NZ618261B2 - Compositions containing, methods involving, and uses of non-natural amino acid linked dolastatin derivatives - Google Patents

Abstract

The disclosure relates to non-natural amino acids and dolastatin analogs (formula (I)) that include at least one non-natural amino acid, and methods for making such non-natural amino acids and polypeptides. The dolastatin analogs can include a wide range of possible functionalities, but typically have at least one oxime, carbonyl, dicarbonyl, and/or hydroxylamine group. Also disclosed herein are non-natural amino acid dolastatin analogs that are further modified post-translationally, methods for effecting such modifications, and methods for purifying such dolastatin analogs. Further disclosed are methods for using such non-natural amino aciddolastatin analogs and modified non-natural amino acid dolastatin analogs, including therapeutic, diagnostic, and other biotechnology use. ve at least one oxime, carbonyl, dicarbonyl, and/or hydroxylamine group. Also disclosed herein are non-natural amino acid dolastatin analogs that are further modified post-translationally, methods for effecting such modifications, and methods for purifying such dolastatin analogs. Further disclosed are methods for using such non-natural amino aciddolastatin analogs and modified non-natural amino acid dolastatin analogs, including therapeutic, diagnostic, and other biotechnology use.

Description

COMPOSITIONS CONTAINING, METHODS INVOLVING, AND USES OF NON-NATURAL AMINO ACID LINKED DOLASTATIN DERIVATIVES CROSS REFERENCE This application claims priority to US. ional ation No. 61/491,076, entitled, “Compositions Containing, Methods Involving, and Uses of Non-Natural Amino Acid Linked Dolastatin Derivatives,” filed May 27, 2011, the contents of Which are incorporated by reference in its entirety.

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

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

SUMMARY OF THE INVENTION sed herein are toxic moieties with one or more linker(s), toxic groups linked to non-natural amino acids, and methods for making such non-natural amino acids and polypeptides.

Some embodiments of the present invention describe a compound, or salt thereof, comprising Formula (1): Me Me 0M9 /L\ N Y (I) '1“ a “H Me R7 O /\ Me OMeO Me Me MeO NH wherein: Z has the structure of: ‘7;\KKAr R5 is H, CORg, C1-C6alkyl, or thiazole; R8 is OH; R6 is OH or H; Ar is phenyl or pyridine; R7 is C1-C6alkyl or hydrogen; Y is selected from the group consisting of an hydroxylamine, , aldehyde, protected aldehyde, ketone, protected ketone, thioester, ester, dicarbonyl, hydrazine, amidine, imine, diamine, azide, keto-amine, keto-alkyne, alkyne, cycloalkyne, and ene-dione; L is—alkylene— or —(alkylene—O)n—alkylene—; and n is an integer greater than or equal to one.

In some embodiments, R5 is thiazole. In other embodiments, R6 is H. In certain embodiments, Ar is phenyl. In further or onal embodiments, R7 is methyl. In some embodiments, n is an integer from Ot020,0to 10 orOtoS.

In some embodiments, a compound is described comprising Formula (II): Me Me H N2 L H " \0/ \'\Il Me (11) : N R7 o A Me OMeO Me Me MeO NH In certain embodiments, L is —(alkylene—O)n—alkylene—. In specific embodiments, each alkylene is — CH2CH2—, n is equal to 3, and R7 is methyl. In other embodiments, L is —alkylene—. In specific embodiments, each alkylene is —CH2CH2— and R7 is methyl or hydrogen.

In some ments, Y is azide. In other embodiments, Y is cyclooctyne. In specific ments, the cyclooctyne has a structure of: each R19 is ndently selected from the group consisting of C1-C6 alkyl, C1-C6 , ester, ether, thioether, aminoalkyl, halogen, alkyl ester, aryl ester, amide, aryl amide, alkyl halide, alkyl amine, alkyl sulfonic acid, alkyl nitro, thioester, sulfonyl ester, halosulfonyl, nitrile, alkyl e, and nitro; and qis 0,1, 2, 3, 4, 5, 6, 7, 8, 9,10 or 11.

