoattractants, monocyte chemoattractant protein-2, chemoattractant protein-3 monocyte, monocyte inflammatory alpha-1 protein, monocyte inflammatory beta-1 protein, RANTES, 1309, R83915, R91733, HCCl, T58847, D31065, T64262, CD40, CD40 ligand, c-team ligand, collagen, colony-stimulating factor (CSF), complement factor 5a, complement inhibitor, complement receptor 1, cytokine, peptide-78 that activates the epithelial neutrophil, MIP-16, MCP-I, epidermal growth factor (EGF), peptide that activates the neutrophil epithelial, erythropoietin (EPO), exfoliating toxin, Factor IX, Factor VII, Factor VIII, Factor X, fibroblast growth factor (FGF), fibrinogen, fibronectin, four-helix bundle protein, G-CSF, glp-1, GM -CSF, glucocerebrosidase, gonadotropin, growth factor, growth factor receptor, grf, hedgehog protein, hemoglobin, hepatocyte growth factor (hGF), hirudin, human growth hormone (hGH), human serum albumin, ICAM- I, ICAM-I receptor, LFA-I, LFA-I receptor, insulin, insulin-like growth factor (IGF), IGF-I, IGF-II, interferon (IFN), IFN-alpha, IFN-beta, IFN-gamma, any molecule similar to interferon or members of the IFN family, interleukin (IL), IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL- 12, keratinocyte growth factor (KGF), lactoferrin, leukemia inhibitory factor, luciferase, neurturin, neutrophil inhibitory factor (NIF), oncostatin M, osteogenic protein, product of oncogene, paracytonin, 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, receptor I of soluble supplement, soluble I-CAM 1, soluble interleukin receptor, soluble TNF receptor, somatomedin, somatostatin, somatotropin, streptokinase, superantigens, staphylococcal enterotoxin, SEA, SEB, SEC1, SEC2, SEC3, SED, SEE, hormone receptor spheroid, 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, receptor tumor necrosis factor (TNFR), VLA-4 protein, VCAM-I protein, vascular endothelial growth factor (VEGF), urokinase, mos, ras, raf, met, p53, tat, fos, myc, jun, myb, rei , estrogen receptor or, progesterone receptor, testosterone receptor, aldosterone receptor, LDL receptor and corticosterone. Thus, encompassed by the methods, techniques and compositions described herein are a polypeptide member of the GH supergene family containing one or more unnatural amino acids linker to another member of the GH supergene family or variants themselves or any other polypeptide that is not a member of the GH supergen family or variant thereof, either directly to the fundamental chain of the polypeptide or via a linker. Due to their increased molecular weight compared to the monomers, the dimer or multimer conjugates of the member of the GH supergene family may exhibit new desirable properties or properties, including, but not limited to, different pharmacological, pharmacokinetic, pharmacodynamic properties Modulated therapeutic half-life or plasma half-life modulated in relation to the member of the monomeric GH supergene family. In some embodiments, the dimers of the GH supergene family member described herein will modulate the dimerization of the member receptor of the GH supergene family. In other embodiments, the dimers or multimers of the member of the GH supergene family described herein will act as an antagonist, agonist or receptor modulator of the member of the GH supergene family. In some embodiments, the polypeptides of the GH supergene family member are directly linked, which include but are not limited to, via a bond of Asn-Lys amide or Cys-Cys disulfide bond. In some embodiments, the polypeptides of the linked GH supergene family member and / or the member of the non-GH linked supergen family will comprise different non-natural amino acids to facilitate dimerization, wherein include, but are not limited to, a first member of the GH supergene family and / or the member of the supergen family that is not GH stranded, a polypeptide comprising an unnatural amino acid containing aromatic amine conjugated to a second polypeptide of the member of the GH supergene family comprising an unnatural amino acid containing aldehyde and the polypeptides are reacted via reductive alkylation, forming an amine bond between the two. Alternatively, the two polypeptides the member of the GH supergene family and / or members of the non-GH supergene family are linked via a linker. Any hetero-or homo-bifunctional linker can be linked to link the two members of the GH supergene family and / or the member of the supergen family which is not GH linked, polypeptides, which may have the same or different sequence primary. In some cases, the linker used to hold the member of the GH supergene family, and / or the member of the supergen family that is not GH linked, polypeptides together can be a bifunctional PEG reagent. In some embodiments, the methods and compositions described herein provide water-soluble bifunctional linkers having a thickening structure that includes: a) an aldehyde-containing portion on at least one first end of a polymer backbone; and b) at least one second functional group on a second end of the polymer backbone. The second functional group may be the same or different as the first functional group. The second functional group, in some modalities, is not reactive with the first functional group. The methods and compositions described herein provide, in some embodiments, water soluble compounds comprising at least one arm of a branched molecular structure. For example, the branched molecular structure can be dendritic. In some embodiments, the methods and compositions described herein provide multimers that comprise one or more members of the GH supergene family formed by reactions with water-soluble activated polymers having the structure: R- (CH2CH20) n-0- ( CH2) mX wherein n is from about 5 to about 3,000, m is 2-10, X can be a carbonyl-containing portion (including an aldehyde) and R is a coronation 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, alkoxy, N-hydroxysucmimidyl ester, 1-benzotriazolyl ester, N-hydroxysuccinimidyl carbonate, carbonate 1-ben-otriazolyl, acetal, aldehyde, hydrates of aldehyde, 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.

I. Example of Aggregate Functionality: Facilitation of Isolate Properties of a Polypeptide An amino acid polypeptide that occurs stably in nature or unnatural amino acid polypeptide can be difficult to isolate from a sample for a variety of reasons, in the which include but are not limited to the solubility or polypeptide linkage characteristics. For example, in the preparation of a polypeptide for therapeutic use, such a polypeptide can be isolated from a recombinant system that has been designed to overproduce the polypeptide. However, due to the solubility or binding characteristics of the polypeptide, obtaining a desired level of purity often proves difficult. The methods, compositions, techniques and strategies described herein provide a solution to this situation. Using the methods, compositions, techniques and strategy described herein, an experienced in the art can produce an unnatural amino acid polypeptide containing alkylated amine that is homologous to the desired polypeptide, wherein the non-natural amino acid polypeptide containing alkylated amine has improved isolation characteristics. In one embodiment, a non-natural homologous amino acid polypeptide is produced biosynthetically. In a further embodiment or additional embodiment, the non-natural amino acid has incorporated into its structure one of the non-natural amino acids described herein. In a further embodiment or additional embodiment, the non-natural amino acid is incorporated in a terminal or internal position and is also specifically incorporated in the site. In one embodiment, the resulting non-natural amino acid, as produced biosynthetically, already has the desired improved isolation characteristics. In further embodiments or additional embodiments, the non-natural amino acid comprises an amine bond to a group that provides the improved isolation characteristics. In other embodiments or additional embodiments, the unnatural amino acid is further modified to form an unnatural amino acid polypeptide containing alkylated amine, wherein the modifications provide an amine bond to a group that provides the improved isolation characteristics. In some embodiments, such a group is directly linked to the non-natural amino acid, and in other embodiments, such a group is linked via a linker group to the non-natural amino acid. In certain embodiments, such a group is bound to the non-natural amino acid by a single chemical reaction, in other embodiments a series of chemical reactions is required to bind such a group to the non-natural amino acid. Preferably, the group that imparts improved isolation characteristics is specifically bound at the site to the non-natural amino acid in the unnatural amino acid polypeptide and is not linked to an amino acid that occurs stably in nature under the reaction conditions used. In other embodiments or additional embodiments, the resulting non-natural amino acid polypeptide is homologous to members of the GH supergene family, however, the methods, techniques and compositions described in this section can be applied to virtually any other polypeptide that can be benefit from improved isolation characteristics, which include by way of example only: alpha-1 antitrypsin, angiostatin, antihemolytic factor, antibody, apolipoprotein, apoprotein, atrial natriuretic factor, atrial natriuretic polypeptide, atrial peptide, chemokine CXC, T39765 , NAP-2, ENA-78, gro-a, gro-b, gro-c, IP-10, GCP-2, NAP-4, SDF-I, PF4, M1G, calcitonin, c-kit ligand, cytokine, CBC chemokine, monocyte chemoattractant protein-1, monocyte chemoattractant protein-2, chemoattractant protein-3 monocyte, monocyte inflammatory alpha-1 protein, monocyte inflammatory beta-1 protein, RANTES, 1309, R83915, R91733, HCCl, T58847, D31065, T64262, CD40, CD40 ligand, c-team ligand, collagen, colony-stimulating factor (CSF), complement factor 5a, complement inhibitor, complement receptor 1, cytokine, epithelial neutrophil activation peptide-78, MIP-16, MCP-I, epidermal growth factor (EGF), neutrophil activation peptide epithelial, erythropoietin (EPO), exfoliating toxin, Factor IX, Factor VII, Factor VIII ,. X factor, fibroblast growth factor (FGF), fibrinogen, fibronectin, four-helix 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-I, ICAM-I receptor, LFA-I, LFA-I receptor, insulin, factor of insulin-like growth (IGF), IGF-I, IGF-II, interferon (IFN), IFN-alpha, IFN-beta, IFN-gamma, any interferon-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-II, IL-I2, factor of keratinocyte growth (KGF), lactoferrin, leukemia inhibitory factor, luciferase, neurturin, neutrophil inhibitory factor (NIF), oncoestatin M, osteogenic protein, oncogene product, paracytonin, 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, biosynthetic protein small, soluble complement receptor 1, soluble I-CAM 1, soluble interleukin receptor, soluble TNF receptor, somatomedin, somatostatin, somatotropin, streptokinase, superantigens, staphylococcal enterotoxin, SEA, SEB, SEC1, SEC2, SEC3, SED, SEE Steroid hormone receptor, superoxide dismutase, toxic shock syndrome toxin, thymosin alpha 1, tissue plasminogen activator, tumor growth factor (TGF), tumor necrosis factor, tumor necrosis factor alpha, tumor factor tumor necrosis beta, tumor necrosis factor receptor (TNFR), VLA-4 protein, VCAM-I protein, vascular endothelial growth factor (VEGF), urokinase, mos, ras, raf, met, p53, tat, fos, myc , jun, myb, laugh, estrogen receptor, progesterone receptor, testosterone receptor, aldosterone receptor, LDL receptor and corticosterone. In other embodiments or additional embodiments, the group that imparts improved isolation characteristics improves the water solubility of the polypeptide; in other embodiments, the group improves the binding properties of the polypeptide; in other embodiments, the group provides novel binding properties to the polypeptide (in which are included, example only, a biotin group or a biotin linking group). In embodiments wherein the group improves the water solubility of the polypeptide, the group is selected from the water-soluble polymers described herein, wherein, by way of example only, any of the PEG polymer groups described herein are included. the present.

J. Example of Added Functionality: Detection of Presence of a Polypeptide An amino acid polypeptide that occurs stably in nature or unnatural amino acid polypeptides can be difficult to detect in a sample (in which a sample is included in vivo or an in vi tro sample) by a sample. a variety of reasons, including, but not limited to, the lack of a reagent or label that can be easily linked to the polypeptide. The methods, compositions, techniques and strategies described herein provide a solution to this situation. Using the methods, compositions, techniques and strategy described herein, one skilled in the art can produce an unnatural amino acid polypeptide containing alkylated amine that is homologous to the desired polypeptide, wherein the non-natural amino acid polypeptide containing amine The alkylation allows the detection of the polypeptide in an in vivo sample and an in vi tro sample. In a embodiment, a homologous non-natural amino acid polypeptide is produced biosynthetically. In a further embodiment or additional embodiment, the non-natural amino acid has incorporated into its structure the non-natural amino acids described herein. In a further embodiment or additional embodiment, the non-natural amino acid is incorporated into a terminal potion or internal position and is also specifically incorporated into the site. In one embodiment, the resulting non-natural amino acid, as produced biosynthetically, already has the desired detection characteristics. In other embodiments or additional embodiments, the non-natural amino acid comprises an amine bond to a group that provides the improved detection characteristics. In further embodiments or additional embodiments, the unnatural amino acid is further modified to form an unnatural amino acid polypeptide containing alkylated amine, wherein the modification provides an amine bond to a group that provides the improved detection characteristics. In some embodiments, such a group is directly linked to the non-natural amino acid and in other embodiments, such a group is linked via a linker group to the non-natural amino acid. In certain embodiments, such a group is bound to the non-natural amino acid by a single chemical reaction, in other embodiments a series of chemical reactions are required to bind the group to the non-natural amino acid. natural. Preferably, the group imparting the improved detection characteristics is specifically bound at the site to the non-natural amino acid in the non-natural amino acid polypeptide and is not linked to an amino acid that occurs stably in nature under the reaction conditions used . In other embodiments or additional embodiments the resulting non-natural amino acid polypeptide is homologous to members of the GH supergene family, however, the methods, techniques and compositions described in this section can be applied to virtually any other polypeptide that needs to be detected in an in vivo sample and an in vi tro sample, in which are included by way of example only: alpha-1 antihemolytic factor, antibody, apolipoprotein, apoprotein, atrial natriuretic factor, atrial natriuretic polypeptide, atrial peptide, chemokine CXC, T39765, NAP-2, ENA-78, gro-a, gro-b, gro-c, IP-10, GCP-2, NAP-4, SDF-I, PF4, MIG, calcitonin, c-kit ligand, cytokine , BCC chemokine, monocyte chemoattractant protein-1, monocyte chemoattractant protein-2, monocyte chemoattractant protein-3, monocyte inflammatory protein-1 alpha, monocyte inflammatory beta-1 protein, RANTES, 1309, R83915, R91733, HCCl , T5 8847, D31065, T64262, CD40, CD40 ligand, c-team ligand, collagen, colony stimulating factor (CSF), complement factor 5a, inhibitor of complement, complement 1 receptor, cytokine, epithelial neutrophil activation peptide-78, MIP-16, MCP-I, epidermal growth factor (EGF), epithelial neutrophil activation peptide, erythropoietic (EPO), exfoliating toxin , Factor IX, Factor VII, Factor VIII, Factor X, fibroblast growth factor (FGF), fibrinogen, fibronectm, four-helix bundle protein, G-CSF, glp-1, GM-CSF, glucocerebrosidase, gonadotropin, factor growth, growth factor receptor, grf, hedgehog protein, hemoglobin, hepatocyte growth factor (hGF), hirudma, human growth hormone (hGH), human serum albumin, ICAM-I, ICAM-I receptor, LFA- I, LFA-I receptor, insulin, insulin-like growth factor (IGF), IGF-I, IGF-II, interferon (IFN), IFN-alpha, IFN-beta, IFN-gamma, any molecule similar to myferferone or member of the IFN family, methylleukin (IL), IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10 , IL-II, IL-I2, Kerat growth factor ocito (KGF), lactoferma, leukemia inhibitory factor, luciferase, neurturma, neutrophil inhibitory factor (NIF), oncoestatma M, osteogenic protein, oncogene product, paracytonine, parathyroid hormone, PD-ECSF, PDGF, peptide hormone, pleiotropma, protein A, protein G, pth, pyrogenic exotoxin A, pyrogenic exotoxin B, pyrogenic exotoxam C, pyy, relaxma, reniña, SCF, small biostite protein, complement receptor 1 soluble, soluble I-CAM 1, soluble interleukin receptor, soluble TNF receptor, somatomedin, somatostatin, somatotropin, streptokinase, superantigens, staphylococcal enterotoxin, SEA, SEB, SEC1, SEC2, SEC3, SED, SEE, steroid hormone receptor , superoxide dismutase, toxic shock syndrome toxin, thymosin alpha 1, tissue plasminogen activator, tumor growth factor (TGF), tumor necrosis factor, tumor necrosis factor alpha, tumor necrosis factor beta, receptor tumor necrosis factor (TNFR), VLA-4 protein, VCAM-I protein, vascular endothelial growth factor (VEGF), urokinase, mos, ras, raf, met, p53, tat, fos, myc, jun, myb, rei , estrogen receptor, progesterone receptor, testosterone receptor, aldosterone receptor, LDL receptor and corticosterone. In other embodiments or additional modalities, the group imparting the improved detection characteristics is selected from the group consisting of a marker; a dye; a marker by affinity; a photoaffinity marker; a spin marker; a fluorophore; a radioactive portion; a portion that incorporates a heavy atom; an isotopically labeled portion; a biophysical probe; a phosphorescent group; a chemiluminescent group; a dense group of electrons; a magnetic group; a chromophore; an energy transfer agent; a detectable marker; and any combination of the previous ones.

K. Example of Adding Functionality: Improving the Therapeutic Properties of a Polypeptide An amino acid polypeptide that is stably present in the nature or non-natural amino acid polypeptide will act to provide a therapeutic benefit to a patient with a particular disorder, disease or condition. Such a therapeutic benefit will depend on a variety of factors, which include by way of example only: the safety profile of the polypeptide and the pharmacokinetic, pharmacological and / or pharmacodynamic characteristics of the polypeptide (eg, water solubility, bioavailability, mean serum life, therapeutic half-life, immunogenicity, biological activity or circulation time). In addition, it may be advantageous to provide additional functionality to the polypeptide, such as an attached compound or cytotoxic drug, or it may be desirable to append additional polypeptides to form the homo- and heteromultimers described herein. Such modifications preferably do not destroy the activity and / or tertiary structure of the original polypeptide. The methods, compositions, techniques and strategy described here provide solutions to these issues. Using the methods, compositions, techniques and As described herein, those skilled in the art can produce an unnatural amino acid polypeptide containing alkylated amine that is homologous to the desired polypeptide, wherein the non-natural amino acid polypeptide containing alkylated amine has improved therapeutic characteristics. In one embodiment, a homologous non-natural amino acid polypeptide is biosynthetically produced. In a further embodiment or additional embodiment, the non-natural amino acid has incorporated into its structure one of the non-natural amino acids described in. the present. In a further embodiment or additional embodiment, the non-natural amino acid is incorporated into a terminal or internal function and is also incorporated specifically into the site. In one embodiment, the resulting non-natural amino acid, as produced biosynthetically, already has the desired improved therapeutic characteristics. In other embodiments or additional embodiments, the non-natural amino acid comprises an amine bond to a group that provides the improved therapeutic characteristics. In further embodiments or additional embodiments, the unnatural amino acid is further modified to form an unnatural amino acid polypeptide containing alkylated amine, wherein the purification provides an amine bond to a group that provides the improved therapeutic characteristics. In some embodiments, such a group is directly linked to the non-natural amino acid and in other embodiments, such a group is linked via a linker group to the non-natural amino acid. In certain embodiments, such a group is bound to the non-natural amino acid by a single chemical reaction, in other embodiments a series of chemical reactions is required to bind such a group to the non-natural amino acid. Preferably, the group imparting improved therapeutic characteristics is specifically bound at the site to the non-natural amino acid in the unnatural amino acid polypeptide and is not linked to an amino acid that is stably present in nature under the reaction conditions used. In other embodiments or additional embodiments the resulting non-natural amino acid polypeptide is homologous to members of the GH supergene family, however, the methods, techniques and compositions described in this section can be applied to virtually any other polypeptide that can be benefit from the improved therapeutic features, which include by way of example only: alpha-1 antihemolytic factor, antibody, apolipoprotein, apoprotein, atrial natriuretic factor, atrial natriuretic polypeptide, atrial peptide, chemokine CXC, T39765, NAP-2, ENA-78, gro-a, gro-b, gro-c, IP-10, GCP-2, NAP-4, SDF-I, PF4, MIG, calcitonin, c-kit ligand, cytokine, chemokine CCO, protein- 1 monocyte chemoattractant, monocyte chemoattractant protein-2, protein-3 monocyte chemoattractant, monocyte inflammatory alpha-1 protein, monocyte inflammatory beta-1 protein, RANTES, 1309, R83915, R91733, HCCl, T58847, D31065, T64262, CD40, CD40 ligand, c-team ligand, collagen, stimulating factor of colony (CSF), complement factor 5a, complement inhibitor, complement receptor 1, cytokine, epithelial neutrophil activation peptide-78, MIP-16, MCP-I, epidermal growth factor (EGF), activation peptide of epithelial neutrophil, erythropoietin (EPO), exfoliating toxin, Factor IX, Factor VII, Factor VIII, Factor X, fibroblast growth factor (FGF), fibrinogen, fibronectin, four-helix bundle protein, G-CSF, glp-1 , GM-CSF, glucocerebrosidase, gonadotropin, growth factor, growth factor receptor, grf, hedgehog protein, hemoglobin, hepatocyte growth factor (hGF), hirudin, human growth hormone (hGH), human serum albumin, ICAM-I, receiver IC AM-I, LFA-I, LFA-I receptor, insulin, insulin-like growth factor (IGF), IGF-I, IGF-II, interferon (IFN), IFN-alpha, IFN-beta, IFN-gamma, any molecule similar to interferon 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-II, IL-I2, keratinocyte growth factor (KGF), lactoferrin, leukemia inhibitory factor, luciferase, neurturin, neutrophil inhibitory factor (NIF), oncoestatin M, protein osteogenics, oncogene product, paracytoma, parathyroid hormone, PD-ECSF, PDGF, peptide hormone, pleiotropin, protein A, protein G, pth, pyrogenic exotoxin A, pyrogenic exotoxin B, pyrogenic exotoxin C, pyy, relaxma, reniña, SCF, small therapeutics bios, soluble complement receptor 1, soluble I-CAM 1, soluble mterleukin receptor, soluble TNF receptor, somatomed a, somatostatin, somatotropin, streptoqumase, superantigens, staphylococcal enterotoxin, SEA, SEB, SEC1, SEC2, SEC3 , SED, SEE, steroid hormone receptor, superoxide dismutase, toxic shock syndrome toxin, alpha 1 timosma, 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-I protein, vascular endothelial growth factor (VEGF), urokinase, mos, ras, raf, met, p53, tat , fos, myc, jun, myb, laugh, estrogen receptor, progesterone receptor, testosterone receptor, aldosterone receptor, LDL receptor and corticosterone. In other embodiments or additional modalities, the group imparting improved therapeutic characteristics improves the water solubility of the polypeptide; in other embodiments, the group improves the binding properties of the polypeptide; In other modalities, the group provides new properties of linkage to the polypeptide (in which are included, by way of example only, a biotin group or a biotin linking group). In embodiments wherein the group improves the water solubility of the polypeptide, the group is selected from the water-soluble polymers described herein, in which only the PEG polymer groups are included as examples. In other modalities or additional modalities, the group is a cytotoxic compound, while in other modalities the group is a drug. In additional embodiments, the linked group or cytotoxic compound can be excised from the non-natural amino acid polypeptide to deliver the drug or cytotoxic compound to a desired therapeutic site. In other embodiments, the group is a second polypeptide, which includes, by way of example, an unnatural amino acid polypeptide containing alkylated amine, which further includes, by way of example, a polypeptide having the same amino acid structure as the first non-natural amino acid polypeptide. In further embodiments or additional embodiments, the non-natural amino acid polypeptide containing alkylated amine is an unnatural amino acid polypeptide containing modified alkylated amine. In other embodiments or additional embodiments, the non-natural amino acid polypeptide containing alkylated amine increases the bioavailability of the polypeptide relative to the amino acid polypeptide that occurs stably in the homologous nature. In other embodiments or additional embodiments, the non-natural amino acid polypeptide containing alkylated amine increases the safety profile of the polypeptide relative to the amino acid polypeptide that is stably present in the homologous nature. In further embodiments or embodiments, the non-natural amino acid polypeptide containing alkylated amine increases the water solubility of the polypeptide relative to the amino acid polypeptide that is stably present in the homologous nature. In other embodiments or additional embodiments, the non-natural amino acid polypeptide containing alkylated amine increases the therapeutic half-life of the polypeptide relative to the amino acid polypeptide that is stably present in the homologous nature. In other embodiments or additional embodiments, the non-natural amino acid polypeptide containing alkylated amine increases the half-life in the serum of the polypeptide relative to the amino acid polypeptide that is stably present in the homologous nature. In other embodiments or additional embodiments, the non-natural amino acid polypeptide containing alkylated amine prolongs the circulation time of the polypeptide relative to the amino acid polypeptide that is stably present in the homologous nature. In other modalities or additional modalities, the polypeptide of A non-natural amino acid containing alkylated amine modulates the biological activity of the polypeptide in relation to the amino acid polypeptide that is presented in a stanol manner in the homologous nature. In other embodiments or additional embodiments, the non-natural amino acid polypeptide containing alkylated amine modulates the immunogenicity of the polypeptide in relation to the amino acid polypeptide that is stably present in the homologous nature.

XI. Therapeutic Uses of Modified Polypeptides For convenience, the "modified or unmodified" non-natural polypeptides described in this section have been described generically and / or with specific examples. However, the "modified or unmodified" unnatural polypeptides described in this section should not be limited to only the generic descriptions or specific examples provided in this section, but rather the unmodified "modified or unmodified" polypeptides described in this section. section applies equally well to "unmodified or modified" non-natural polypeptides that comprise at least one amino acid that falls within the scope of the Formulas (A) - (E) and (I) - (XVI), in which any specific sub-formulas or compounds are included that fall within the scope of Formulas (A) - (E) and (I) - (XVI ) that are described in the specification, claims and figures herein.

The modified or unmodified non-natural amino acid polypeptides described herein, wherein homo- and hetero-multimers thereof are included, find multiple uses, including, but not limited to: therapeutic uses, diagnostics, base of analysis, industrial, cosmetics, plant biology, environmental, energy production and / or military. As a non-limiting illustration, the following therapeutic uses of modified and unmodified non-natural amino acid polypeptides are provided. The modified or unmodified non-natural amino acid polypeptides described herein are useful for treating a wide range of disorders, conditions or diseases. Administration of the modified or unmodified non-natural amino acid polypeptide products described herein results in any of the activities demonstrated by the preparations of polypeptides commercially available in humans. The average amounts of the modified or unmodified modified amino acid polypeptide product may vary and in particular should be based on the recommendations and prescription of a qualified physician. The exact amount of the modified or unmodified non-natural amino acid polypeptide is a matter of preference subject to factors such as the exact type of condition being treated, the condition of the patient who is treated, as well as the other ingredients in the composition. The amount to be used can be easily determined by one skilled in the art based on therapy with the modified or unmodified modified non-natural amino acid polypeptide.

A. Administration and Pharmaceutical Compositions The "non-modified or unmodified" non-natural amino acid polypeptides described herein, wherein homo- and hetero-multimers thereof are found to be used in multiple ways, including, but not limited to, : therapeutic use, diaknostic, based on analysis, industrial, cosmetic, plant biology, environmental, energy production and / or military. As a non-limiting illustration, the following therapeutic uses of the "modified or unmodified" non-natural amino acid polypeptides are provided. The "modified or unmodified" non-natural amino acid polypeptides described herein are useful for treating a wide range of alterations. The administration of the "modified or unmodified" unnatural amino acid polypeptides described herein results in any of the activities demonstrated by the preparations of polypeptides commercially available in humans. The average amounts of the polypeptide product of Non-natural amino acid "modified or unmodified" may vary and in particular should be based on the recommendations and prescription of a qualified doctor. The exact amount of the "modified or unmodified" non-natural amino acid polypeptide is a matter of preference subject to factors such as the exact type of condition being treated, the condition of the patient being treated, as well as the other ingredients in the composition . The amount to be given can easily be determined by one skilled in the art based on therapy with the "modified or unmodified" non-natural amino acid polypeptide. Polypeptides of non-natural, modified or unmodified amino acids, as described herein (in which, but not limited to, synthetases, proteins comprising one or more non-natural amino acids, etc.) are optionally employed for therapeutic uses, including, but not limited to, in combination with an appropriate pharmaceutical carrier . Such compositions, for example, comprise a therapeutically effective amount of the unnatural, modified or unmodified amino acid polypeptides, as described herein, and a pharmaceutically acceptable carrier or excipient. Such a carrier or excipient includes, but is not limited to, saline, pH regulated saline, dextrose, water, glycerol, ethanol and / or combinations thereof. The formulation is made appropriate to the mode of administration. In general, protein delivery methods are well known in the art and can be applied to the administration of unnatural, modified or unmodified amino acid polypeptides, as described herein. Therapeutic compositions comprising one or more of the unnatural, modified or unmodified amino acid polypeptides, as described herein are optionally tested in one or more models of amical disease m vi tro and / or vivo, aoropiados to confirm efficacy, tissue metabolism and to estimate dosages, according to methods well known in the art. In particular, the dosages may be determined micially by activity, stability or other appropriate measures of non-natural to natural amino acid homologs (including, but not limited to, a comparison of a modified polypeptide to include one or more non-natural amino acids). a natural amino acid polypeptide), that is, in relevant analysis. Administration by any of the routes normally used to introduce a molecule in intimate contact with blood or tissue cells. The unnatural, modified or unmodified amino acid polypeptides, as described herein, are administered in any appropriate manner, optionally with one or more acceptable carriers. pharmaceutically Suitable methods of administration of the unnatural, modified or unmodified amino acid polypeptides, as described herein, to a patient are available, and, although more than one route may be used to administer a particular composition, a particular route may be used. frequently provide an immediate or more selective action or reaction from another route. The pharmaceutically acceptable carriers are determined in part by the particular composition that is administered, as well as by the particular method used to administer the composition. Thus, there is a wide variety of suitable formulations of pharmaceutical compositions described herein. The polypeptides of non-natural amino acids described herein and compositions comprising such polypeptides can be administered by any conventional route appropriate for proteins or peptides, which include, but are not limited to, parenterally, for example injections that include , but not limited to, subcutaneously or intravenously or any other form of injections or infusions. Pharmaceutical compositions of polypeptides comprising non-natural amino acid polypeptides described herein, can be administered by a variety of routes in which they include, but are not limited to, oral, intravenous, intraperitoneal, intramuscular, transdermal, subcutaneous, topical, sublingual or rectal media. Compositions comprising unnatural, modified or unmodified amino acid polypeptides, as described herein, may also be administered via liposomes. Such routes of administration and appropriate formulations are generally known to those skilled in the art. The non-natural amino acid polypeptides described herein may be used alone or in combination with other appropriate components, including, but not limited to, a pharmaceutical carrier. Unnatural, modified or unmodified amino acid polypeptides, as described herein, alone or in combination with other appropriate components, can also be made into aerosol formulations (ie, they can be "nebulized") to be administered via inhalation. The aerosol formulations can be placed in acceptable pressurized propellants, such as dichlorodifluoromethane, propane, nitrogen and the like. Appropriate formulations for parenteral administration, such as, for example, by intraarticular (in the joints), intravenous, intramuscular, intradermal, intraperitoneal and subcutaneous routes, include sterile aqueous and non-aqueous isotonic injection solutions, which may contain anti-oxidants, regulatory solutions of pH, bacteriostats and solutes that return to the isotonic formulation with the blood of the proposed container, and sterile aqueous and non-aqueous suspensions which may include suspending agents, solubilizers, thickening agents, stabilizers and preservatives. Packaged nucleic acid formulations can be presented in sealed multi-dose unit dose containers, such as ampoules and flasks. Parenteral administration and intravenous administration are preferred administration methods. In particular, the administration routes already in use for homologous therapeutics of natural amino acids (in which are included but not limited to, those commonly used for EPO, IFN, GH, G-CSF, GM-CSF, IFNs, interleukins, antibodies and / or any other pharmaceutically administered protein), together with formulations in current use, provide preferred routes of administration and formulation for unnatural, modified or unmodified amino acid polypeptides, as described herein. The dose administered to a patient, in the context of compositions and methods described herein, is sufficient to have a beneficial therapeutic response in the patient with the passage of time. Dosages are determined by the efficacy of the particular formulation and the activity, stability or half-life in the serum of the unnatural, modified or unmodified amino acid polypeptides, employees in the condition of the patient, also as body weight or surface area of the patient to be treated. The size of the dose is also determined by the existence, nature and extent of any adverse side effects that accompany the administration of a particular formulation, or the like in a particular patient. By determining the effective amount of the formulation to be administered in the treatment or prophylaxis of disease (including, but not limited to, cancers, hereditary diseases, diabetes, AIDS or the like), the physician evaluates circulating plasma levels , formulation toxicities, disease progression and / or where relevant, the production of anti-non-natural amino acid polypeptide antibodies. The dose administered, for example, to a patient of 70 kilograms is typically in the range equivalent to dosages of therapeutic proteins currently used, adjusted for the altered activity or serum half-life of the relevant composition. The pharmaceutical formulations described herein may supplement treatment conditions by any known conventional therapy, in which they include antibody administration, administration of vaccine, administration of cytotoxic agents, natural amino acid polypeptides, nucleic acids, nucleotide analogs, modifiers of answer biological and the like. For administration, the pharmaceutical formulations described herein are administered at a rate determined by the LD-50 or ED-50 of the relevant formulation and / or observation of any side effects of unnatural, modified or unmodified amino acid polypeptides, at various concentrations, which include but are not limited to, as they are applied to the mass and overall health of the patient. The administration can be carried out via a single dose or divided doses. If a patient suffering from an infusion of a formulation develops fever, chills or muscle pain, he receives the appropriate dose of aspirin, ibuprofen, acetaminophen or another drug that controls pain / fever. Patients who experience infusion reactions such as fever, muscle aches and chills are premedicated 30 minutes before future infusions with either aspirin, acetaminophen, or, in, but not limited to, diphenhydramine. Meperidine is used for chills and more severe muscle pains that do not respond quickly to antipyretics and antihistamines. The cellular infusion is stopped or discontinued depending on the severity of the reaction. Non-natural, modified or unmodified amino acid polypeptides, as described herein, can be administered directly to a mammalian subject. The administration is by any of the routes normally used to introduce a polypeptide to a subject. Unnatural, modified or unmodified amino acid polypeptides, as described herein, include those suitable for oral, rectal, topical, inhalation (including, but not limited to, an aerosol), buccal (in those which include, but are not limited to, sublingual), vaginal, parenteral (including, but not limited to, subcutaneous, intramuscular, intradermal, intrarenal, mtrapleural, medial, medial, medial, or intravenous), topical (ie , both skin and mucosal surfaces, in which airway surfaces are included) and transdermal administration, although the most appropriate route in any case will depend on the nature and severity of the condition being treated. The administration can be either local or systemic. The formulations may be presented in sealed unit dose or multiple dose containers, such as ampoules or flasks. Unnatural, modified or unmodified amino acid polypeptides, as described herein, can be prepared in an injectable unit dosage form (in which they include but are not limited to, solution, suspension or emulsion) with an acceptable carrier pharmaceutically Polypeptides of non-natural, modified or unmodified amino acids, as described in present, they can also be administered by continuous infusion (in which are included but not limited to, pumps such as osmotic pumps), single bolus deposit formulations or slow release tank combinations. Formulations suitable for administration include aqueous and non-aqueous solutions, sterile isotonic solutions, which may contain anti-oxidants, pH-regulating solutions, bacteriostats and solutes that return to the isotonic formulation and sterile aqueous and non-aqueous suspensions which may include suspending agents, solubilizers, thickening agents, stabilizers and preservatives. The solutions and suspensions can be prepared from sterile powders, granules and tablets of the kind previously described. Freeze drying is a technique commonly used to present proteins that serve to remove water from the protein preparation of interest. Chilled by freezing or freeze-drying, it is a process by which the material to be dried is frozen first and then the ice or frozen solvent is removed by sublimation in a vacuum environment. An excipient can be included in the pre-lyophilized formulations to improve the stability during the freeze drying process and / or to improve the stability of the lyophilized product in storage.