Certain embodiments of the present invention describe a compound sing Formula (VIII): wherein: A is optional, and when present is lower ne, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene; B is al, and when present is a linker selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower alkylene, substituted lower heteroalkylene, -O-, -O-(alkylene or substituted alkylene)-, - S-, -S-(alkylene or substituted ne)-, -S(O)k- where k is l, 2, or 3, -S(O)k(alkylene or substituted alkylene)-, -C(O)—, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)- (alkylene or substituted alkylene)-, -N(R’)-, -NR’-(alkylene or substituted alkylene)-, -C(O)N(R’)-, ’)-(alkylene or substituted alkylene)-, -CSN(R’)-, ’)-(alkylene or substituted ne)-, -N(R’)CO-(alkylene or substituted alkylene)-, -N(R’)C(O)O-, 'S(O)kN(R’)'a -N(R’)C(0)N(R’)-, _N(R’)C(S)N(R’)_a -N(R’)S(O)kN(R’)-, -N(R’)-N=, - N—, -C(R’)=N—N(R’)-, -C(R’)=N—N=, -C(R’)2-N=N-, and -C(R’)2-N(R’)-N(R’)-, where each R’ is independently H, alkyl, or substituted alkyl, R is H, alkyl, substituted alkyl, cycloalkyl, or tuted cycloalkyl; R1 is H, an amino protecting group, resin, at least one amino acid, polypeptide, or polynucleotide; R2 is OH, an ester protecting group, resin, at least one amino acid, polypeptide, or cleotide; R3 and R4 are each independently H, n, lower alkyl, or substituted lower alkyl, or R3 and R4 or two R3 groups optionally form a cycloalkyl or a heterocycloalkyl; Z has the structure of: ‘7;\KKAr R5 ; R5 is H, COZH, C1-C6alkyl, or thiazole; R6 is OH or H; Ar is phenyl or pyridine; R7 is C1-C6alkyl or hydrogen; and L is—alkylene— or —(alkylene—O)n—alkylene—; and n is an r greater than or equal to one; or an active metabolite, or a pharmaceutically acceptable prodrug or solvate thereof.

In some embodiments, R1 is a polypeptide. In specific embodiments, the polypeptide is an antibody. In certain specific embodiments, the dy is tin. In other embodiments, R2 is a polypeptide. In specific embodiments, the polypeptide is an antibody. In certain specific embodiments, the antibody is herceptin.

In some ments, provided herein is a method for tizing a dolastatin analog sing Formula (I), the method comprising contacting the dolastatin analog With a t of Formula (XXXVII), wherein Formula (I) correspond to: wax,R70 /\ Me OMeO wherein: Z has the structure of: €Yé\(KAr R5 ; R5 is H, CORg, C1-C6alkyl, or thiazole; R8 is OH; R6 is OH or H; Ar is phenyl or pyridine; R7 is C1-C6 alkyl or hydrogen; Y is NHg—O— or methyl; L is—alkylene— or —(alkylene—O)n—alkylene—; and n is an integer greater than or equal to one; wherein Formula (XXXVII) corresponds to: R3 A\ /K\ R3 B R (XXXVII) R1\ R2 wherein: A is optional, and when t is lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, e, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene; B is optional, and when present is a linker selected from the group ting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted ne)-, -S(O)k- where k is 1, 2, or 3, -S(O)k(alkylene or substituted alkylene)-, -C(O)-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R’)-, -NR’-(alkylene or substituted alkylene)-, -C(O)N(R’)-, -CON(R’)-(alkylene or substituted alkylene)-, -CSN(R’)-, -CSN(R’)-(alkylene or substituted alkylene)-, CO-(alkylene or substituted alkylene)-, C(0)0-, -S(0)kN(R’)-, -N(R’)C(0)N(R’)-, _N(R’)C(S)N(R’)_a S(O)kN(R’)-, -N(R’)-N=, =N—, -C(R’)=N—N(R’)-, -C(R’)=N—N=, -C(R’)2-N=N-, and -C(R’)2-N(R’)-N(R’)-, where each R’ is independently H, alkyl, or substituted alkyl, each R’ is independently H, alkyl, or substituted alkyl, R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; R1 is H, an amino protecting group, resin, at least one amino acid, or cleotide; R2 is OH, an ester ting group, resin, at least one amino acid, or polynucleotide; and R3 and R4 are each independently H, halogen, lower alkyl, or substituted lower alkyl, or R3 and R4 or two R3 groups optionally form a cycloalkyl or a heterocycloalkyl.

In some embodiments, the derivatized dolastatin analog comprises at least one oxime containing amino acid haVing the structure of Formula (VIII): In specific embodiments, the dolastatin analog is ted With the reagent of Formula (XXXVII) in aqueous solution under mildly acidic conditions.

Certain embodiments of the present invention describe a compound comprising Formula (XXV): (R16)n_/TqNJ/L\NNIR;ETMMi%Ne%| R7 OMeAMeMe OMeOM (XXV) wherein: Z has the structure of: Jew/KN R5 ; R5 is H, COZH, lkyl, or thiazole; R6 is OH or H; Ar is phenyl or pyridine; R1 is H, an amino protecting group, resin, at least one amino acid, polypeptide, or polynucleotide; R2 is OH, an ester protecting group, resin, at least one amino acid, polypeptide, or polynucleotide; R4 is H, halogen, lower alkyl, or substituted lower alkyl; R7 is C1-C6alkyl or hydrogen; L is—alkylene— or —(alkylene—O)n—alkylene—; and n is an integer r than or equal to one; each R16 is independently selected from the group consisting of hydrogen, halogen, alkyl, N02, CN, and substituted alkyl.