Pikal, M. Biopharm. 3 (9) 26-30 (1990) and Arakawa et al., Pharm. Res. 8 (3): 285-291 (1991). Pharmaceutical spray drying is also known to those of ordinary skill in the art. For example, see Broadhead, J. et al., "The Spray Dryinq of Pharmaceuticals", in Drug Dev. Ind. Pharm, 18 (11 &12), 1169-1206 (1992). In addition to small molecule pharmaceuticals, a variety of biological materials have been spray dried and these include: enzymes, sera, plasma, micro-organisms and yeasts. Spray drying is a useful technique because it can convert a liquid pharmaceutical preparation to a fine powder, free of powder or agglomerate in a one-step process. The basic technique comprises the following four steps: a) atomization of the feed solution to a spray; b) air atomization contact; c) atomization drying; and d) separation of the dry product from the atomization air. U.S. Patent Nos. 6,235,710 and 6,001,800, which are incorporated herein by reference in their entirety, describe the preparation of recombinant erythropoietin by spray drying. The pharmaceutical compositions described herein may comprise a pharmaceutically acceptable carrier, excipient or stabilizer. The pharmaceutically acceptable carriers are determined in part by the particular composition that is administered, as well as by the particular method used to administer the composition. Thus, there is a wide variety of suitable formulations of pharmaceutical compositions (in which pharmaceutically optional acceptable carriers, excipients or stabilizers are included) for the unnatural, modified or unmodified amino acid polypeptides, described herein, (see, for example. , in Remmgton: The Sci ence and Practice of Pharma cy, Nmeteenth Ed (Easton, Pa .: Mack Publishing Company, 1995), Hoover, John E., Remington's Pharma ceuti ca l Sciences, Mack Publpshmg Co., Easton , Pennsylvania 1975; Liberman, HA and Lachman, L., Eds., Pharmaceuti cal Dosage Forms, Marcel Decker, New York, NY, 1980; and Clinical Pharma Dosage Forms and Drug Delivery Sys tems, Seventh Edition. (Lippmcott Williams & Wilkins, 1999)). Suitable carriers include pH regulating solutions containing succinate, phosphate, borate, HEPES, citrate, imidazole, acetate, bicarbonate and other organic acids; antioxidants which include, but are not limited to, ascorbic acid; low molecular weight polypeptides which include but are not limited to those less than about 10 residues; proteins, which include, but are not limited to, serum albumin, gelatin or immunoglobulins; hydrophilic polymers including, but not limited to, polyvinylpyrrolidone; amino acids which include, but are not limited to, glycine, glutamine, asparagma, argmam, histidine or histidine, metionma, glutamate or lysine derivatives; monosaccharides, disaccharides and other carbohydrates, which include but are not limited to, trehalose, sucrose, glucose, mannose or dextrm; chelating agents including, but not limited to, EDTA and disodium edentate; divalent metal ions in which, but not limited to, zinc, cobalt or sachet; sugar alcohols which include, but are not limited to, mannitol or sorbitol; against salt-forming ions in which, but not limited to, sodium is included; and / or non-ionic surfactants, wherein they are included but not limited to Tween ™ (including, but not limited to, Tween 80 (polysorbate 80) and Tween 20 (polysorbate 20), Pluron ™ csMR and other pluronic acids, wherein, but not limited to, and other pluronic acids, including, but not limited to, pluronic acid F68 (poloxamer 188) or PEG, suitable surfactants include, but are not limited to, poly ( ethylene oxide) -poly (propylene oxide) -poly (ethylene oxide), that is, (PEO-PPO-PEO) or poly (propylene oxide) -poly (ethylene oxide) -poly (propylene oxide) , ie, (PPO-PEO-PPO) or a combination of the mimes, PEO-PPO-PEO and PPO-PEO-PPO are commercially available under the tradenames Pluromcs ™, R-Pluron ™ csMR, Tetron ™ cs ™ and R- Tetron ™ csMR (BASF Wyandotte Corp., Wyandotte, Mich.) And are further described in U.S. Patent No. 4,820,352 incorporated herein by reference in its entirety. Other ethylene / polypropylene block polymers may be suitable surfactants. A surfactant or a combination of surfactants can be used to stabilize non-natural amino acid polypeptides coated with PEG against one or more stresses including, but not limited to, stresses resulting from agitation. Some of the above can be determined as "volume agents". Some may also be determined as "tonicity modifiers". Antimicrobial preservatives can also be applied for product stability and antimicrobial effectiveness; Suitable preservatives include but are not limited to, benzyl alcohol, benzalkonium chloride, metacresol, methyl / propyl) paraben, cresol and phenol or a combination thereof. Non-natural, modified or unmodified amino acid polypeptides, as described herein, in which those linked to water-soluble polymers such as PEG are included can also be administered by or as part of the sustained release system. Sustained release compositions, which include but are not limited to, semi-permeable polymer matrices in the form of shaped articles, which include but are not limited to, films or microcapsules. Sustained-release matrices include biocompatible materials such as poly (2-hydroxyethyl methacrylate) (Langer et al., J. Biomed, Ma ter. Res., 15: 267-277 (1981); Langer, Chem. Tech., 12: 98-105 (1982), ethylene acetate of vinyl (Langer et al., supra) or poly-D- (-) -3-hydroxybutyric acid (EP 133,988), polylactides (polylactic acid) (U.S. Patent No. 3,773,919; EP 58,881), polyglycolide (glycolic acid polymer) , co-glycolide polylactide (copolymers of lactic acid and glycolic acid) polyanhydrides, copolymers of L-glutamic acid t gamma-ethyl-L-glutamate (U. Sidman et al., Biopolymers, 22, 547-556 (1983), poly ( ortho) esters, polypeptides, hyaluronic acid, collagen, chondroitin sulfate, carboxylic acids, fatty acids, phospholipids, polysaccharides, nucleic acids, polyamino acids, amino acids such as phenylalanine, tyrosine, isoleucine, polynucleotides, polyvinyl propylene, polyvinyl pyrrolidone and silicone. sustained release tamó They also include a liposomally entrapped compound. The liposomes containing the compound are prepared by methods known per se: DE 3,218,121; Eppstein et al., Proc. Na ti. Acad. Scí E. U. A , 82: 3688-3692 (1985); Hwang et al., Proc. Na ti. Acad. Sci. E. U. A , 77: 4030-4034 (1980); EP 52,322; EP 36,676; EP 143,949; Japanese Patent Application 83-118008; U.S. Patent Nos. 4,485,045, 4,619,794, 5,021,234 and 4, 544, 545; and EP 102, 324.

The liposomally entrapped polypeptides can be prepared by methods described in, for example, DE 3,218,121; Eppstein et al., Proc. Na ti. Acad. Sci E. U. A , 82: 3688-3692 (1985); Hwang et al., Proc. Na ti. Acad. Sci E. U. A , 77: 4030-4034 (1980); EP 52,322; EP 36,676; EP 143,949; Japanese Patent Application 83-118008; U.S. Patent Nos. 4,485,045, 4,619,794, 5,021,234 and 4,544,545; and EP 102,324. The composition and size of liposomes are well known or are apt to be easily revealed empirically by one skilled in the art. Some examples of liposomes as described in, for example, Park JW, et al., Proc. Na ti. Acad. Sci. USA 92: 1327-1331 (1995); Lasic D and Papahadjopoulos D (eds): MEDICAL APPLICATIONS OF LIPOSOMES (1998); Drummond DC et al., Liposomal drug delivery systems for cancer therapy, in Teicher B (ed): CANCER DRUG DISCOVERY AND DEVELOPMENT (2002); Park JW et al., Cl in. Cancer Res. 8: 1172-1181 (2002); Nielsen UB and collaborators, Biochim. Biophys. Acta 1591 (1-3): 109-118 (2002); Mamot C et al. Cancer Res. 63: 3154-3161 (2003). The dose administered to a patient in the context of the compositions, formulations and methods described herein, should be sufficient to elicit a beneficial response in the subject over time. In general, the total pharmaceutically effective amount of non-natural, modified or unmodified amino acid polypeptides, such as are described herein, parenterally administered per dose is in the range of about 0.01 μg / kg / day to about 100 μg / kg, or about 0.05 mg / kg to about 1 mg / kg of the patient's body weight, although this is subject to therapeutic discretion. The dosage frequency is also subject to therapeutic discretion and may be more frequent or less frequent than commercially available products approved for human use. In general, a polymer: polypeptide conjugate, in which are included by way of example only, a PEG coated polypeptide, as described herein, can be administered by any of the administration routes described above. In some embodiments, there is found a method for the treatment of an alteration, condition or disease comprising administering a therapeutically effective amount of a polypeptide comprising at least one unnatural amino acid selected from the group consisting of: wherein each Rn is independently selected from the group consisting of H, halogen, alkyl, -N02, -CN, substituted alkyl, -N (R ') 2, -C (0) kR', -C (0) N (R ') 2, -OR' and -S (0) kR ', where k is 1, 2 or 3; M is H or -CH2R5; or the portion M-N-C (R5) can form a ring structure of 4 to 7 members; Ri is H, an amino protecting group, resin, amino acid, polypeptide or polynucleotide; R2 is OH, an ester, ream, amino acid, polypeptide or polynucleotide protecting group; R5 is alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkyl, alkoxy, substituted alkoxy, alkylalkoxy, substituted alkylalkoxy, polyalkylene oxide, substituted polyalkylene oxide, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl , heterocycle, substituted heterocycle, substituted alkaryl alkaryl, aralkyl, substituted aralkyl, -C (0) R ", C (0) 0R", -C (0) N (R ") 2, -C (0) NHCH (R 'J 2, - (alkylene or substituted alkylene) -N (R ") 2, - (alkenylene or substituted alkenylene) -N (R'J 2, - (alkylene or substituted alkylene) - (aryl or substituted aryl), - (alkenylene or substituted alkenylene) - (substituted aryl or aryl), - (substituted alkylene or alkylene) -ON (R'J 2, - (alkylene or substituted alkylene) -C (0) SR ", - (alkylene or alkylene replaced) -S- S- (aryl or substituted aryl), wherein each R "is independently hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocycle, substituted heterocycle, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl or C (O) OR '; is LX, wherein, X is a group selected from the group consisting of a label, a dye, a polymer, a water soluble polymer, a polyethylene glycol derivative; a photoreticulator, a cytotoxic compound, a drug, a marker by affinity, a marker by photoaffinity, a reactive compound, a resin, a second protein or polypeptide or polypeptide analogue, an antibody or antibody fragment, a metal chelate; cofactor, a fatty acid, a carbohydrate, a polynucleotide, a DNA, an RNA, an antisense polynucleotide, a saccharide, a water-soluble dendrimer, a cyclodextrin, a biomaterial, a nanopart icle, a spin marker; a fluorophore, a portion containing metal; a radioactive portion; a novel functional group; a group that interacts covalently or non-covalently with other molecules; a photo-charged portion; an excitable portion of actinic radiation; a ligand; a photoisomerizable portion; biotin; a biotin analogue; a portion that incorporates a heavy atom; a group chemically excised him; a photocleavable group; an elongated side chain; a sugar linked to carbon; a redox active agent; an amino thioacid; non-toxic portion; an isotopically labeled portion; a biophysical probe; a phosphorescent group; a chemiluminium group; a dense group of electrons; a magnetic group; an interleaving group; a chromophore; an agent that transfers energy; a biologically active agent; a detectable marker; a small molecule; an inhibitory ribonucleic acid; a radionucleotide; a neutron capture agent; a biotin derivative; quantum dot (s); a nanotransmitter; aradiotransmitter; an enzyme, an activated complex activator, a virus, an adjuvant, an aglycan, an allergen, an angioestatin, an antihormone, an antioxidant, an aptamer, a grumed RNA, a saponin, a shuttle vector, a macromolecule, a mimotope , a receptor, a reverse micella and any combination thereof; and L is optional, and when present is a linker selected from the group consisting of alkylene, substituted alkylene, alkenylene, substituted alkenylene, -O-, -O- (alkylene or substituted alkylene) -, - (alkylene or substituted alkylene) -O-, -C (0) -, -C (0) - (alkylene or substituted alkylene) -, - (alkylene or substituted alkylene) -C (0) -, -C (0) N (R ') - , -C (0) N (R ') - (alkylene or substituted alkylene) -, - (alkylene or substituted alkylene) -C (0) N (R '), -OC (0) N (R') -, -OC (0) N (R ') - (alkylene or alkylene substituted) -, - (alkylene or substituted alkylene) -0C (0) N (R ') -, -N (R') C (0) -, -NR 'C (0) - (alkylene or substituted alkylene) - , - (alkylene or substituted alkylene) -NR 'C (0) -, -S-, -S- (alkylene or substituted alkylene) -, -S (0) k- where k is 1, 2 or 3, -S (0) (alkylene or substituted alkylene) -, -C (S) -, -C (S) - (alkylene or substituted alkylene) -, -CSN (R ') -, -CSN (R') - (alkylene or substituted alkylene) -, -N (R ') CO- (alkylene or substituted alkylene) -, -N (R') C (0) 0-, - (alkylene or substituted alkylene) -0-N = CR '-, - (alkylene or substituted alkylene) - C (0) NR '- (alkylene or substituted alkylene) -, - (alkylene or substituted alkylene) -S (0) k- (alkylene or substituted alkylene) -S-, - (alkylene) or substituted alkylene) -SS-, -S (0) kN (R), -N (R ') C (O) N (R') -, -N (R ') C (S) N (R') -, N (R ') S (0) kN (R), -N (R') - N =, -C (R ') = N-, -C (R') = NN (R ') ) -, C (R ') = N-N =, -C (R') 2-N = N- and -C (R ') 2-N (R') -N (R ') -; or R5 and any R3 optionally forms a cycloalkyl or a heterocycloalkyl; and each R 'is independently H, alkyl or substituted alkyl with the proviso that when Ri is H then R2 is not OH, or when R2 is OH then Ri is not H. In other embodiments a method is found for the treatment of a alteration, condition or illness, that further comprises administering a pharmaceutically acceptable carrier with the therapeutically effective amount of the polypeptide. In further embodiments a method for the treatment of an alteration, condition or disease is found, wherein Ri and R2 are both polypeptides. In some embodiments, a method for the treatment of an alteration, condition or disease is found, wherein X is a water-soluble polymer. In other embodiments, a method for the treatment of an alteration, condition or disease is found, wherein X is a derivative of polyethylene glycol. In a further embodiment there is found a method for the treatment of an alteration, condition or disease, wherein X is a cytotoxic compound. In some embodiments, a method is found for the treatment of an alteration, condition or disease, wherein X is a drug. In some embodiments, a method for the treatment of an alteration, condition or disease is found, wherein X is a second polypeptide. In other embodiments, there is found a method for the treatment of an alteration, condition or disease, wherein the second polypeptide is a peptide that contains an unnatural amino acid polypeptide. In some embodiments, a method for the treatment of an alteration, condition or disease is found, wherein the polypeptide is a protein homologous to a protein.

Therapeutics selected from the group consisting of: alpha-1 antihemolytic factor, antibody, apolipoprotein, apoprotein, atrial natriuretic factor, atrial natriuretic polypeptide, atrial peptide, chemokine CXC, T39765, NAP-2, ENA-78, gro-a, gro- b, gro-c, IP-10, GCP-2, NAP-4, SDF-I, PF4, MIG, calcitonin, c-kit ligand, cytokine, CCO chemokine, monocyte chemoattractant protein-1, chemoattractant protein-2 monocyte, monocyte chemoattractant protein-3, monocyte inflammatory alpha-1 protein, monocyte inflammatory beta-1 protein, RANTES, 1309, R83915, R91733, HCCl, T58847, D31065, T64262, CD40, CD40 ligand, PC ligand , Collagen, Colony Stimulating Factor (CSF), Complement Factor 5a, Complement Inhibitor, Complement Receptor 1, Cytokine, Epithelial Neutrophil Activation Peptide-78, MIP-16, MCP-I, Epidermal Growth Factor (EGF) ), epithelial neutrophil activation peptide, erythropoietin (EPO) Exfoliating toxin, Factor IX, Factor VII, Factor VIII, Factor X, Fioblasto growth factor (FGF), fibrinogen, fibronectin, four-helix 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-I, ICAM-I receptor , LFA-I, LFA-I receptor, insulin, insulin-like growth factor (IGF), IGF-I, IGF-II, interferon (IFN), IFN-alpha, IFN-beta, IFN-gamma, any molecule similar to interferon 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-II, IL- I2, keratinocyte growth factor (KGF), lactoferrin, leukemia inhibitory factor, luciferase, neurturin, neutrophil inhibitory factor (NIF), oncoestatin M, osteogenic protein, oncogene product, paracytonine, 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 1, soluble I-CAM 1, soluble interleukin receptor, soluble TNF receptor, somatomedin, somatostatin, somatotropin, streptokinase, superantíqenos, staphylococcal enterotoxin, SEA, SEB, SEC1, SEC2, SEC 3, SED, SEE, steroid hormone receptor, superoxide dismutase, toxic shock syndrome toxin, thymosin alpha 1, tissue plasminogen activator, tumor growth factor (TGF), tumor necrosis factor, necrosis factor tumor, beta tumor necrosis factor, tumor necrosis factor receptor (TNFR), VLA-4 protein, VCAM-I protein, vascular endothelial growth factor (VEGF), urokinase, mos, ras, raf, met, p53, tat, fos, myc, jun, myb, laugh, receiver of estrogen, progesterone receptor, testosterone receptor, aldosterone receptor, LDL receptor and corticosterone. In other embodiments, a method for the treatment of an alteration, condition or disease is found, wherein the at least one non-natural amino acid is incorporated into a specific site within the polypeptide. In some embodiments, a method for the treatment of an alteration, condition or disease is found, wherein the polypeptide is synthesized by a ribosome. In some embodiments, there is found a method for the treatment of an alteration, condition or disease, wherein the polypeptide comprising at least one unnatural amino acid is stable in aqueous solution for at least 1 month. In other embodiments, a method for the treatment of an alteration, condition or disease is found, wherein the polypeptide comprising at least one unnatural amino acid is stable for at least 2 weeks. In some embodiments, a method for the treatment of an alteration, condition or disease is found, wherein the polypeptide comprising at least one unnatural amino acid is stable for at least 5 days. In a further embodiment there is found a method for detecting the presence of a polypeptide in a patient, the method comprising administering an effective amount of a homologous non-natural amino acid polypeptide comprising at least one non-natural amino acid selected from the group consisting of: wherein each Ra is independently selected from the group consisting of H. halogen, alkyl, -N02, -CN, substituted alkyl, -N (R ') 2, -C (0) kR', -C (0) N ( R ') 2, -OR' and -S (0) kR ', where k is 1, 2 or 3; M is H or -CH2R5; or the portion M-N-C (Rs) can form a ring structure of 4 to 7 members; Ri is H, an amino protecting group, resin, amino acid, polypeptide or polynucleotide; R2 is OH, an ester, resin, amino acid, polypeptide or polynucleotide protecting group; R5 is alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, alkylalkoxy, substituted alkylalkoxy, polyalkylene oxide, substituted polyalkylene oxide, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl , heterocycle, substituted heterocycle, alkaryl, alkaryl substituted, aralkyl, substituted aralkyl, -C (0) R ", C (0) OR", -C (0) N (R'J 2, -C (0) NHCH (R ") 2, - (alkylene or substituted alkylene) -N (R'J 2, - (alkenylene or substituted alkenyl) -N (R'J 2, - (alkylene or substituted alkylene) - (aryl or substituted aryl), - (substituted alkenylene or alkenylene) - (substituted aryl or aryl), - (alkylene or substituted alkylene) -ON (R ") 2, - (alkylene or substituted alkylene) -C (0) SR ", - (alkylene or substituted alkylene) -SS- (substituted aryl or aryl), wherein each R" is independently hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, alkoxy substituted, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocycle, substituted heterocycle, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl, or -C (0) 0R '; or R5 is L-X, where X is a group selected from the group consisting of a label; a dye; a polymer; a water soluble polymer; a polyethylene glycol derivative; a photo-reticulator; a cytotoxic compound; a drug; a marker by affinity; a marker by photoaffinity; a reactive compound; a resin; a second protein or polypeptide or polypeptide analogue; an antibody or antibody fragment; a metal chelate; a cofactor; a fatty acid; a carbohydrate; a polynucleotide; a DNA; an RNA; an antisense polynucleotide; a saccharide, a dendrimer soluble in water, a cyclodextrin, a biomaterial; a nanoparticle; a spin marker; a fluorophore, a portion containing metal; a radioactive portion; a new functional group; a group that interacts covalently or non-covalently with other molecules; a photo-charged portion; an excitable portion of actinic radiation; a ligand; a photoisomerizable portion; biotin; a biotin analogue; a portion that incorporates a heavy atom; a group chemically excised him; a photocleavable group; an elongated side chain; a sugar linked to carbon; a redox active agent; an amino thioacid; a toxic portion; an isotopically labeled portion; a biophysical probe; a phosphorescent group; a chemiluminescent group; a dense group of electrons; a magnetic group; an interleaving group; a chromophore; an agent that transfers energy; a biologically active agent; a detectable marker; a small molecule; an inhibitory ribonucleic acid; a radionucleotide; a neutron capture agent; a biotin derivative; quantum dot (s); a nanotransmitter; a radio transmitter; an abzyme, an activated complex activator, a virus, an adjuvant, an aglycan, an allergen, an angioestatin, an antihormone, an antioxidant, an aptamer, a guided RNA, a saponin, a shuttle vector, a macromolecule, a mimotope , a receiver, a reverse micelle and any combination thereof; and L is optional, and when present is a linker selected from the group consisting of alkylene, substituted alkylene, alkenylene, substituted alkenylene, -O-, -O- (alkylene or substituted alkylene) -, -O (alkylene or substituted alkylene) ) -O-, C (O) -, -C (0) - (alkylene or substituted alkylene) -, (alkylene or substituted alkylene) -C (O) -, -C (0) N (R ') -, C (O) N (R ') - (alkylene or substituted alkylene) -, - (alkylene or substituted alkylene) -C (O) N (R'), -OC (O) N (R ') -, OC ( 0) N (R ') - (alkylene or substituted alkylene) -, - (alkylene or substituted alkylene) -OC (O) N (R') -, -N (R ') C (0) -, -NR' C (O) - (alkylene or substituted alkylene) -, - (alkylene or substituted alkylene) -NR 'C (0) -, -S-, -S- (alkylene or substituted alkylene) -, -S (0) k - where k is 1, 2 or 3, S (0) (alkylene or substituted alkylene) -, -C (S) -, -C (S) - (alkylene or substituted alkylene) -, -CSN (R ') - , -CSN (R ') - (alkylene or substituted alkylene) -, -N (R') CO- (alkylene or substituted alkylene) -, -N (R ') C (0) 0-, - (alkylene or substituted alkylene) -0-N = CR '-, - (alkylene or substituted alkylene) -C (0) NR' - (alkylene or substituted alkylene) -, (alkylene or substituted alkylene) -S (0) k- (alkylene or substituted alkylene) -S-, - (alkylene or substituted alkylene) -SS-, -S (0) kN (R '), -N (R') C (0) N (R ') - , N (R ') C (S) N (R') -, 0-N (R ') S (0) kN (R') -, -N (R ') - N =, -C (R') ) = N-, -C (R ') = NN (R), -C (R') = NN =, -C (R ') 2-N = N- and -C (R') 2-N ( R ') - N (R ') -; or R5 and any Rn optionally forms a cycloalkyl or a heterocycloalkyl; each R 'is independently H, alkyl or substituted alkyl; and with the proviso that when Ri is H then R2 is not OH, or when R2 is OH then Ri is not H. In other embodiments a method is found for detecting the presence of a polypeptide in a patient wherein at least A non-natural amino acid is incorporated at a specific site within the polypeptide. In some embodiments, a method for detecting the presence of a polypeptide in a patient is found, wherein the non-natural amino acid is incorporated using a translation system. In some embodiments, a method for detecting the presence of a polypeptide in a patient is found, wherein the non-natural amino acid is incorporated into the polypeptide using a translation system and a post-translation modification system. In other embodiments, a method for detecting the presence of a polypeptide in a patient is found, wherein the at least one non-natural amino acid is stable in aqueous solution for at least 1 month. In some embodiments, there is found a method for detecting the presence of a polypeptide in a patient, wherein the at least one unnatural amino acid is stable for at least 2 weeks. In other modalities there is a method to detect the presence of a polypeptide in a patient, wherein the at least one unnatural amino acid is stable for at least 5 days. In some embodiments, a method for detecting the presence of a polypeptide in a patient is found, wherein the polypeptide is a protein homologue to a therapeutic protein selected from the group consisting of: ** alpha-1 antihemolytic factor, antibody, apolipoprotein, apoprotein, atrial natriuretic factor, atrial natriuretic polypeptide, atrial peptide, chemokine CXC, T39765, NAP-2, ENA-78, gro-a, gro-b, gro-c, IP-10, GCP-2, NAP-4, SDF-I, PF4, MIG, calcitonin, c-kit ligand, cytokine, chemokine CCO, monocyte chemoattractant protein-1, monocyte chemoattractant protein-2, monocyte chemoattractant protein-3, monocyte-inflammatory alpha-1 protein, protein -1 monocyte inflammatory beta, RANTES, 1309, R83915, R91733, HCCl, T58847, D31065, T64262, CD40, CD40 ligand, c-team ligand, collagen, colony stimulating factor (CSF), complement factor 5a, inhibitor of complement, complement receptor 1, cytokine , epithelial neutrophil activation peptide-78, MIP-16, MCP-I, epidermal growth factor (EGF), epithelial neutrophil activation peptide, erythropoietin (EPO), exfoliating toxin, Factor IX, Factor VII, Factor VIII, X factor, fibroblast growth factor (FGF), fibrinogen, fibronectin, four-helix bundle protein, G-CSF, glp-1, GM-CSF, glucocerebrosidase, gonadotrop a, growth factor, growth factor receptor, grf, hedgehog protein, hemoglobin, hepatocyte growth factor (hGF), hirudin, human growth hormone (hGH), human serum albumin, ICAM-I, ICAM-I receptor, LFA-I, LFA-I receptor, insulin, insulin-like growth factor (IGF), IGF-I, IGF-II, interferon (IFN), IFN-alpha, IFN-beta, IFN-gamma , any molecule similar to mterferone or member of the IFN family, methylleucine (IL), IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-II, IL-I2, keratinocyte growth factor (KGF), lactoferrin, leukemia inhibitory factor, luciferase, neurturin, neutrophil inhibitory factor (NIF), oncoestatin M, osteogenic protein, product of oncogene, parathomonas, paratrroid hormone, PD-ECSF, PDGF, peptide hormone, pleiotropma, protein A, proterna G, pth, pyrogenic exotoxin A, pyrogenic exotoxin B, pyrogenic exotoxin C, pyy, relaxma, reniñ a, SCF, small biostatic protein, soluble complement receptor 1, soluble I-CAM 1, soluble metherleucine receptor, soluble TNF receptor, somatomed a, somatostatic, somatotropy, streptokinase, superantigen, staphylococcal enterotox, SEA, SEB, SEC1, SEC2, SEC3, SED, SEE, steroid hormone receptor, superoxide dismutase, toxic shock syndrome toxin, alpha 1 timosma, tissue plasminogen activator, tumor growth factor (TGF), tumor necrosis factor, tumor necrosis factor alpha, tumor necrosis factor beta, tumor necrosis factor receptor (TNFR), protein VLA-4, protein VCAM-I, vascular endothelial growth factor (VEGF), urokinase, mos, ras, raf, met, p53, tat, fos, myc, jun, myb, rei, estrogen receptor, progesterone receptor, testosterone receptor, aldosterone receptor, LDL receptor and corticosterone.

EXAMPLES The following examples describe methods for synthesis of amino acids containing aromatic amine.

Example: Synthesis of 3-chloro-4-amino-phenylalanine The 3-chloro-4-amino-phenylalanine was synthesized according to the method of FIG. eleven.

Example Ib: Synthesis of 3-iodo-4-amino-phenylalanine The 3-iodo-4-amino-phenylalanine was synthesized according to the method of FIG. eleven.

Example: Synthesis of 3-methoxy-4-amino-phenylalanine The 3-methoxy-4-amino-phenylalanine was synthesized according to the method of FIG. 12 Example Id: Synthesis of 3-fluoro-4-amino-phenylalanine The 3-fluoro-4-amino-phenylalanine was synthesized according to the method of FIG. 12 Example: Synthesis of N-methyl-p-amino-phenylalanine The N-methyl-p-amino-phenylalanine was synthesized according to the method of FIG. 13 Example If: Synthesis of N-ethyl-p-amino-phenylalanine N-ethyl-p-amino-phenylalanine was synthesized according to the method of FIG. 13. The following examples describe methods for the cloning and expression of a modified polypeptide followed by post-translational modification.

Example 2: PEG coating of hGH This example details the cloning and expression of a modified polypeptide in E. coli An introduced translation system comprising an orthogonal tRNA (O-tRNA) and an orthogonal amino acid tRNA N synthetase (O-RS) is used to express the polypeptide containing an unnatural amino acid.

O-RS preferentially aminoacylates O-tRNA with a non-natural amino acid. In turn, the translation system inserts the non-natural amino acid into the polypeptide, in response to a coding selector codon. The transformation of E. coli with plasmids containing the modified gene and the aminoacyl tARN synthetase / orthogonal rRNA pair (specific for the desired non-natural amino acid) allows site-specific incorporation of the non-natural amino acid into the polypeptide. The E Col i transformed, cultured at 37 ° C in a medium containing 0.01-100 mM of the particular non-natural amino acid, expresses the modified polypeptide with high fidelity and efficiency. The His-tagged polypeptide containing an unnatural amino acid is produced by E host cells. col i as inclusion bodies or aggregates. The aggregates are solubilized and purified by affinity under denaturing conditions in 6 M guanidine HCl. The folded damage is carried out by dialysis, at 4 ° C overnight in 50 mM TRIS-HC1, pH 8.0, 40 uM CUS04 and 2 Sarcosine. % (p / v). The material is then dialyzed against 20 mM TRIS-HC1, pH 8.0, 100 mM NaCl, 2 mM CaCl2, followed by the removal of His-tag. See Boissel et al., (1993) 268: 15983-93. Methods for purification of polypeptides are well known in the art and are confirmed by SDS-PAGE analysis, Western Blot or electro-ionization-ionization trap and mass spectrometry and the like. By way of example, the hGH polypeptide with p-aminophenylalanine substituted by tyrosine at position 35 (H6-hGH Y35pAF2) was generated in E cells. col i using constructs that code for hGH and a pair of tRNA synthetase orthogonal-orthogonal tRNA for the non-natural amino acid. The expressed protein was purified using IMAC and IEX chromatography. The protein was dialyzed to 10 mM Sodium Phosphate, glycine 20 g / L, mannitol 5 g / L, pH 7.0 and then concentrated to 350 uM. The pH of the sample was adjusted to pH 4.0 using 10% acetic acid. The post-translation modification of H6-hGH Y35pAF2 was demonstrated by coating H6-hGH Y35pAF2 by reductive alkylation of p-aminophenylalan with several PEG-aldehydes. A non-limiting process for such a coating with PEG is summarized in FIG. 39 and is described herein as a sequence: coating reactions with PEG 100 ul were adjusted using PEG 20 K, 30 K and 40 K aldehydes. The PEG aldehyde was a buffer solution of 20 mM acetate pH 4.0. Each reaction had a molar ratio of 1: 1 of the PEG to protein, and a molar ratio of 5: 1 of NaCNBH3 solubilized in DMF: protein. The reactions were incubated either at 4 degrees or at room temperature and were analyzed by SDS-PAGE after 3, 4, 6 and 16 hrs. Gel electrophoresis of the resulting peptides are shown in FIG. 40. The following examples describe methods for modifying polypeptides by post-translational modification as shown in FIGS. 20-34, where X is p-aminophenylamine.

Example 3a: Reducing alkylations of urotensin-II (UT-H-SH) with propionaldehyde and benzaldehyde. A mixture of 3 equivalents of NaBCNH3 at pH 4 was added to a mixture of 0.25 mM propionaldehyde (or 0.25 mM benzaldehyde) and 0.25 mM reduced urotensin and allowed to react for 2 hours. The analysis of the product mixture was by HPLC with the following conditions: 0-50% of B (A, 0.05% of TFA in water, B, 60% of acetonitrile and 0.05% of TFA in water), flow rate: 1.5 mL / min; column: Zorbax Extend C 18, 3.5 m, 4.6x.50mm.

Example 3b: Reducing alkylations of urotensin-II (UT-II) with propionaldehyde and benzaldehyde A mixture of 3 equivalents of NaBCNH3 at pH 4 was added to a mixture of 0.25 mM propionaldehyde (or 0.25 mM benzaldehyde) and 0.25 mM urotensin and Allows it to react for 2 hours. The analysis of the product mixture was by HPLC with the following conditions: 5-60% of B (A, 0.05% of TFA in water, B, 60% of acetonitrile and 0.05% of TFA in water), flow rate: 1.5 mL / min; column: Zorbax Extend C 18, 3.5 m, 4.6x50 mm.

Example 3c: Reducing Alkylations of the XT-S Peptide with Propionaldehyde, Benzaldehyde, Isobutaldehyde and Pivalaldehyde A mixture of 3 equivalents of NaBCNH3 at pH 4 was added to a mixture of 0.25 mM propionaldehyde (or 0.25 mM benzaldehyde or 0.25 mM isobutaldehyde or pivalaldehyde 0. 25 mM) and XT-8 0.25 mM is allowed to react for 2 hours. The analysis of the product mixture was by HPLC with the following conditions: 5-60% of B (A, 0.05% of TFA in water, B, 60% of acetonitrile and 0.05% of TFA in water), flow rate: 1.5 mL / min; column: Zorbax Extend C 18, 3.5 m, 4. 6x50 mm Example 3d: Reducing alkylations of the SXT-9 peptide with propionaldehyde, benzaldehyde, isobutaldehyde and pivalaldehyde A mixture of 3 equivalents of NaBCNH3 at pH 4 was added to a mixture of 0.25 mM propionaldehyde (or 0.25 mM benzaldehyde, or 0.25 mM isobutaldehyde or 0.25 pivalaldehyde. mM) and SXT-9 0.25 mM are allowed to react during 2 hours. The analysis of the product mix was through HPLC with the following conditions: 5-60% B (A, 0.05% of TFA in water; B, 60% acetonitrile and 0.05% TFA in water), flow rate: 1.5 mL / min; column: a Zorbax Extend C 18, 3.5 m, 4.6x50 mm.

Example 3e: Reducing Alkylations of the HXT-9 Peptide with Propionaldehyde, Benzaldehyde, Isobutaldehyde and Pivalaldehyde A mixture of 3 equivalents of NaBCNH3 at pH 4 was added to a mixture of 0.25 mM propionaldehyde (or 0.25 mM benzaldehyde, or 0.25 mM isobutaldehyde or pivalaldehyde 0. 25 mM) and 0.25 mM HXT-9 is allowed to react for 2 hours. The analysis of the product mixture was by HPLC with the following conditions: 5-60% of B (A, 0.05% of TFA in water, B, 60% of acetonitrile and 0.05% of TFA in water), flow rate: 1.5 mL / min; column: Zorbax Extend C 18, 3.5 m, 4. 6x50 mm Example 3f: Reducing alkylations of WXT-9 peptide with propionaldehyde, benzaldehyde and isobutaldehyde A mixture of 3 equivalents of NaBCNH3 at pH 4 was added to a mixture of 0.25 mM propionaldehyde (or 0.25 mM benzaldehyde, or 0.25 mM isobutaldehyde) and WXT-9 0.25 mM is allowed to react for 2 hours. The analysis of the product mixture was by HPLC with the following conditions: 5-60% of B (A, 0.05% of TFA in water, B, 60% of acetonitrile and 0.05% of TFA in water), flow rate: 1.5 mL / min; column: Zorbax Extend C18, 3.5 m, 4.6x50 mm.