In some embodiments, R1 is a polypeptide. In specific embodiments, the polypeptide is an antibody. In certain specific embodiments, the antibody is herceptin. In other embodiments, R2 is a polypeptide. In specific embodiments, the polypeptide is an antibody. In certain specific embodiments, the antibody is tin.

Some embodiments of the present invention be a compound comprising a (XXXI): \/L\:gr\)LRRH 33 R7 O /\ Me OMeO R2 MeO NH (XXXI) wherein: Z has the structure of: €Yé\(KAr R5 ; R5 is H, COZH, C1-C6alkyl, or thiazole; R6 is OH or H; Ar is phenyl or pyridine; R1 is H, an amino protecting group, resin, at least one amino acid, polypeptide, or polynucleotide; R2 is OH, an ester protecting group, resin, at least one amino acid, polypeptide, or polynucleotide; R4 is H, halogen, lower alkyl, or substituted lower alkyl; R7 is C1-C6alkyl or hydrogen; L is—alkylene— or —(alkylene—O)n—alkylene—; and n is an integer greater than or equal to one; D has the structure of: R I | ) , $2 17 R17 R17 R17 R17 R N\ R N\ ‘zL ”Y $1 ”Y .5 M N n R17/T3 , , R17/T3 , H H H N N N a— \_5 s1 9—; g— /2 K / N N N H H ’ H each R17 is independently selected from the group consisting of H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, alkylalkoxy, substituted lkoxy, polyalkylene oxide, substituted polyalkylene oxide, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkaryl, substituted l, l, substituted l, - (alkylene or substituted alkylene)-ON(R”)2, -(alkylene or substituted alkylene)-C(O)SR”, - (alkylene or substituted alkylene)-S-S-(aryl or substituted aryl), -C(O)R”, R”, or -C(O)N(R”)2, Wherein each R” is independently hydrogen, alkyl, substituted alkyl, alkenyl, tuted alkenyl, alkoxy, substituted , aryl, substituted aryl, heteroaryl, alkaryl, substituted alkaryl, aralkyl, or substituted aralkyl; each 21 is a bond, CR17R17, O, S, NR’, CR17R17-CR17R17, CR17R17-O, O-CR17R17, CR17R17-S, S- CR17R17, 7-NR’, or NR’-CR17R17; each R’ is H, alkyl, or substituted alkyl; each 22 is selected from the group consisting of a bond, -C(O)-, , optionally substituted C1-C3 alkylene, optionally substituted C1-C3 alkenylene, and optionally substituted heteroalkyl; each Z3 are independently selected from the group consisting of a bond, optionally substituted C1-C4 alkylene, optionally substituted C1-C4 alkenylene, optionally substituted heteroalkyl, -O-, -S-, -C(O)-, -C(S)-, and -N(R’)-; each T3 is a bond, C(R”)(R”), O, or S; With the proviso that When T3 is O or S, R” cannot be halogen; each R” is H, halogen, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; m and p are 0, l, 2, or 3, provided that at least one ofm or p is not 0; b b “T” ,3 as) 03) 45,3" I m m b) /C—C\_§(b) (202$R4 § 03) /C\—O—E(b)E/R—S—g (b) M2 is (21) R3 R4 (J‘s R4 @2114 (a) (b) as) (b) (b) aw R3 JJJ‘ R3 “’W' Rs I \ / \ / i—d—E /c=(|:—E (b) O—T—E (b) S_T—§ (b) (b) R3/ \R4 >55 R4 m m m (a) (a) (a) or (a) Where (a) indicates g a , , , to the B group and (b) indicates bonding to respective positions Within the heterocycle group; (b) (b) (b) “if” /R3 Ego) /|c|—C/R—3§<b> )‘3—0‘EG’ j—S—EG’) E—ng(b) M3 is (21:1; 00%? \R4 (3)2 ? 4 or Where , , (a) tes bonding to the B group and (b) indicates bonding to respective positions Within the heterocycle group; (b) (b) £3, (b) (b) q'“ “1"" “I“ “I“ /C\—<;=§ (b) Rs—C_C=g (b) o—c: (b) s—c: (b) 19/0—g (b) l, (J; (f (g3 M41s (a) (a) R3 R3 (a) (a) or (a) Where , , , , , (a) indicates bonding to the B group and (b) indicates bonding to tive ons Within the heterocycle group; each R19 is independently selected from the group consisting of C1-C6 alkyl, C1-C6 alkoxy, ester, ether, thioether, aminoalkyl, halogen, alkyl ester, aryl ester, amide, aryl amide, alkyl halide, alkyl amine, alkyl sulfonic acid, alkyl nitro, thioester, sulfonyl ester, halosulfonyl, nitrile, alkyl nitrile, and nitro; qis 0,1,2, 3, 4, 5, 6, 7, 8, 9,10 or 11; and each R16 is independently selected from the group consisting of hydrogen, halogen, alkyl, N02, CN, and substituted alkyl.