Example 3g: Reducing Alkylations of NXT-9 Peptide with Propionaldehyde and Benzaldehyde A mixture of 3 equivalents of NaBCNH3 at pH 4 was added to a mixture of 0.25 mM propionaldehyde (or 0.25 mM benzaldehyde) and 0.25 mM NXT-9 is allowed to react for 2 hours. The analysis of the product mixture was by HPLC with the following conditions: 5-60% of B (A, 0. 05% of TFA in water; B, 60% acetonitrile and 0.05% TFA in water), flow rate: 1.5 mL / min; column: Zorbax Extend C 18, 3.5 m, 4.6x50 mm.

Example 3h: Reducing Alkylations of the RXT-10 Peptide with Propionaldehyde or Benzaldehyde A mixture of 3 equivalents of NaBCNH3 at pH 4 was added to a mixture of 0.25 mM propionaldehyde (or 0.25 mM benzaldehyde) and 0.25 mM RXT-10 is allowed to react for 2 hours. The analysis of the product mixture was by HPLC with the following conditions: 5-60% of B (A, 0.05% of TFA in water, B, 60% of acetonitrile and 0.05% of TFA in water), flow rate: 1.5 mL / min; column: Zorbax Extend C 18, 3.5 m, 4.6x50 mm.

Example 3i: Reducing Alkylations of the AXT-11 Peptide with Propionaldehyde or Benzaldehyde A mixture of 3 equivalents of NaBCNH3 at pH 4 was added to a mixture of 0.25 mM propionaldehyde (or 0.25 mM benzaldehyde) and 0.25 mM AXT-11 is allowed to react for 2 hours. The analysis of the product mixture was by HPLC with the following conditions: 5-60% of B (A, 0.05% of TFA in water, B, 60% of acetonitrile and 0.05% of TFA in water), flow rate: 1.5 mL / min; column: Zorbax Extend C 18, 3.5 m, 4.6x50 mm.

Example 3j: Reducing Alkylations of the AXT-11 Peptide with 3-phenylpropanal, 2-phenylacetaldehyde or cinnamaldehyde A mixture of 3 equivalents of NaBCNH3 at pH 4 was added to a mixture of 0.25 mM 3-phenylpropanal (or 0.25 mM 2-phenylacetaldehyde or cinnamaldehyde 0.25 mM) and AXT-11 0.25 mM is allowed to react for 5 hours. The analysis of the product mixture was by HPLC with the following conditions: 5-60% of B (A, 0.05% of TFA in water, B, 60% of acetonitrile and 0.05% of TFA in water), flow rate: 1.5 mL / min; column: Zorbax Extend C 18, 3.5 m, 4.6x50 mm.

Example 3k: Reducing Alkylations of the AXT-11 Peptide with lH-imidazole-5-carbaldehyde, thiophen-2-carbaldehyde, picolinaldehyde or quinoline-4-carbaldehyde A mixture of 3 equivalents of NaBCNH3 at pH 4 was added to a mixture of 0.25 mM lH-imidazole-5-carbaldehyde (or 0.25 mM thiophen-2-carbaldehyde or 0.25 mM picolinaldehyde, or 0.25 mM quinolin-4-carbaldehyde) and 0.25 mM AXT-11 is allowed to react for 5 hours . The analysis of the product mixture was by HPLC with the following conditions: 5-60% of B (A, 0.05% of TFA in water, B, 60% of acetonitrile and 0.05% of TFA in water), flow rate: 1.5 mL / min; column: Zorbax Extend C 18, 3.5 m, 4.6x50 mm.

Example 31: Reductive alkylations of the peptide AXT-11 with benzaldehyde, 1-phenylbutan-1,3-dione or a mixture of benzaldehyde and 1-phenylbutan-1,3-dione A mixture of 3 equivalents of NaBCNH3 at pH 4 was added to a mixture of 0.20 mM benzaldehyde, or 0.20 mM 1-phenylbutan-1, 3-dione or a mixture of benzaldehyde 0. 20 mM and 0.20 mM 1-phenylbutan-1,3-dione) and 0.20 mM AXT-11 are allowed to react for 2 hours. The analysis of the product mixture was by HPLC with the following conditions: -60% B (A, 0.05% TFA in water, B, 60% acetonitrile and 0.05% TFA in water), flow rate: 1.5 mL / min; column: Zorbax Extend C 18, 3.5 m, 4.6x50 mm.

Example 3m: Reductive alkylations of peptide NXT-9 with benzaldehyde, 1-phenylpropan-1,2-dione or a mixture of benzaldehyde and 1-phenylpropan-1,2-dione A mixture of 3 equivalents of NaBCNH3 at pH 4 was added to a mixture of 0.20 mM benzaldehyde, (or 0.20 mM 1-phenylpropan-1,2-dione or a mixture of 0.20 mM benzaldehyde and 0.20 mM 1-phenylpropan-1,2-dione) and 0.20 mM NXT-9 is allowed to react for 2 hours. hours. The analysis of the product mixture was by HPLC with the following conditions: 5-60% of B (A, 0.05% of TFA in water, B, 60% of acetonitrile and 0. 05% of TFA in water), flow rate: 1.5 mL / min; column: Zorbax Extend C 18, 3.5 m, 4.6x50 mm.

Example 3n Reducing alkylations of the peptide MXT-9 with benzaldehyde, acetophenone, a mixture of benzaldehyde and acetophenone, a mixture of benzaldehyde and propionaldehyde, or a mixture of benzaldehyde and butan-2-one A mixture of 3 equivalents of NaBCNH3 at pH 4 was added to a mixture of 0.20 mM benzaldehyde, (or acetophenone) 0. 20 mM or a mixture of 0.20 mM benzaldehyde and 0.20 mM acetophenone, or a mixture of 0.20 mM benzaldehyde and 0.20 mM propionaldehyde or a mixture of 0.20 mM benzaldehyde and 0.20 mM butan-2-one) and 0.20 mM MXT-9 is allowed react for 2 hours. The analysis of the product mixture was by HPLC with the following conditions: 5-60% B (A, 0.05% TFA in water, B, 60% acetonitrile and 0.05% TFA in water), flow rate: 1.5 mL / min; column: Zorbax Extend C 18, 3.5 m, 4.6x50 mm.

Example 3o: Reduction of the peptide MXT-9-N3 followed by reductive alkylations of the peptide MXT-9-NH2 with propionaldehyde or benzaldehyde A mixture of MXT-9-N3 was reduced with TCEP to give the peptide MXT-9-NH2. A mixture of 3 db NaBCNH3 equivalents at pH 4 was added to a mixture of 0.20 mM propionaldehyde, (or 0.20 mM benzaldehyde) 0.20 mM MXT-9-NH2 is allowed to react for 1 hour. The analysis of the product mixture was by HPLC with the following conditions: 5-60% of B (A, 0.05% of TFA in water, B, 60% of acetonitrile and 0.05% of TFA in water), flow rate: 1.5 mL / min; column: Zorbax Extend C 18, 3.5 m, 4.6x50 mm. The following examples describe methods for measuring and comparing the in vi tro and in vivo activity of a modified therapeutically active unnatural amino acid polypeptide of a therapeutically active natural amino acid polypeptide.

Example 4: Measurement of the activity and affinity of the unnatural amino acid polypeptide This example details the measurement of the activity and affinity of the unnatural amino acid polypeptide of unnatural amino acid polypeptides in terms of their receptor, binding partner or ligand. The protein for the non-natural amino acid polypeptide receptor, binding partner or ligand is expressed and isolated according to methods known to those of ordinary skill in the art. The B? OcoreMR system is used to analyze the binding of the unnatural amino acid polypeptide to its receptor. Similarly, a link partner or ligand can be used in ecte analysis. Approximately 600-800 RUs of the soluble receptor are immobilized on a CM5 chip of B? Acore ™, using a standard amine coupling procedure, as recommended by the manufacturer. Various concentrations of the unnatural amino acid polypeptide and by wild type (or modified) in pH buffer HBS-EP (B? Acore ™, Pharmacia) are injected onto the surface at a flow rate of 40 μl / rnin for 4-5. minutes and the dissociation was monitored for 15 minutes post-injection. The surface is regenerated by a 15 second pulse of 4.5 M MgCl2. Only a minimal loss of binding affinity (1-5%) is observed after at least 100 regeneration cycles. A reference cell without immobilized receptor is used to provide any volume effect of regulatory solution and non-specific binding.

Kinetic linkage data obtained from unnatural amino acid polypeptide (modified) titration experiments are processed with the programming elements of BiaEvaluation 4.1 (BIACOREMR). Equilibrium dissociation constants (Kd) are calculated as proportions of rate constants (kapagado / kencend? Do) • Stable cell lines are established that express receptor, binding partner or ligand for the non-natural amino acid polypeptide. The cells are subjected to electroporation with a construct containing the receptor, link partner or liganao cDNA. The transfected cells are allowed to recover for 48 hours before cloning. Transfectants that express receptor, binding partner or ligand are identified by surface staining with antibody against the receptor and are analyzed on a FACS Array (BD Biosciences, San Diego, CA). Stably transfected cell clones are established after repeated rounds of repeated subcloning of the desired transfectants. Such cells are used in cell binding analysis. The cells (3xl06) are incubated in duplicate in PBS / 1% BSA (100 μl) in the absence or presence of various concentrations (volume: 10 μl) of unlabeled natural amino acid polypeptide or a negative control polypeptide and in the presence of non-natural amino acid polypeptide 125I (modified) (approximately 100,000 cpm or 1 ng) at 0 ° C for 90 minutes (total volume: 120 μl). The cells are then resuspended and certified on 200 μl of ice cold FCS in a centrifuged 350 μl plastic centrifuge tube (1000 g; 1 minute) . The pellet is collected by cutting the end of the tube and the pellet and supernatant counted separately in a gamma counter (Packard). The specific link (cpm) is determined as the total link in the absence of a competitor (mean of duplicates) minus the non-specific link. The non-specific binding is measured for each of the types of cells used. The experiments are run on separate days using the same 125I- (modified) non-natural amino acid polypeptide preparation and must exhibit internal consistency. The unnatural amino acid polypeptide 125I- (modified) demonstrates binding to the cells that produce receptor, binding protein or ligand. The linkage is inhibited in a dose-dependent manner by the unlabeled unnatural amino acid polypeptide, but not by a negative control polypeptide.

Example 5: In vivo studies of the therapeutically active modified non-naturally occurring amino acid polypeptide The therapeutically active modified non-naturally occurring amino acid polypeptide, amino acid polypeptide Therapeutically active unnatural and pH regulating solution are administered to mice or rats. The results will show superior activity and prolonged half-life of the modified therapeutically active unnatural amino acid polypeptide compared to the therapeutically active natural amino acid polypeptide.

Example 6: Measurement of the Average Life in Vivo of the modified therapeutically active non-natural amino acid polypeptide conjugated and without .Conjugar and variants thereof. All animal experimentation is facilitated in a facility accredited by AAALAC and under the protocols approved by the Institutional Animal Care and Use Committee of the University of St. Louis The rats are housed individually in cages in rooms with a light / safety cycle of 12 hrs. Access to Purina rodent chow 5001 and water ad libi tum are provided to the visitors.

Example 7: Pharmacokinetic Studies The quality of each modified therapeutically active unnatural amino acid polypeptide is evaluated by three tests before entering animal experiments. The purity of the modified therapeutically active unnatural amino acid polypeptide is examined by running a Bis-Tps NuPAGE gel of 4-12% acrylamide with run buffer pH of MES SDS ba or non-reducing conditions (Invitrogen, Carlsbad, CA). The gels are stained with Coomassie blue. The modified therapeutically active unnatural amino acid polypeptide band is greater than 95% pure based on the densitometry scan. The level of endotox in each modified therapeutically active unnatural amino acid polypeptide is tested by an EIA LAL assay using the KTA2 equipment from Charles River Laboratories (Wilmington, MA) and is less than 5 EU per dose. The biological activity of the modified therapeutically active unnatural amino acid polypeptide is determined by the analysis of cells that characterize the bioactivity of the polypeptide. The pharmaco-ether properties of the modified therapeutically active unnatural amino acid polypeptide compounds are compared with each other and with the therapeutically active natural amino acid polypeptide in male Sprague-Dawley rats (261-425 g) obtained from Charles River Laboratories. The catheters are surgically installed to the carotid artery for blood collection. Following the successful installation of the catheter, the animals are treated in treatment groups (three to six per group) before dosing. The animals are dosed subcutaneously with 1 mg / kg of compound in a dose volume of 0.41-0.55 ml / kg.

Blood samples are collected at several points in time via the probe to permanence and to microcentrígufa tubes coated with EDTA. The plasma is collected after centrifugation and stored at -80 ° C until analysis. Compound concentrations are measured using antibody sandwich ELISA equipment either from BioSource Internationa! (Camarillo, CA) or Diagnostic Systems Laboratories (Webster, TX). The concentrations are calculated using standards corresponding to the analog that is used. Pharmacokinetic parameters are estimated using the WinNonlin modeling program (Pharsight, version 4.1). Non-compartmental analysis with trapezoidal upward / downward logarithmic integration and uniformly weighted concentration data is used. Then the data is plotted to obtain Cmax: maximum concentration; terminal: terminal half-life; AUCo. _ > ,? nf: area under the concentration curve - time extrapolated to infinity; MRT: average residence time; Cl / f: plasma clearance, apparent total; and Vz / f: apparent volume of distribution during the terminal phase.

Example 8: Pharmacodynamic Studies Male Sprague-Dawley Rats are obtained from Charles River Laboratories. The animals are allowed to acclimate for a period of three weeks, during which time the Associated biological characteristics tonel polypeptide of natural amino acid are monitored. Animals with an acceptable level of change in these biological characteristics are randomized to treatment groups. The rats are administered either a bolus dose or subcutaneously daily dose of the modified non-natural amino acid polypeptide. Throughout the study the rats are anesthetized daily and sequentially, bled and dosed (when applicable) and the biological correlation characteristics are measured. Blood is collected from the sinus orbital using a heparmized capillary tube and placed in a microfuge tube coated with EDTA. The plasma is isolated by centrifugation and stored at -80 ° C until analysis. Plasma concentrations following a single subcutaneous dose in the rats are obtained.

Example 9: Human Clinical Trial of the Safety and / or Efficacy of Modified Active Therapeutically Active Non-Natural Amino Acid Polypeptide. Objective To compare the safety and pharmacokinetics of a therapeutically modified non-naturally occurring amino acid polypeptide with the safety and pharmacokinetics of a therapeutically active natural amino acid polypeptide. Patients Eighteen healthy volunteers who fluctuate between 20-40 years of age and weigh between 60-90 kg are enrolled in the studio. Subjects will have no clinically effective abnormal laboratory value in terms of serum chemistry or hematology and a selection of negative urine toxicology, HIV screening and hepatitis B surface antigen. They should not have any evidence of the following: hypertension; history of any primary hematologic disease; history of significant liver disease, renal, cardiovascular, gastrointestinal, genitourinary, metabolic, neurological; a history of anemia or attack alteration; a known sensitivity to bacterial products or derivatives of mammals, PEG or human serum albumin; habitual and strong consumer to drinks that contain caffeine; participation in any other clinical study or donated blood transfusion or donated within 30 days of entry into the study; have been exposed to a therapeutically active natural amino acid polypeptide within three months of entry into the study; have had an illness within seven days of entering the study; and have significant abnormalities in the pre-study physical examination or clinical laboratory evaluations within 14 days of entry into the study. All subjects are assessable for safety and all blood collections for pharmacokinetic analysis are performed as scheduled. All studies are carried out with approval of institutional ethics and patient consent.

Study design. This will be a randomized, open-label, single-center, Phase I study of two periods in healthy human volunteers. Eighteen subjects are randomly assigned to one of two treatment sequence groups (nine subjects / group). A therapeutically active natural amino acid polypeptide is administered in two separate dosing periods as a s.c. of bolus in the upper thigh using equivalent doses of the modified therapeutically active unnatural amino acid polypeptide. Additional dosing, dosing frequency or other parameters as desired may be added to the study by including additional groups of subjects. Each dosing period is separated by a washout period of 14 days. Subjects are confined to the study center at least 12 hours before and 72 hours after dosing for each of the two dosing periods, but not between dosing periods. Additional groups of subjects can be added if there is to be additional dosage, frequency, or other parameters, to be tested by the modified therapeutically active unnatural amino acid polypeptide as well. Blood Sample Collection. Serum blood is extracted by direct vein function before and after administration of the modified therapeutically active unnatural amino acid polypeptide or amino acid polypeptide Therapeutically active natural Venous blood samples (5 mL) for the determination of the concentrations of the modified therapeutically active unnatural amino acid polypeptide or the therapeutically active natural amino acid polypeptide in the serum are obtained at about 30, 20 and 10 minutes before dosing (3 reference samples) and approximately the following times after dosing: 30 minutes and 1, 2, 5, 8, 12, 15, 18, 24, 30, 36, 48, 60 and 72 hours. Each serum sample is divided into two aliquots. All serum samples are stored at -20 ° C. The serum samples are packed on dry ice. Clinical fasting laboratory tests (hematology, serum chemistry and urinalysis) are performed immediately before the initial dose on day 1, the morning of day 4, immediately before dosing on day 16 and the morning of day 19. Bioanalytical methods. An ELISA equipment procedure (Diagnostic Systems Laboratory [DSL], Webster TX), is used for the determination of serum concentrations. Security Determinations. Vital signs are recorded immediately before each dosage (Days 1 and 16) and at 6, 24, 48 and 72 hours after each dosage. The safety determinations are based on the incidence and type of adverse events and the changes in the clinical laboratory tests of the reference. In addition, changes in Pre-study on vital sign measurements, in which blood pressure is included and physical examination results are evaluated. Analysis of data. The concentration values in the post-dose serum are corrected by the pre-dose reference concentrations, subtracting from each of the post-dose values the average reference concentration determined from the premediation of the levels of the three samples collected at 30 , 20 and 10 minutes before dosing. The concentrations in the ore-dose are not included in the calculation of the average value if they are below the quantification level of the analysis. The pharmacokinetic parameters of the serum concentration data corrected by the reference concentrations are determined. The pharmacokinetic parameters are calculated by independent model methods in a VAX 8600 computer system from Digital Equipment Corporation using the latest version of the BIOAVL programming elements. The following pharmacokinetic parameters are determined: maximum serum concentration (Cmax); time at the maximum serum concentration (tmax); area under the concentration-time curve (AUC) from time zero to last sample taken (AUQo-72) calculated with the use of the linear trapezoidal rule; and terminal elimination half life (tv.), calculated from the elimination rate constant. The speed constant of Elimination is estimated by linear regression of consecutive data points in the terminal linear region of the log-concentration-time-lmeal plot. The mean, standard deviation (SD) and coefficient of variation (CV) of the pharmacokinetic parameters are calculated for each treatment. The proportion of the parameter means (conserved formulation / formulation without preservation) is calculated. Security Results The incidence of adverse events is equally distributed across the treatment groups. There were no clinically significant changes of the reference or clinical laboratory tests pre-study or blood pressures and no noticeable change of the pre-study in physical examination results and vital signs measurements. The security profiles for the two treatment groups appear similar. Pharmacokinetic results. The concentration-time profiles of the therapeutically active modified non-natural amino acid polypeptide or the therapeutically active natural amino acid polypeptide in the average serum (uncorrected for the reference levels) in all 18 subjects after receiving a single dose of the amino acid polypeptide Therapeutically active modified non-natural or therapeutically active natural amino acid polypeptide are compared at each point in time measured. All subjects should have pre-dose reference concentrations within the normal physiological interval. The pharmacokinetic parameters are determined from corrected serum data for the average pre-dose reference concentrations and the Cmax and tmax are determined. The mean tmax for the modified therapeutically active natural amino acid polypeptide is significantly shorter than the tmax for the modified therapeutically active unnatural amino acid polypeptide. The terminal half-life values are significantly shorter for the modified therapeutically active natural amino acid polypeptide compared to the terminal half-life for the modified therapeutically active unnatural amino acid polypeptide. Although the present study is performed on healthy baron subjects, similar absorption characteristics and similar safety profiles would be anticipated in other patient populations; such as patient barons or female with cancer or chronic renal failure, patients with pediatric renal failure, patients in autologous or patient predeposit programs programmed for elective surgery. In conclusion, individual doses administered subcutaneously of the modified therapeutically active unnatural amino acid polypeptide will be safe and well tolerated by healthy baron subjects. Based on a comparative incidence of adverse events, clinical laboratory values, vital signs and physical examination results, Safety profiles of the modified therapeutically active non-natural amino acid polypeptide and the therapeutically active natural amino acid polypeptide will be equivalent. The modified therapeutically active unnatural amino acid polypeptide potentially provides clinical utility to patients and health care providers. It will be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and will be included within the spirit and scope of this application and scope. of the appended claims. All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety for all purposes.

Claims (64)