In some ments, R1 is a polypeptide. In specific embodiments, the polypeptide is an antibody. In certain specific ments, the antibody is tin. In other embodiments, R2 is a polypeptide. In specific embodiments, the polypeptide is an antibody. In certain specific ments, the antibody is herceptin.

In some embodiments, a compound is described comprising Formula (XXXI-A): ,EQI21M%%=N R7 OMe/\M Me OMeO H'?‘ R1 R2 In n embodiments, a pharmaceutical composition is provided comprising any of the compounds described and a pharmaceutically acceptable carrier, excipient, or binder.

In further or alternative embodiments are methods for detecting the presence of a polypeptide in a patient, the method comprising stering a ptide comprising at least one heterocycle- containing non—natural amino acid and the resulting heterocycle-containing non-natural amino acid polypeptide modulates the immunogenicity of the polypeptide relative to the gous naturally- occurring amino acid polypeptide.

It is to be understood that the methods and compositions described herein are not limited to the particular methodology, protocols, cell lines, constructs, and reagents bed herein and as such may vary. It is also to be understood that the terminology used herein is for the purpose of describing ular 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 and1n the appended claims, the singular forms 44a 39 ccan,” and “the” include plural reference unless the t clearly indicates otherwise.

Unless defined otherwise, all technical and scientific terms used herein have the same g as commonly understood to one of ordinary skill in the art to which the inventions described herein belong.

Although any methods, devices, and materials similar or lent to those described herein can be used in the practice or testing of the inventions described herein, the preferred methods, devices and materials are now described.

All publications and patents mentioned herein are incorporated herein by reference in their entirety for the purpose of describing and disclosing, for example, the constructs and methodologies that are bed in the publications, which might be used in connection with the presently described inventions. The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the inventors described herein are not entitled to antedate such disclosure by Virtue of prior invention or for any other reason.

The terms “aldol-based linkage” or “mixed aldol-based linkage” refers to the acid— or base- zed condensation of one carbonyl compound with the enolate/enol of another carbonyl nd, which may or may not be the same, to generate a B-hydroxy carbonyl compound—an aldol.

The term “affinity label,” as used herein, refers to a label which reversibly or irreversibly binds r molecule, either to modify it, destroy it, or form a compound with it. By way of example, affinity labels e enzymes and their substrates, or antibodies and their antigens.

The terms “alkoxy,” amino” and “alkylthio” (or thioalkoxy) are used in their tional sense, and refer to those alkyl groups linked to molecules Via an oxygen atom, an amino group, or a sulfur atom, respectively.

The term “alkyl,” by itself or as part of another molecule means, unless otherwise stated, a ht or branched chain, or cyclic arbon radical, or combination thereof, which may be fully saturated, mono- or polyunsaturated and can include di- and multivalent radicals, having the number of carbon atoms designated (Le. C1-C10 means one to ten carbons). Examples of saturated hydrocarbon ls include, but are not limited to, groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, t-butyl, isobutyl, tyl, cyclohexyl, (cyclohexyl)methyl, cyclopropylmethyl, homologs and isomers of, for example, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. An unsaturated alkyl group is one having one or more double bonds or triple bonds. Examples of unsaturated alkyl groups include, but are not limited to, vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(l,4-pentadienyl), ethynyl, l- and 3-propynyl, 3-butynyl, and the higher homologs and isomers. The term “alkyl,” unless otherwise noted, is also meant to include those derivatives of alkyl defined in more detail herein, 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, n n may be 1 to about 24. By way of example only, such groups include, but are not d to, groups having 10 or fewer carbon atoms such as the structures — CH2CH2— and —CH2CH2CH2CH2—. A “lower alkyl” or “lower alkylene” is a shorter chain alkyl or alkylene group, generally having eight or fewer carbon atoms. The term ene,” unless otherwise noted, is also meant to include those groups described herein as “heteroalkylene.” The term “amino acid” refers to naturally occurring and non-natural amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the lly ing amino acids. Naturally encoded amino acids are the 20 common amino acids (alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, ic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and ) and pyrolysine and selenocysteine. Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally ing amino acid, by way of example only, an (x-carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group. Such analogs may have modified R groups (by way of example, norleucine) or may have modified peptide backbones while still retaining the same basic chemical structure as a naturally occurring amino acid. Non-limiting examples of amino acid analogs include homoserine, cine, methionine sulfoxide, methionine methyl sulfonium.