1. CLAIMS 1. A compound or salt thereof selected from the group consisting of: wherein each Ra is independently selected from the group consisting of H, halogen, alkyl, -N02, -CN, substituted alkyl, - (NR ') 2, -C (0) kR', -C (0) N (R ') 2, -OR and -S (0) kR', where is 1, 2 or 3; M is H or -CH2R5; or the portion M-N-C (R5) which can form a ring structure of 4 to 7 members; Ri is H, an amino protecting group, resin, amino acid, polypeptide or polynucleotide and R2 is OH, an ester, resin, amino acid, polypeptide or polynucleotide protecting group; Rs is alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, substituted alkoxy, alkylalkoxy, substituted alkylalkoxy, polyalkylene oxide, substituted polyalkylene oxide, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heteroaryl, heteroaryl substituted, heterocycle, substituted heterocycle, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl,, C (0) R ", -C (0) OR", -C (0) N (R ") 2, -C (0) NHCH (R ") 2, - (alkylene or alkylene) -N (R") 2> - (alkenylene or substituted alkenylene) -N (R ") 2, - (alkylene or substituted alkylene) - (aryl or substituted aryl), - (substituted alkenylene or alkenylene) - (substituted aryl or aryl), - (substituted alkylene or alkylene) -ON (R ") 2, - (substituted alkylene or alkylene) -C (0) SR", - (alkylene or substituted alkylene) -SS- (aryl or substituted aryl), wherein each R "is independently hydrogen, alkyl, substituted alsuyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocycle, substituted heterocycle, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl or C (0) 0R '; or R5 is LX, wherein X is selected from the group consisting of a label, a dye, a polymer "; a polymer solved in water; a polyethylene glycol derivative; a photocrosslinker; a cytotoxic compound; a drug; an affinity marker; a photoaffinity marker; a reactive compound; a resin; a second protein or polypeptide or polypeptide analogue; an antibody or antibody fragment; a metal chelator; a co-factor; a fatty acid; a carohydrate; a polynucleotide; a DNA; an RNA; an antisense polynucleotide; a saccharide, a dendrimero solule in water, a cyclodextrin, a biomaterial; a nanoparticle; a spin marker; a fluorophore, a portion containing metal; a radioactive portion; a new functional group; a group that interacts covalently or non-covalently with other molecules; a photoenaged portion; a portion excitable by actinic radiation; a ligand; a photoisomerizable portion; biotin; a biotin analogue; a portion that incorporates a heavy atom; a group chemically cleaved it; a photocleavable group; an elongated side chain; a carbon-bound sugar: a redox-active agent; an amino thioacid; a toxic portion; an isotopically labeled portion; a biophysical probe; a phosphorescent group; a chemiluminescent group; a dense group of electrons; a magnetic group; an intercalary group; a chromophore; an energy transfer agent; a biologically active agent; a detectable marker; a small molecule; an inhibitory ribonucleic acid; a radionucleotide; an electron capture agent; a biotin derivative; quantum dot (s); a nanotransmitter; a radio transmitter; an abzyme, an activated complex activator, a virus, an adjuvant, an aglycan, an allergen, an angiostatin, an antihormone, an antioxidant, an aptamer, a guide RNA, a saponin, a shuttle vector, a macromolecule, an mimotope, a receptor, a reverse micelle and any combination thereof and L is optional and when present is a selected linker from the group consisting of alkylene, substituted alkylene, alkenylene, substituted alkenylene, -0-, -0- (alkylene or substituted alkylene), - (alkylene or substituted alkylene) -0-, -C (0) -, -C (0) - (alkylene or substituted alkylene), (alkylene or substituted alkylene) -C (0) -, -C (0) N (R ') -, -C (0) N (R') - (alkylene or substituted alkylene) -, - (alkylene or substituted alkylene) -0C (0) N (R ') -, -N (R') ) C (0) -, -N (R ') C (0) - (alkylene or substituted alkylene) -, (alkylene or substituted alkylene) -NR' C (0) -, -S-, -S- (alkylene) or substituted alkylene) -, -S (0) k- where k is 1,? or 3, -S (0) y- (substituted alkylene or alkynylene) -, -C (S) -, C (S) - (alkylene or substituted alkylene) -, CSN (R ') -, CSN (R') - (alkylene or substituted alkylene) -, -N (R ') CO- (alkylene or substituted alkylene) -, -N (R ') C (0) 0-, - (alkylene or substituted alkylene) -0-N = CR' -, - (alkylene or substituted alkylene) -C (0) NR '- (alkylene or substituted alkylene) -, (alkylene or substituted alkylene) -S (0) k- (alkylene or substituted alkylene) -S-, - (alkylene or substituted alkylene) -SS-, S (0) kN (R ') -, K (R ') C (0) N (R') -, -N (R ') C (S) N (R') -, -N (R ') S (0) kN (R') -, -N ( R ') - N =, -C (R') = N-, -C (R ') = NN (R') -, -C (R ') = NN =, -C (R') 2-N = N and -C (R ') 2 ~ N (R') -N (R ') -; or R5 and any Ra optionally form a cycloalkyl or a heterocycloalkyl and each R 'is independently H, alkyl or substituted alkyl.
2. 2. The compound according to claim 1, characterized in that Ri is H and R2 is OH.
3. 3. The compound according to claim 1, characterized in that Ra is a halogen.
4. 4. The compound according to claim 1, characterized in that both Ri and R2 are polypeptides. The compound according to claim 1, characterized in that X is a biologically active agent selected from the group consisting of a peptide, protein, enzyme, antibody, drug, dye, lipid, nucleosides, oligonucleotide. cell, virus, lipocoma, microparticle and micelle. 6. The compound according to claim 5, characterized in that X is a drug selected from the group consisting of an antibiotic, fungicide, antiviral agent, anti-inflammatory agent, antitumor agent, cardiovascular agent, anti-anxiety agent, hormone, growth factor and agent. steroidal The compound according to claim 5, characterized in that X is an enzyme selected from the group consisting of horseradish peroxidase, alkaline phosphatase, β-galactosidase and glucose oxidase. The compound according to claim 1, characterized in that X is a detectable marker selected from the group consisting of a fluorescent, phosphorescent, chemiluminescent, chelating, dense portion of electrons, magnetic, intercalante, radioactive, chromophoric and energy transfer. The compound according to claim 1, characterized in that X is a polymer comprising alkyl, substituted alkyl, alkenyl, substituted alkenyl, substituted alkynyl, alkoxy, substituted alkoxy, alkylalkoxy, substituted alkylalkoxy, polyalkylene oxide, polyalkylene oxide substituted , aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkaryl, substituted alkaryl, aralkyl or substituted aralkyl. 10. The compound according to claim 9, characterized in that the polymer comprises polyalkylene oxide or substituted polyalkylene oxide. 11. The compound according to claim 9, characterized in that the polymer comprises - [(alkylene or substituted alkylene) -O- (hydrogen, alkyl or substituted alkyl)] x, wherein x is 20-10,000. 12. The compound according to claim 9, characterized in that the polymer is m-PEG having a molecular weight ranging from 2 to about 40 KDa. 13. A polypeptide or salt thereof, characterized in that it contains at least one compound according to claim 1, selected from the group consisting of: where X is a halogen. 14. A polypeptide or salt thereof, characterized in that it contains at least one compound according to claim 1, selected from the group consisting of: -.r 1 or X J- or,.; X. At- < -'- .-? -x? "C > .. > H} ! OR • - 15. A polypeptide or salt thereof, characterized in that it contains at least one compound according to claim 1, selected from the group consisting of: It is a halogen. n compound or salt thereof, which has the structure: characterized in that each Ra is independently selected from the group consisting of H, halogen, alkyl, -N02, -CN, substituted alkyl, - (NR ') 2, -C (0) kR', C (0) N (R ' ) 2, -OR and -S (0) kR ', where k is 1, 2 or 3; R; is H, an amino protecting group, resin, amino acid, polypeptide or polynucleotide and R2 is OH, an ester, resin, amino acid, polypeptide or polynucleotide protecting group; each R3 and R4 is independently H, halogen, lower alkyl or substituted lower alkyl or R3 and R4 or two R3 groups optionally form a cycloalkyl or a heterocycloalkyl; M is -CH2R5; R5 is alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, substituted alkoxy, alkylalkoxy, substituted alkylalkoxy, polyalkylene oxide, substituted polyalkylene oxide, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocycle , substituted heterocycle, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl,, C (0) R ", -C (0) OR", -C (0) N (R ") 2, -C (0) NHCH (R ") 2, - (alkylene or alkylene) -N (R ") 2" - (alkenylene or substituted alkenylene) -N (R ") 2, - (alkylene or substituted alkylene) - (aryl or substituted aryl), - (substituted alkenylene or alkenylene) - (aryl or substituted aryl), - (alkylene or substituted alkylene) -ON (R ") 2, - (alkylene or substituted alkylene) -C (0) SR", - (alkylene or substituted alkylene) -SS- (aryl or substituted aryl ), wherein each R "is independently hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, substituted heterocycle, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl or C (0) 0R '; or R5 is LX, wherein X is selected from the group consisting of a marker, a dye, a polymer "; a polymer soluble in water; a polyethylene glycol derivative; a photocrosslinker; a cytotoxic compound; a drug; an affinity marker; a photoaffinity marker; a reactive compound; a resin; a second protein or polypeptide or polypeptide analogue; an antibody or antibody fragment; a metal chelator; a co-factor; a fatty acid; a carbohydrate; a polynucleotide; a DNA; an RNA; an antisense polynucleotide; a saccharide, a water-soluble dendrimer, a cyclodextrin, a biomaterial; a nanoparticle; a spin marker; a fluorophore, a portion containing metal; a radioactive portion; a new functional group; a group that interacts covalently or non-covalently with other molecules; a photoenaged portion; a portion excitable by actinic radiation; a ligand; a photoisomerizable portion; biotin; a biotin analogue; a portion that incorporates a heavy atom; a group chemically cleaved it; a photocleavable group; an elongated side chain; a carbon-bonded sugar; a redox-active agent; an amino thioacid; a toxic portion; an isotopically labeled portion; a biophysical probe; a phosphorescent group; a chemiluminescent group; a dense group of electrons; a magnetic group; an intercalary group; a chromophore; an energy transfer agent; an oxiologically active agent; a marker detected; a small molecule; an inhibitory ribonucleic acid; a radionucleotide; an electron capture agent; an oiotine derivative; quantum dot (s); a nanotransmitter; a radio transmitter; an abzyme, an activated complex activator, a virus, an adjuvant, an aglycan, an allergen, an angiostatin, an antihormone, an antioxidant, an aptamer, a guide RNA, a saponin, a shuttle vector, a macromolecule, an mimotope, a receptor, a reverse micelle and any combination thereof and L is optional and when present is a linker selected from the group consisting of alkylene, substituted alkylene, alkenylene, substituted alkenylene, -O-, -0- (alkylene) or substituted alkylene), - (alkylene or substituted alkylene) -0-, -C (0) -, -C (O) - (alkylene or substituted alkylene), (alkylene or substituted alkylene) -C (0) -, -C (0) N (R ') -, -C (0) N (R ') - (alkylene or substituted alkylene) -, - (alkylene or substituted alkylene) -0C (0) N (R') -, -N (R ') C (0) -, -N (R') ) C (0) - (alkylene or substituted alkylene) -, (alkylene or substituted alkylene) -NR 'C (0) -, -S-, -S- (alkylene or substituted alkylene) -, -S (0) k_ wherein k is 1, 2 or 3, -S (0) _ (alkylene or substituted alkylene) -, -C (S) -, -C (S) - (alkylene or substituted alkylene) -, CSN (R ') -, CSN (R ') - (alkylene or substituted alkylene) -, -N (R') CO- (alkylene or substituted alkylene) -. -N (R ') C (0) 0-, - (alkyl or substituted alkylene) -0-N = CR' -, - (alkylene or substituted alkylene) -C (0) NR '- (alkylene or alkylene substituted) -, (alkylene or substituted alkylene) -S (0) k_ (alkylene or substituted alkylene) -S-, - (alkylene or substituted alkylene) -SS-, S (0) kN (R ') -, N ( R ') C (0) N (R') -, -N (R ') C (S) N (R') -, -N (R ') S (0) kN (R') -, -N (R ') - N =, -C (R') = N-, -C (R ') = NN (R') -, -C (R ') = NN =, -C (R') 2- N = N and -C (R ') 2-N (R') -N (R ') -; or R5 and any Ra optionally forms a cycloalkyl or heterocycloalkyl; with the proviso that when Ri is H, then R2 is not OH or when R2 is OH, then Ri is not H; and G is an amine protecting group. 17. The compound according to claim 16, characterized in that the amine protecting group is selected from the group consisting of: siMti 18. The compound according to claim 16, characterized in that both Ri and R2 are polypeptides. 19. A compound or salt thereof having the structure of: R 1 i K • R < l characterized in that L 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 lower heteroalkylene, alkarylene, substituted alkarylene, substituted aralkylene or aralkylene; Q is optional and when present is a linker selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O- (alkylene or substituted alkylene), -S- (alkylene or substituted alkylene), wherein k is 1, 2 or 3, -S (0) k (alkylene or substituted alkylene), -C (0) - (alkylene or substituted alkylene), -C (S) - ( alkylene or substituted alkylene), -NR '- (alkylene or substituted alkylene) - , -CON (R'J - (alkylene or substituted alkylene) -, -CSN (R ') - (alkylene or substituted alkylene) -, -N (R') CO- (alkylene or substituted alkylene) - and wherein each R 'is independently H, alkyl or substituted alkyl, Ri is H, an amino protecting group, resin, amino acid, polypeptide or polynucleotide and R2 is OH, an ester, resin, amino acid, polypeptide or polynucleotide protecting group, each R and R4 is independently H, halogen, lower alkyl or substituted lower alkyl or P3 and R4 or two R3 groups optionally form a cycloalkyl or a heterocycloalkyl, each R a is independently selected from the group consisting of H, halogen, alkyl, -N02, -CN, substituted alkyl, - (NR ') 2, -C (0) kR', -C (0) N (R ') 2, -OR and -S (O) kR', where k is 1, 2 or 3 R6 is a protected aldehyde or a masked aldehyde, wherein the protecting group includes but is not limited to, . < *, where each X is independently selected from the group consisting of -O. S-, N (ii) -, -NtR} -. -N (? C) - Y -N (O) -. X, it is OK. - ?? c. -SK, -N (R) -. -N (R) (A -N; () (0 \ í «) or N- and wherein each R 'and R is independently H, alkyl or substituted alkyl: The compound according to claim 19, because Xi is O. The compound according to claim 19, characterized in that both Ri and R2 are polypeptides. 22. A polypeptide characterized in that it contains at least one compound according to claim 19, selected from the group consisting of: 23. A method of manufacturing a compound containing at least one non-natural amino acid selected from the group consisting of: wherein the compound is formed by an alkylation reducing an aromatic amino portion on at least one unnatural amino acid with at least one reagent comprising at least a portion of aldehyde; wherein each Ra is independently selected from the group consisting of H, halogen, alkyl, -N02, -CN, substituted alkyl, - (NR ') 2, -C (0) kR', -C (0) N (R ') 2, -OR and -S (0) kR', where k is 1, 2 or 3; M is H or -CH2R5; or the portion M-N-C (R5) which can form a ring structure of 4 to 7 members; RL is H, an amino protecting group, resin, amino acid, polypeptide or polynucleotide and R? is OH, a protecting group ester, resin, amino acid, polypeptide or polynucleotide; R5 is alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, substituted alkoxy, alkylalkoxy, substituted alkylalkoxy, polyalkylene oxide, substituted polyalkylene oxide, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocycle , substituted heterocycle, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl,, C (0) R ", -C (0) 0R", -C (0) N (R ") 2, -C (0) NHCH (R") 2, - (alkylene or alkylene) -N (R " ) 2 - (alkenylene or substituted alkenylene) -N (R ") 2, - (alkylene or substituted alkylene) - (aryl or substituted aryl), - (substituted alkenylene or alkenylene) - (substituted aryl or aryl), - ( alkylene or substituted alkylene) -ON (R ") 2, - (alkylene or substituted alkylene) -C (0) SR ", - (alkylene or substituted alkylene) -SS- (aryl or substituted aryl), wherein each R" is independently hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl , alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocycle, substituted heterocycle, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl or C (0) 0R '; or R5 is L-X, wherein X is selected from the group consisting of a label; a dye; a polymer ", a water-soluble polymer, a polyethylene glycol derivative, a photocrosslinker, a cytotoxic compound, a drug, an affinity tag, a photoaffinity tag, a reactive compound, a resin, a second protein or polypeptide or analogue polypeptide, an antibody or fragment of antibodies, a metal chelator, a co-factor, a fatty acid, a carbohydrate, a polynucleotide, a DNA, an RNA, an antisense polynucleotide, a saccharide, a water-soluble dendrimer, a cyclodextrin a biomaterial, a nanoparticle, a spin marker, a fluorophore, a metal-containing portion, a radioactive portion, a new functional group, a group that interacts covalently or non-covalently with other molecules, a photoenaged portion, a portion excitable by actinic radiation, a ligand, a portion fotoisomerizaóle; óiotina; a analogue of biotin; a portion that incorporates a heavy atom; a chemically cleavable group; a photocleavable group; an elongated side chain; a carbon-bonded sugar; a redox-active agent; an amino thioacid; a toxic portion; an isotopically labeled portion; a biophysical probe; a phosphorescent group; a chemiluminescent group; a dense group of electrons; a magnetic group; an intercalary group; a chromophore; an energy transfer agent; an oxiologically active agent; a detectable marker; a small molecule; an inhibitory ribonucleic acid; a radiucleotide; an electron capture agent; an oiotine derivative; quantum dot (s); a nanotransmitter; a radio transmitter; an abzyme, an activated complex activator, a virus, an adjuvant, an aglycan, an allergen, an angiostatin, an antihormone, an antioxidant, an aptamer, a guide RNA, a saponin, a shuttle vector, a macromolecule, an mimotope, a receptor, a reverse micelle and any combination thereof and L is optional and when present is a linker selected from the group consisting of alkylene, substituted alkylene, alkenylene, substituted alkenylene, -0-, -0- (alkylene) or substituted alkylene), - (alkylene or substituted alkylene) -0-, -C (0) -, -C (0) - (alkylene or substituted alkylene), (alkylene or substituted alkylene) -C (0) -, - C (0) N (R ') -, -C (0) N (R') - (alkylene or substituted alkylene) -, - (alkylene or substituted alkylene) -0C (0) N (R ') -, - N (R ') C (0) -, -N (R') C (0) - (alkylene or alkylene substituted) -, (alkylene or substituted alkylene) -NR 'C (0) -, -S-, -S- (alkylene or substituted alkylene) -, -S (0) k_ where k is 1, 2 or 3, -S (0) k- (alkylene or substituted alkylene) -, -C (S) -, - C (S) - (alkylene or substituted alkylene) -, CSN (R ') -, CSN (R') - (alkylene or substituted alkylene) -, -N (R ') CO- (alkylene or substituted alkylene) -, -N (R') C (0) 0-, - (alkylene or substituted alkylene) -0-N = CR '-, - (alkylene or substituted alkylene) - C (0) NR' - (alkylene or substituted alkylene) -, (alkylene or substituted alkylene) -S (0) k- (alkylene or substituted alkylene) -] C S- , - (alkylene or substituted alkylene) -SS-, (0 kN (R ') -, - N (R') C (0) N (R ') -, - N (R') C (S) N ( R ') -, -N (R') S (0) kN (R ') -, -N (R') - N =, -C (R ') = N-, -C (R') = NN (R ') -, -C (R') = NN =, -C (R ') 2 -N = N and - C (R') 2-N (R ') -N (R') -; R5 and any Ra optionally form a cycloalkyl or A heterocycloalkyl and each R 'is independently H, alkyl or substituted alkyl. 24. The method according to claim 23, characterized in that Ri and R2 of at least one amino acid are not 20 natural is H and OH respectively. 25. The method according to claim 23, characterized in that Ra of at least one non-natural amino acid is a halogen. 26. The method according to claim 23, characterized in that amomers of Ri and R2 of at least one non-natural amino acid are polypeptides. The method according to claim 23, characterized in that X of at least one non-natural amino acid is an oxiologically active agent selected from the group consisting of a peptide, protein, enzyme, antibody, drug, dye, lipid, nucleosides, oligonucleotide, cell, virus, liposome, microparticle and micelle. The method according to claim 27, characterized in that X of at least one non-natural amino acid is a drug selected from the srupc consisting of an antibiotic, fungicide, antiviral agent, anti-inflammatory agent, antitumor agent, cardiovascular agent, anti-anxiety agent , hormone, growth factor and steroidal agent. 29. The method according to claim 27, characterized in that X of at least one non-natural amino acid is an enzyme selected from the group consisting of horseradish peroxidase, alkaline phosphatase, β-galactosidase and glucose oxidase. 30. The method according to claim 23, characterized in that X of at least one non-natural amino acid is a detectable label selected from the group consisting of a fluorescent, phosphorescent, chemiluminescent, chelating, electron dense, magnetic, intercalant, radioactive, chromophoric and energy transfer. 31. The method according to claim 23, characterized in that X of the non-natural amino acid is a polymer comprising 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 or substituted aralkyl. 32. The method according to claim 31, characterized in that the polymer comprises polyalkylene oxide or substituted polyalkylene oxide. 33. The method according to claim 31, characterized in that the polymer comprises [(alkylene or substituted alkylene) -0- (hydrogen, alkyl or substituted alkyl)] x, wherein x is 20-10,000. 34. The method according to claim 31, characterized in that the polymer is m-PEG having a molecular weight ranging from about 2 to about 40 KDa. 35. The method according to claim 23, characterized in that the at least one non-natural amino acid is selected from the group consisting of: 36. The method according to claim 23, characterized in that the at least one non-natural amino acid is selected from the group consisting of: 37. The method according to claim 23, characterized in that the at least one non-natural amino acid is selected from the group consisting of: 38. A method for the manufacture of a polypeptide containing at least one non-natural amino acid selected from the group consisting of: characterized in that the polypeptide is formed by a reductive alkylation of an aromatic amino moiety on at least one unnatural amino acid with at least one reagent containing at least a portion of aldehyde; each Ra is independently selected from the group consisting of H, halogen, alkyl, -N02, -CN, substituted alkyl, - (NR ') 2, -C (0) kR', -C (0) N (R ') 2, -OR and -S (0) kR ', where k is 1, 2 or 3; Ri is H, an amino protecting group, resin, amino acid, polypeptide or polynucleotide and R2 is OH, an ester, resin, amino acid, polypeptide or polynucleotide protecting group; each R3 and R < Is independently H, halogen, lower alkyl or substituted lower alkyl or R3 and R4 or two R3 groups optionally form a cycloalkyl or a heterocycloalkyl; M is -CH2R5; R5 is alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, substituted alkoxy, alkylalkoxy, substituted alkylalkoxy, polyalkylene oxide, substituted polyalkylene oxide, cycloalkyl, cycloalkyl substituted, aplo, substituted substituted, heteroaryl, substituted heteroaryl, heterocycle, substituted heterocycle, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl,, C (0) R ", -C (0) OR", -C (0) N ( R ") 2, -C (0) NHCH (R'J 2, - (alkylene or alkylene) -N (R") 2 '- (alkenylene or substituted alkenylene) -N (R ") 2, - (alkylene or substituted alkylene) - (substituted aryl or aryl), - (substituted alkenylene or alkenylene) - (substituted aryl or substituted), - (substituted alkylene or alkylene) -ON (R ") 2, - (substituted alkylene or alkylene) -C (0) SR ", - (alkylene or substituted alkylene) -SS- (aryl or substituted aplo), wherein each R" is independently hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, aryl, aplo substituted, heteroaryl, substituted heteroaryl, heterocycle, substituted heterocycle, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl or C (0) 0R '; or R5 is LX, wherein X is selected from the group consisting of a marker; a dye; a polymer, a water-soluble polymer, a polyethylene glycol derivative, a photocrosslinker, a cytotoxic compound, a drug, an affinity tag, a photoprint tag, a reactive compound, a ream, a second protein or polypeptide or analogue. polypeptide, an antibody or fragment of antibodies, a chelator of metals, a co-factor, a fatty acid, a carbohydrate, a polynucleotide, a DNA, an RNA, a antisense polynucleotide; a saccharide, a water-soluble dendrimer, a cyclodextrin, a biomaterial; a nanoparticle; a spin marker; a fluorophore, a portion containing metal; a radioactive portion; a new functional group; a group that interacts covalently or non-covalently with other molecules; a photoenaged portion; a portion excitable by actinic radiation; a ligand; a photoisomerizable portion; biotin; a biotin analogue; a portion that incorporates a heavy atom; a group chemically cleaved it; a group photoe = cindible; an elongated side chain; a carbo-linked sugar; a redox-active agent; an amino thioacid; a toxic portion; an isotopically labeled portion; a biophysical probe; a phosphorescent group; a chemiluminescent group; a dense group of electrons; a magnetic group; an intercalary group; a chromophore; an energy transfer agent; an oxiologically active agent; a marker detected; a small molecule; an inhibitory ribonucleic acid; a radionucleotide; an electron capture agent; a biotin derivative; quantum dot (s); a nanotransmitter; a radio transmitter; an abzyme, an activated complex activator, a virus, an adjuvant, an aglycan, an allergen, an angiostatin, an antihormone, an antioxidant, an aptamer, a guide RNA, a saponin, a shuttle vector, a macromolecule, an mimotope, a receptor, a reverse micelle and any combination thereof and L is optional and when present is a linker selected from the group consisting of alkylene, substituted alkylene, alkenylene, substituted alkenylene, -0-, -0- (alkylene or substituted alkylene), - (alkylene or substituted alkylene) -0-, - C (0) -, -C (0) - (alkylene or substituted alkylene), (alkylene or substituted alkylene) -C (0) -, -C (0) N (R ') -, -C (0) N (R ') - (alkylene or substituted alkylene) -, - (alkylene or substituted alkylene) -0C (0) N (R') -, -N (R ') C (0) -, -N (R') C (0) - (alkylene or substituted alkylene) -, (alkylene or substituted alkylene) -NR 'C (0) -, -S-, -S- (alkylene or substituted alkylene) -, -S (0) k- wherein k is 1, 2 or 3, -S (0) k- (alkylene or substituted alkylene) -, -C (S) -, -C (S) - (alkylene or substituted alkylene) -, CSN (R ' ) -, CSN (R ') - (alkylene or substituted alkylene) -, -N (R') C0- (alkylene or substituted alkylene) -, -N (R ') C (0) 0-, - (alkylene or substituted alkylene) -0-N = CR '-, - (alkylene or substituted alkylene) -C (0) NR' - (alkylene or alkylene) ituido) -, (alkylene or substituted alkylene) -S (0) k- (alkylene or substituted alkylene) -S-, - (alkylene or substituted alkylene) -SS-, S (0) kN (R ') -, N (R ') C (0) N (R') -, - N (R ') C (S) N (R') -, - N (R ') S (0) kN (R') -, - N (R ') - N =, -C (R') = N-, -C (R ') = NN (R') -, -C (R ') = NN =, -C (R') 2 -N = N and -C (R ') 2-N (R') -N (R ') -; or two R5 groups optionally form a cycloalkyl or a heterocycloalkyl; or R5 and any Ra optionally forms a cycloalkyl or heterocycloalkyl; with the proviso that when Ri is H, then R2 is not OH or when R2 is not OH, then Ri is not H; and G is an amine protecting group. 39. The method according to claim 38, characterized in that the protective amine group is selected from the group consisting of: 40. The method according to claim 38, characterized in that both of Ri and R2 are polypeptides. 41. The method according to any of claims 23 and 38, characterized in that the at least one portion of aldehyde has the structure corresponding to: wherein: Rs is alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, substituted alkoxy, alkylalkoxy, substituted alkylalkoxy, polyalkylene oxide, substituted polyalkylene oxide, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heteroaryl, heteroaryl substituted, heterocycle, substituted heterocycle, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl,, C (0) R ", -C (0) 0R", -C (0) N (R ") 2, -C (0) NHCH (R ") 2, - (alkylene or alkylene) -N (R ") 2" - (alkenylene or substituted alkenylene) -N (R ") 2, - (alkylene or substituted alkylene) - (aryl or substituted aryl), - (substituted alkenylene or alkenylene) - (aryl or substituted aryl), - (alkylene or substituted alkylene) -ON (R ") 2, - (alkylene or substituted alkylene) -C (O) SR", - (alkylene or substituted alkylene) -SS- (aryl or substituted aryl ), wherein each R "is independently hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocycle, substituted heterocycle, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl or C (0) 0R '; or R5 is LX, wherein X is selected from the group consisting of a marker, a dye, a polymer "; a polymer soluble in water; a polyethylene glycol derivative; a photocrosslinker; a cytotoxic compound; a drug; an affinity marker; a photoaffinity marker; a reactive compound; a resin; a second protein or polypeptide or polypeptide analogue; an antibody or antibody fragment; a metal chelator; a co-factor; a fatty acid; a carohydrate; a polynucleotide; a DNA; an RNA; an antisense polynucleotide; a saccharide, a water-soluble dendrimer, a cyclodextrin, a biomaterial; a nanoparticle; a spin marker; a fluorophore, a portion containing metal; a radioactive portion; a new functional group; a group that interacts covalently or non-covalently with other molecules; a photoenaged portion; an excitatory portion by actinic radiation; a ligand; a photoisomerizable portion; biotin; a biotin analogue; a portion that incorporates a heavy atom; a chemically cleavable group; a photocleavable group; an elongated side chain; a carbon-bonded sugar; a redox-active agent; an amino thioacid; a toxic portion; an isotopically labeled portion; a biophysical probe; a phosphorescent group; a chemiluminescent group; a dense group of electrons; a magnetic group; an intercalary group; a chromophore; an energy transfer agent; a biologically active agent; a detectable marker; a small molecule; an inhibitory ribonucleic acid; a radionucleotide; an electron capture agent; a biotin derivative; quantum dot (s); a nanotransmitter; a radio transmitter; an abzyme, an activated complex activator, a virus, an adjuvant, an aglycan, an allergan, an angiostatin, an antihormone, an antioxidant, an aptamer, a guide RNA, a saponin, a shuttle vector, a macromolecule, an mimotope, a receptor, a reverse micelle and any combination thereof and L is optional and when present is a linker selected from the group consisting of alkylene, substituted alkylene, alkenylene, substituted alkenylene, -0-, -0- (alkylene) or substituted alkylene), - (alkylene or substituted alkylene) -0-, -C (O) -, -C (O) - (alkylene or substituted alkylene), (alkylene or substituted alkylene) -C (0) -, -C (0) N (R ') -, -C (0) N (R ') - (alkylene or substituted alkylene) -, - (alkylene or substituted alkylene) -0C (0) N (R') -, -N (R ') C (0) -, -N (R') ) C (0) - (alkylene or substituted alkylene) -, (alkylene or substituted alkylene) -NR 'C (0) -, -S-, -S- (alkylene or substituted alkylene) -, -S (0) k - wherein k is 1, 2 or 3, -S (0) - (alkylene or substituted alkylene) -, -C (S) -, -C (S) - (alkylene or substituted alkylene) -, CSN (R ') ) -, CSN (R ') - (alkylene or substituted alkylene) -, -N (R') C0- (alkylene or substituted alkylene) -, -N (R ') CJO) 0-, - (alkylene or substituted alkylene) ) -0-N = CR '-, - (alkylene or substituted alkylene) -C (0) NR' - (alkylene or substituted alkylene) -, (alkylene or substituted alkylene) -S (0) k ~ (alkylene or alkylene) substituted) -S-, - (alkylene or substituted alkylene) -SS-, S (0) kN (R ') -, N (R') C (0) N (R ') -, -N (R') C (S) N (R ') -, -N (R') S (0) k (R ') -, -N (R') - N =, -C (R ') = N-, -C (R ' ) = NN (R ') -, -C (R') = NN =, -C (R ') 2-N = N and -C (R') 2-N (R ') -N (R') -; 42. A method for the treatment of an alteration, condition or disease characterized in that it comprises administering a therapeutically effective amount of a polypeptide comprising at least one unnatural amino acid selected from the group consisting of: wherein each Ra is independently selected from the group consisting of H, halogen, alkyl, -N02, -CN, substituted alkyl, - (NR ') 2, -C (0) kR', -C (0) N (R ') 2, -OR and -S (0) kR', where k is 1, 2 or 3; M is H or -CH2R5; or the -N-C (R5) portion which can form a ring structure of 4 to 7 members; Ri is H, an amino protecting group, resin, amino acid, polypeptide or polynucleotide and R2 is OH, an ester, resin, amino acid, polypeptide or polynucleotide protecting group; R5 is alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, substituted alkoxy, alkylalkoxy, substituted alkylalkoxy, polyalkylene oxide, substituted polyalkylene oxide, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heteroaryl, heteroaryl substituted, heterocycle, substituted heterocycle, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl,, C (0) R ", -C (0) OR", -C (0) N (R ") 2, -C (0) NHCH (R ") 2, - (alkylene or alkylene) -N (R") 2 '- (alkenylene or substituted alkenylene) -N (R'J 2, - (alkylene or substituted alkylene) - (aryl or substituted aryl) , - (alkenylene or substituted alkenylene) - (aryl or substituted aryl), - (alkylene or substituted alkylene) -ON (R ") 2, - (alkylene or substituted alkylene) -C (0) SR", - (alkylene or substituted alkylene) -SS- (aryl or substituted aryl), wherein each R "is independently hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocycle, heterocycle substituted, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl or C (O) 0R '; or R5 is LX, wherein X is selected from the group consisting of a label, a dye, a "polymer", a polymer soluble in Water; a polyethylene glycol derivative; a photocrosslinker; a cytotoxic compound; a drug; an affinity marker; a photoaffinity marker; a reactive compound; a resin; a second protein or polypeptide or polypeptide analogue; an antibody or antibody fragment; a metal chelator; a co-factor; a fatty acid; a carohydrate; a polynucleotide; a DNA; an RNA; an antisense polynucleotide; a saccharide, a soluble dendrimer in water, a cyclodextrin, a biomaterial; a nanoparticle; a spin marker; a fluorophore, a portion containing metal; a radioactive portion; a new functional group; a group that interacts covalently or non-covalently with other molecules; a photoenaged portion; a portion excitable by actinic radiation; a ligand; a photoisomerizable portion; biotin; a biotin analog; a portion that incorporates a heavy atom; a chemically cleavable group; a photocleavable group; an elongated side chain; a carbon-bonded sugar; a redox-active agent; an amino thioacid; a toxic portion; an isotopically labeled portion; a biophysical probe; a phosphorescent group; a chemiluminescent group; a dense group of electrons; a magnetic group; an intercalary group; a chromophore; an energy transfer agent; a biologically active agent; a detectable marker; a small molecule; an inhibitory ribonucleic acid; a radionucleotide; an electron capture agent; a biotin derivative; quantum dot (s); a nanotransmitter; a radio transmitter; an abzyme, an activated complex activator, a virus, an adjuvant, an aglycan, an allergen, an angiostatin, an antihormone, an antioxidant, an aptamer, a guide RNA, a saponin, a shuttle vector, a macromolecule, an mimotope, a receptor, a reverse micelle and any combination thereof and L is optional and when present is a selected linker of the group consisting of alkylene, substituted alkylene, alkenylene, substituted alkenylene, -0-, -0- (alkylene or substituted alkylene), - (alkylene or substituted alkylene) -0-, -C (0) -, -C ( 0) - (alkylene or substituted alkylene), (alkylene or substituted alkylene) -C (0) -, -C (0) N (R ') -, -C (0) N (R') - (alkylene or alkylene substituted) -, - (alkylene or substituted alkylene) -0C (0) N (R ') -, -N (R') C (0) -, -N (R ') C (0) - (alkylene or alkylene substituted) -, (alkylene or substituted alkylene) -NR 'C (0) -, -S-, -S- (alkylene or substituted alkylene) -, -S (0) k- where k is 1, 2 or 3 , -S (O1 * - (II alkylene or substituted alkylene) -, -C (S) -, -C (S) - (alkylene or substituted alkylene) -, CSN (R ') -, CSN (R') - ( alkylene or substituted alkylene) -, -N (R ') C0- (alkylene or substituted alkylene) -, -N (R') C (0) 0-, - (substituted alkylene or alkylene) -0-N = CR '-, - (alkylene or substituted alkylene) -C (0) NR' - (alkylene or substituted alkylene) -, (alkylene or substituted alkylene) -S (0) k- (alkyl nickel or substituted alkylene) -S-, - (alkylene or substituted alkylene) -SS-, S (0) kN (R ') -, N (R') C (0) N (R ') -, -N ( R ') C (S) N (R') -, -N (R ') S (0) kN (R') -, -N (R ') - N =, -C (R') = N- , -C (R ') = NN (R') -, -C (R ') = NN =, -C (R') 2 -N = N and -C (R ') 2-N (R') -N (R ') -; or R5 and any Ra optionally form a cycloalkyl or a heterocycloalkyl and each R 'is independently H, alkyl or substituted alkyl; with the proviso that when Ri is H, then R2 is not OH or when R2 is OH, then Ri is not H 43. The method according to claim 42, characterized in that it further comprises administering a pharmaceutically acceptable carrier with the therapeutically effective amount of the polypeptide. 44. The method according to claim 42, characterized in that Ri and R2 are both polypeptides. 45. The method according to claim 42, characterized in that X is a solule polymer in water. 46. The method according to claim 42, characterized in that X is a polyethylene glycol derivative. 47. The method according to claim 42, characterized in that X is a cytotoxic compound. 48. The method according to claim 42, characterized in that X is a drug. 49. The method according to claim 42, characterized in that X is a second polypeptide. 50. The method according to claim 49, characterized in that the second polypeptide is a peptide that contains an unnatural amino acid polypeptide. 51. The method according to claim 42, characterized in that the polypeptide is a protein homologue to a therapeutic protein selected from the group consisting of: alpha-1, antitrypsin, angiostatin, antihemolytic factor, antibody, apolipoprotein, apoprotein, natruiritic factor atrial, atrial natriuretic polypeptide, atrial peptide, chemokine CXC, T39765, NAP-2, ENA-78, gro-a, gro-o, gro-c, IP-10, GCP-2, NAP-4, SDF-1, PF4, M1G, calcitonin, ligand c-kit, cytosine, chemokine CC, protein-1 alpha chemoattractant monocyte, protein-2 alpha monocyte chemoattractant, protein-3 alpha chemoattractant monocyte, protein-1 alpha-monocyte inflammatory, protein- 1 Inflammatory monocyte beta, RANTES, 1309, R83915, R91733, HCCl, T58847, D31065, T64262, CD40, CD40 ligand, c-kit ligand, collagen, colony stimulating factor (CSF), complement factor 5a inhibitor, receptor 1 complement, cytosine, peptide-78 epithelial neutrophil activator, MIP-16, MCP-1, epithelial 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-helix beam protein, GC SF, 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-1, IGF-II, interferon (IFN) , IFN-alpha, IFN-beta, IFN-gamma, any interferon-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, keratinocyte growth factor (KGF), lactoferrin, leukemia inhibitory factor, luciferase, neurturin, neutrophil inhibiting factor (NIF), oncostatin M, osteogenic protein, oncogene product, paracytonine, 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 ICAM-1, soluble interleukin receptor, soluble TNF receptor, somatomedin , somatostatin, somatotropin, streptokinase, superantigen, staphylococcal enterotoxin, SEA, SEB, SEC1, SEC2, SEC3, SED, SEE, steroid hormone receptor, superoxide dismutase, toxic shock syndrome toxin, alpha limosine 1, tissue plasminogen scavenger tumor growth factor (TFR), tumor necrosis factor, tumor necrosis factor alpha, tumor necrosis factor beta, tumor necrosis factor receptor (TNRF), VLA-4 protein, VCAM-1 protein Vascular endothelial growth factor (VEGF), urokinase, mos, ras, raf, met, p53, tat, fos, myc, jun, myb, rei, estrogen receptor, progesterone receptor, testosterone receptor, aldosterone receptor, receptor LDL and corticosterone. 52. The method according to claim 42, characterized in that the at least one non-natural amino acid is incorporated at a specific site within the polypeptide. 53. The method according to claim 42, characterized in that the non-natural amino acid is incorporated using a translation system. 54. The method according to claim 42, characterized in that the non-natural amino acid is incorporated into the polypeptide using a translation system and a post-translation modification system. 55. The method according to claim 42, characterized in that the polypeptide comprising at least one unnatural amino acid is stanol for at least one month. 56. The method according to claim 42, characterized in that the polypeptide comprising at least one unnatural amino acid is stanol for at least two weeks. 57. A method for detecting the presence of a polypeptide in a patient, the method is characterized in that it comprises administering an effective amount of a homologous non-natural amino acid polypeptide comprising at least unnatural amino acid selected from the group consisting of: and detecting an indicator in the selected polypeptide consisting of: a marker; a dye; an affinity marker; a photoaffinity marker; a spin marker, a second protein or polypeptide analogue; a fluorogote, a portion that incorporates a heavy atom; an isotopically labeled portion; an osophysical probe; a phosphorescent group, a chemiluminescent group; Oiotine, an analogue of Oiotine; a dense group of electrons; a magnetic group; a chromophore; an energy transfer agent; a detectable marker; a radio transmitter and any combination thereof. wherein each Ra is independently selected from the group consisting of H, halogen, alkyl, -N02, -CN, substituted alkyl, - (NR ') 2, -C (0) kR', -C (0) N (R ') 2, -OR and -S (0) kR', where k is 1, 2 or 3; M is H or -CH2R5; or the portion M-N-C (R5) which can form a ring structure of 4 to 7 members; Ri is H, an amino protecting group, resin, amino acid, polypeptide or polynucleotide and R2 is OH, an ester, resin, amino acid, polypeptide or polynucleotide protecting group; Rs is alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, substituted alkoxy, alkylalkoxy, substituted alkylalkoxy, polyalkylene oxide, substituted polyalkylene oxide, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocycle, substituted heterocycle, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl,, C (O) R ", -C (0) OR", -C (0) N (R ") 2, - C (0) NHCH (R'J 2, - (alkylene or alkylene) -N (R ") 2» - (alkenylene or substituted alkenylene) -N (R ") 2 F - (alkylene or substituted alkylene) - ( aryl or substituted aryl), - (substituted alkenylene or alkenylene) - (substituted aryl or aryl), - (substituted alkylene or alkylene) -ON (R ") 2, - (substituted alkylene or alkylene) -C (0) SR" , - (alkylene or substituted alkylene) -SS- (aryl or substituted aryl), wherein each R "is independently hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, heteroaryl substituted, heterocycle, substituted heterocycle, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl or C (0) 0R '; or R5 is L-X, wherein X is selected from the group consisting of a label; a dye; a "polymer"; a polymer solved in water; a polyethylene glycol derivative; a photocrosslinker; a cytotoxic compound; a drug; an affinity marker; a photoaffinity marker; a compound reagent; a resin; a second protein or polypeptide or polypeptide analogue; an antibody or antibody fragment; a metal chelator; a co-factor; a fatty acid; a carbohydrate; a polynucleotide; a DNA; an RNA; an antisense polynucleotide; a saccharide, a dendrimer solved in water, a cyclodextrin, a biomaterial; a nanoparticle; a spin marker; a fluorophore, a portion containing metal; a radioactive portion; a new functional group; a group that interacts covalently or non-covalently with other molecules; a photoenaged portion; an excitatory portion by actinic radiation; a ligand; a photoisomerizable portion; biotin; a biotin analogue; a portion that incorporates a heavy atom; a group chemically cleaved it; a group photographed him; an elongated side chain; a carbon-bonded sugar; a redox-active agent; an amino thioacid; a toxic portion; an isotopically labeled portion; a biophysical probe; a phosphorescent group; a chemiluminescent group; a dense group of electrons; a magnetic group; an intercalary group; a chromophore; an energy transfer agent; a biologically active agent; a detectable marker; a small molecule; an inhibitory ribonucleic acid; a radionucleotide; an electron capture agent; a biotin derivative; quantum dot (s); a nanotransmitter; a radio transmitter; an abzyme, an activated complex activator, a virus, an adjuvant, an aglican, an allergana, an angiostatin, an antihormone, an antioxidant, an aptamer, a guide RNA, a saponin, a shuttle vector, a macromolecule, a mimotope, a receptor, a reverse micelle and any combination thereof and L is optional and when present is a linker selected from the group consisting of alkylene, substituted alkylene, alkenylene, substituted alkenylene, -O-, -0- (alkylene or substituted alkylene), - (alkylene or substituted alkylene) -0-, -C ( 0) -, -C (0) - (alkylene or substituted alkylene), (alkylene or substituted alkylene) -C (0) -, -C (0) N (R '^ -, -C (0) N (R ') - (alkylene or substituted alkylene) -, - (alkylene or substituted alkylene) -0C (0) N (R') -, -N (R ') C (0) -, -N (R') C ( 0) - (alkylene or substituted alkylene) -, (alkylene or substituted alkylene) -NR 'C (0) -, -S-, -S- (alkylene or substituted alkylene) -, -S (0) - wherein k is 1, 2 or 3, -S (0) k- (alkylene or substituted alkylene) -, -C (S) -, -C (S) - (alkylene or substituted alkylene) -, CSN (R ') -, CSN (R ') - (alkylene or substituted alkylene) -, -N (R') CO- (alkylene or substituted alkylene) -, -N (R ') C (0) 0-, - (alkylene or alkylene substituted) -0-N = CR '-, - (alkylene or substituted alkylene) -C (0) NR' - (alkylene or substituted alkylene) -, (alkylene or substituted alkylene) -S (0) k- (alkylene or substituted alkylene) -S-, - (alkylene or substituted alkylene) -SS-, S (0) kN (R ') -, N (R') C (0) N (R ') -, - N (R') ) C (S) N (R ') -, -N (R') S (0) kN (R ') -, -N (R') - N =, -C (R ') = N-, - C (R ') = NN (R') -, -C (R ') = NN =, -C (R') 2 -N = N and -C (R ') 2-N (R') -N (R ') -; or R5 and any Ra optionally form a cycloalkyl or a heterocycloalkyl; each R 'is independently H, alkyl or substituted alkyl and with the proviso that when Ri is H, then R2 is not OH or when R2 is OH, then Ri is not H. 58. The method according to claim 57, characterized because at least one non-natural amino acid is incorporated at a specific site within the polypeptide. 59. The method according to claim 57, characterized in that the non-natural amino acid is incorporated using a translation system. 60. The method according to claim 57, characterized in that the non-natural amino acid is incorporated into the polypeptide using a translation system, a post-translation modification system. 61. The method according to claim 57, characterized in that the non-natural amino acid is stable in aqueous solution for at least one month. 62. The method according to claim 57, characterized in that the unnatural amino acid is stable in aqueous solution for at least two weeks. 63. The method according to claim 57, characterized in that the unnatural amino acid is stable in aqueous solution for at least five days. 64. The method according to claim 57, characterized in that the polypeptide is a protein homologue for a therapeutic protein selected from the group consisting of: alpha-1, antitrypsin, angiostatma, antihemolytic factor, antibody, apolipoprotein, apoprotein, atrial natri-retic factor, atrial natriuretic polypeptide, atrial peptide, chemokine CXC, T39765, NAP-2, ENA-78, gro-a, gro-b, gro-c, IP-10, GCP-2, NAP-4, SDF-1, PF4, M1G, calcitonin, c-kit ligand, cytosm, CC chemokine, monocyte chemoattractant alpha-protein, monocyte chemoattractant-2 alpha protein, monocyte chemoattractant-3 alpha protein, monocyte-inflammatory alpha-1 protein, beta-1 protein Inflammatory monocyte, RANTES, 1309, R83915, R91733, HCCl, T58847, D31065, T64262, CD40, CD40 ligand, c-kit ligand, collagen, colony stimulating factor (CSF), complement factor 5a inhibitor, receptor 1 complement, cytosm, peptide-78 neutrophil activator epi telial, MIP-16, MCP-1, epithelial growth factor (EGF), epithelial neutrophil activating peptide, erythropoietic (EPO), exfoliating toxin, Factor IX, Factor VII, Factor VIII, Factor X, and fioblasts growth factor ( FGF), fibrinogen, fibronectm, four-helix bundle protein, G-CSF, glp-1, GM-CSF, glucocerebrosidase, gonadotropma, growth factor, growth factor receptor, grf, hedgehog protein, hemoglobin, factor hepatocyte growth (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-1, IGF-II, interferon (IFN), IFN-alpha, IFN-beta, IFN-gamma, any molecule similar to interferon or miemóro of the family of IFN, interleukin (IL), IL-1, IL- 2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, keratinocyte growth factor (KGF) , lactoferrin, leukemia inhibitory factor, luciferase, neurturin, neutrophil inhibiting factor (NIF), oncostatin M, osteogenic protein, oncogene product, paracytonine, 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 ICAM-1, soluble interleukin receptor, soluble TNF receptor, somatomedin , somatostatin, somatotropin, streptokinase, superantigen, staphylococcal enterotoxin, SEA, SEB, SEC1, SEC2, SEC3, SED, SEE, steroid hormone receptor, superoxide dismutase, toxic shock syndrome toxin, limosine alpha 1, tissue plasminogen activator tumor growth factor (TFR), tumor necrosis factor, tumor necrosis factor alpha, tumor necrosis factor beta, tumor necrosis factor receptor (TNRF), VLA-4 protein, VCAM-1 protein Vascular endothelial growth factor (VEGF), urokinase, mos, ras, raf, met, p53, tat, fos, myc, jun, myb, rei, estrogen receptor, progesterone receptor, testosterone receptor, aldosterone receptor, LDL receptor and corticosterone.