Amino acids may be referred to herein by either their name, their commonly known three letter s or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. onally, nucleotides, may be referred to by their ly accepted single-letter codes.

An “amino terminus ation group” refers to any molecule that can be attached to a terminal amine group. By way of example, such terminal amine groups may be at the end of polymeric molecules, wherein such ric molecules include, but are not limited to, polypeptides, polynucleotides, and polysaccharides. Terminus modification groups include but are not limited to, various water soluble polymers, es or proteins. By way of example only, terminus modification groups include polyethylene glycol or serum albumin. Terminus modification groups may be used to modify therapeutic characteristics of the polymeric molecule, including but not limited to increasing the serum half-life of peptides.

By “antibody fragment” is meant any form of an antibody other than the full-length form. dy fragments herein include dies that are r components that exist within full-length antibodies, and antibodies that have been engineered. Antibody fragments include but are not limited to Fv, Fc, Fab, and (Fab')2, single chain Fv (scFv), diabodies, triabodies, tetrabodies, bifunctional hybrid antibodies, CDRl, CDR2, CDR3, combinations of CDR’s, variable regions, framework regions, constant regions, heavy , light chains, and variable regions, and alternative scaffold non-antibody molecules, bispecific antibodies, and the like (Maynard & Georgiou, 2000, Annu. Rev. Biomed. Eng. 2:339-76; Hudson, 1998, Curr. Opin. Biotechnol. 402). Another functional substructure is a single chain Fv (scFv), comprised of the variable regions of the immunoglobulin heavy and light chain, ntly connected by a peptide linker (S-z Hu et al., 1996, Cancer Research, 56, 3055-3061). These small (Mr ,000) proteins generally retain specificity and affinity for antigen in a single polypeptide and can provide a convenient building block for larger, antigen-specific molecules. Unless specifically noted otherwise, statements and claims that use the term “antibody” or “antibodies” specifically includes “antibody fragment” and “antibody fragments.” By “antibody-drug conjugate, or “ADC”, as used herein, refers to an antibody molecule, or nt thereof, that is covalently bonded to one or more biologically active molecule(s). The biologically active molecule may be conjugated to the antibody through a linker, polymer, or other covalent bond.

The term “aromatic” or “aryl”, as used herein, refers to a closed ring structure which has at least one ring having a conjugated pi electron system and includes both carbocyclic aryl and heterocyclic aryl (or “heteroaryl” or “heteroaromatic”) groups. The carbocyclic or heterocyclic aromatic group may contain from 5 to 20 ring atoms. The term includes monocyclic rings linked covalently or fused-ring polycyclic (i.e., rings which share adjacent pairs of carbon atoms) groups. An aromatic group can be tituted or substituted. Non-limiting examples of “aromatic” or “aryl”, groups include phenyl, 1-naphthyl, 2- naphthyl, 4-biphenyl, anthracenyl, and phenanthracenyl. tuents for each of the above noted aryl and heteroaryl ring s are selected from the group of acceptable substituents described herein.

For brevity, the term “aromatic” or “aryl” when used in combination with other terms ding but not limited to, aryloxy, arylthioxy, aralkyl) includes both aryl and heteroaryl rings as defined above.

Thus, the term “aralkyl” or “alkaryl” is meant to include those radicals in which an aryl group is attached to an alkyl group (including but not limited to, benzyl, phenethyl, pyridylmethyl and the like) including those alkyl groups in which a carbon atom (including but not limited to, a methylene group) has been replaced by a heteroatom, by way of example only, by an oxygen atom. Examples of such aryl groups include, but are not limited to, phenoxymethyl, dyloxymethyl, 3-(1-naphthyloxy)propyl, and the like.

The term “arylene”, as used herein, refers to a divalent aryl radical. miting examples of ne” include phenylene, pyridinylene, pyrimidinylene and thiophenylene. Substituents for arylene groups are selected from the group of acceptable substituents bed herein.

A “bifunctional polymer”, also referred to as a “bifunctional linker”, refers to a polymer comprising two functional groups that are capable of reacting specifically with other moieties to form nt or non-covalent linkages. Such moieties may include, but are not limited to, the side groups on natural or non-natural amino acids or peptides which contain such natural or non-natural amino acids. The other es that may be linked to the bifunctional linker or bifunctional polymer may be the same or ent moieties. By way of example only, a bifunctional linker may have a functional group ve with a group on a first peptide, and another functional group which is reactive with a group on a second peptide, whereby forming a conjugate that includes the first peptide, the bifunctional linker and the second peptide. Many procedures and linker molecules for attachment of various compounds to peptides are known. See, e.g., European Patent Application No. 188,256; US. Patent Nos. 958, 4,659,839, 4,414,148, 4,699,784; 4,680,338; and 4,569,789 which are incorporated by reference herein in their entirety. A -functional polymer” also referred to as a “multi-functional linker”, refers to a polymer comprising two or more functional groups that are capable of reacting with other moieties. Such moieties may e, but are not limited to, the side groups on natural or tural amino acids or es which contain such natural or non-natural amino acids. (including but not limited to, amino acid side groups) to form covalent or non-covalent linkages. A bi-functional polymer or functional polymer may be any d length or molecular weight, and may be selected to provide a particular desired spacing or conformation between one or more molecules linked to a compound and molecules it binds to or the compound.

The term “bioavailability,” as used herein, refers to the rate and extent to which a substance or its active moiety is delivered from a pharmaceutical dosage form and becomes available at the site of action or in the general ation. Increases in ilability refers to increasing the rate and extent a substance or its active moiety is delivered from a pharmaceutical dosage form and becomes available at the site of action or in the general circulation. By way of example, an increase in bioavailability may be indicated as an increase in concentration of the substance or its active moiety in the blood when compared to other substances or active moieties. A non-limiting example of a method to evaluate increases in ilability is given in examples 21-25. This method may be used for ting the ilability of any polypeptide.

The term “biologically active molecule”, “biologically active moiety” or “biologically active agent” when used herein means any substance which can affect any physical or biochemical properties of a biological system, y, molecule, or interaction relating to an organism, ing but not limited to, Viruses, bacteria, bacteriophage, oson, prion, insects, fungi, plants, animals, and humans. In particular, as used herein, biologically active molecules include but are not limited to any substance intended for diagnosis, cure, mitigation, treatment, or prevention of e in humans or other animals, or to otherwise enhance physical or mental well-being of humans or animals. Examples of biologically active molecules include, but are not limited to, peptides, proteins, enzymes, small le drugs, hard drugs, soft drugs, prodrugs, carbohydrates, inorganic atoms or molecules, dyes, lipids, nucleosides, radionuclides, oligonucleotides, toxins, cells, Viruses, liposomes, microparticles and micelles. Classes of biologically active agents that are suitable for use with the methods and compositions described herein include, but are not limited to, drugs, gs, radionuclides, imaging agents, polymers, antibiotics, fungicides, anti-Viral agents, anti-inflammatory agents, anti-tumor agents, cardiovascular , anti- anxiety agents, hormones, grth factors, steroidal agents, microbially derived toxins, and the like.

By “modulating biological actiVity” is meant increasing or decreasing the reactiVity of a polypeptide, altering the iVity of the polypeptide, enhancing or decreasing the substrate selectiVity of the polypeptide. Analysis of modified biological actiVity can be performed by comparing the biological ty of the non-natural ptide to that of the natural ptide.

The term “biomaterial,” as used herein, refers to a biologically-derived material, including but not limited to material obtained from bioreactors and/or from recombinant methods and techniques.

The term ysical probe,” as used herein, refers to probes which can detect or monitor structural s in molecules. Such les include, but are not limited to, proteins and the “biophysical probe” may be used to detect or monitor interaction of proteins with other macromolecules.

Examples of biophysical probes include, but are not limited to, spin-labels, a fluorophores, and photoactivatible groups.

The term “biosynthetically,” as used herein, refers to any method utilizing a translation system (cellular or non-cellular), including use of at least one of the following components: a polynucleotide, a codon, a tRNA, and a ribosome. By way of example, non-natural amino acids may be “biosynthetically incorporated” into non-natural amino acid polypeptides using the s and techniques described herein, “In vivo tion of polypeptides comprising non-natural amino acids”, and in the miting example 20. Additionally, the methods for the selection of useful non-natural amino acids which may be “biosynthetically incorporated” into non-natural amino acid polypeptides are described in the non-limiting examples 20.

The term “biotin analogue,” or also referred to as “biotin mimic”, as used herein, is any molecule, other than biotin, which bind with high affinity to avidin and/or streptavidin.

The term “carbonyl” as used herein refers to a group containing at a moiety ing from the group consisting of -C(O)-, -S(O)-, -S(O)2—, and —C(S)-, including, but not limited to, groups containing a least one ketone group, and/or at least one aldehyde groups, and/or at least one ester group, and/or at least one carboxylic acid group, and/or at least one thioester group. Such yl groups include s, aldehydes, carboxylic acids, esters, and ters. In addition, such groups may be part of linear, branched, or cyclic molecules.

The term “carboxy terminus modification group” refers to any molecule that can be attached to a terminal carboxy group. By way of example, such terminal carboxy groups may be at the end of ric molecules, wherein such polymeric molecules include, but are not limited to, polypeptides, polynucleotides, and polysaccharides. Terminus modification groups e but are not limited to, various water soluble polymers, peptides or proteins. By way of e only, terminus modification groups include polyethylene glycol or serum albumin. Terminus modification groups may be used to modify therapeutic characteristics of the polymeric molecule, including but not limited to increasing the serum half-life of peptides.

The term “chemically cleavable ” also referred to as “chemically labile”, as used herein, refers to a group which breaks or cleaves upon exposure to acid, base, oxidizing agents, reducing agents, chemical inititiators, or radical initiators.

The term “chemiluminescent group,” as used herein, refers to a group which emits light as a result of a chemical reaction without the addition of heat. By way of e only, luminol (5-amino-2,3- dihydro-l,4-phthalazinedione) reacts with oxidants like hydrogen de (H202) in the presence of a base and a metal catalyst to produce an excited state product (3-aminophthalate, 3-APA).

The term “chromophore,” as used herein, refers to a molecule which absorbs light of e wavelengths, UV wavelengths or IR wavelengths.

The term “cofactor,” as used herein, refers to an atom or molecule ial for the action of a large le. Cofactors include, but are not limited to, inorganic ions, mes, proteins, or some other factor necessary for the ty of enzymes. Examples include heme in obin, magnesium in chlorophyll, and metal ions for proteins.

“Cofolding,” as used herein, refers to ing processes, reactions, or methods which employ at least two molecules which interact with each other and result in the transformation of unfolded or improperly folded molecules to properly folded molecules. By way of example only, “cofolding,” employ at least two polypeptides which interact with each other and result in the transformation of unfolded or improperly folded polypeptides to native, properly folded polypeptides. Such polypeptides may contain natural amino acids and/or at least one tural amino acid.

A “comparison window,” as used herein, refers a t of any one of contiguous positions used to compare a sequence to a nce sequence of the same number of contiguous positions after the two sequences are optimally aligned. Such contiguous ons include, but are not limited to a group consisting of from about 20 to about 600 sequential units, including 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 containing non-natural amino acids, with the sequential units include, but are not limited to natural and non-natural amino acids. In addition, by way of example only, such ces include polynucleotides with nucleotides being the corresponding sequential units. Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison can be conducted, including but not limited to, by the local homology algorithm of Smith and Waterman (1970) Adv. Appl. Math. 2:482c, by the gy alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. , by the search for rity method of Pearson and Lipman (1988) Proc. Nat’l. Acad. Sci. USA 852444, by computerized entations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI), or by manual ent and Visual inspection (see, e. g., Ausubel et al., t Protocols in Molecular Biology (1995 supplement)).

By way of example, an algorithm which may be used to ine percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1997) Nuc. Acids Res. 25:3389-3402, and Altschul et al. (1990) J. Mol. Biol. 215:403-410, respectively.

Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. The BLAST algorithm parameters W, T, and X determine the iVity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a wordlength (W) of 11, an expectation (E) or 10, M=5, N=-4 and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a wordlength of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see ff and Henikoff (1992) Proc. Natl. Acad. Sci. USA 89:10915) alignments (B) of 50, expectation (E) of 10, M=5, N=-4, and a comparison of both strands. The BLAST algorithm is typically performed with the “low complexity” filter turned off. 2012/039468 The BLAST algorithm also performs a statistical analysis of the rity between two sequences (see, e. g., Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5787). One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a reference ce if the smallest sum probability in a ison of the test nucleic acid to the reference nucleic acid is less than about 0.2, or less than about 0.01, or less than about 0.001.

The term “conservatively modified variants” applies to both natural and non-natural amino acid and natural and non-natural nucleic acid ces, and combinations thereof With respect to particular nucleic acid sequences, “conservatively modified variants” refers to those l and non-natural nucleic acids which encode identical or essentially identical natural and non-natural amino acid sequences, or where the natural and non-natural nucleic acid does not encode a natural and non-natural amino acid sequence, to essentially identical sequences. By way of e, because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an e is specified by a codon, the codon can be altered to any of the corresponding codons described t altering the encoded polypeptide. Such nucleic acid variations are “silent variations,” which are one species of vatively d variations. Thus by way of example every natural or non-natural nucleic acid sequence herein which encodes a natural or tural polypeptide also describes every le silent variation of the natural or non-natural nucleic acid. One of ry skill in the art will recognize that each codon in a natural or non-natural nucleic acid (except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan) can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a natural and tural c acid which encodes a natural and non-natural polypeptide is implicit in each bed sequence.

As to amino acid sequences, individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single natural and non-natural amino acid or a small tage of natural and non-natural amino acids in the encoded sequence is a “conservatively modified t” where the alteration results in the deletion 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 providing 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 gs, and alleles of the methods and compositions described herein.

Conservative substitution tables providing 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 for one another: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); ) cine (I), Leucine (L), nine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), phan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M) (see, e. g., ton, Proteins:Structures and Molecular Properties (W H Freeman & Co.; 2nd edition (December 1993) The terms “cycloalkyl” and “heterocycloalkyl”, by themselves or in combination with other terms, represent, unless otherwise stated, cyclic ns of “alkyl” and “heteroalkyl”, respectively. Thus, a cycloalkyl or heterocycloalkyl include saturated, partially rated and fully unsaturated ring linkages. Additionally, for heterocycloalkyl, a heteroatom can occupy the position at which the heterocycle is attached to the remainder of the molecule. The heteroatom may include, but is not limited to, oxygen, nitrogen or sulfur. Examples of cycloalkyl e, but are not limited to, cyclopentyl, cyclohexyl, l-cyclohexenyl, 3-cyclohexenyl, eptyl, and the like. es of heterocycloalkyl include, but are not limited to, l—(l,2,5,6-tetrahydropyridyl), l-piperidinyl, 2-piperidinyl, 3-piperidinyl, 4- morpholinyl, holinyl, tetrahydrofuran—2-yl, tetrahydrofuran—3-yl, tetrahydrothienyl, ydrothien—3-yl, l—piperazinyl, 2-piperazinyl, and the like. Additionally, the term encompasses multicyclic structures, ing but 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 ydrates consisting of at least six to eight glucose molecules in a ring formation. The outer part of the ring contains water soluble groups; at the center of the ring is a vely nonpolar caVity able to accommodate small les.

The term “cytotoxic,” as used herein, refers to a compound which harms cells.

“Denaturing agent” or “denaturant,” as used herein, refers to any compound or material which will cause a reversible unfolding of a polymer. By way of example only, “denaturing agent” or “denaturants,39 may cause a reversible unfolding of a protein. The strength of a denaturing agent or denaturant will be determined both by the properties and the concentration of the particular denaturing agent or denaturant. By way of example, denaturing agents or denaturants include, but are not limited to, chaotropes, detergents, organic, water le solvents, phospholipids, or a combination thereof. Nonlimiting 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 e, or polyoxyethylene ethers (e. g. Tween or Triton detergents), Sarkosyl, mild non-ionic detergents (e.g., digitonin), mild cationic detergents such as -(Dioleyoxy)-propyl-N,N,N- trimethylammonium, mild ionic detergents (e. g. sodium cholate or sodium holate) or zwitterionic detergents including, but not limited to, sulfobetaines (Zwittergent), 3-(3- chlolamidopropyl)dimethylammonio-l -propane sulfate (CHAPS), and 3 -(3 - chlolamidopropyl)dimethylammoniohydroxy-l-propane sulfonate (CHAPSO). 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 alkandiols (C2 - C4 alkandiols such as ne-glycol) may be used as denaturants. Non-limiting examples of phospholipids include, but are not limited to, naturally ing phospholipids such as phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, and phosphatidylinositol or synthetic phospholipid derivatives or variants such as dihexanoylphosphatidylcholine or diheptanoylphosphatidylcholine.

The term “desired functionality” as used herein refers to any group selected from a label; a dye; a polymer; a soluble polymer; a derivative of polyethylene glycol; a photocrosslinker; a cytotoxic compound; a drug; an affinity label; a photoaff1nity label; a reactive compound; a resin; a second protein or polypeptide or polypeptide analog; an antibody or antibody fragment; a metal chelator; a cofactor; a fatty acid; a carbohydrate; a polynucleotide; a DNA; a RNA; an antisense polynucleotide; a saccharide, a water-soluble dendrimer, a cyclodextrin, a biomaterial; a nanoparticle; a spin label; a fluorophore; a metal-containing moiety; a ctive moiety; a novel functional group; a group that covalently or noncovalently interacts with other molecules; a photocaged moiety; an actinic radiation excitable ; a ligand; a photoisomerizable moiety; biotin; a biotin analogue; a moiety incorporating a heavy atom; a chemically ble group; a leavable group; an elongated side chain; a carbon-linked sugar; a redox-active agent; an amino thioacid; a toxic moiety; an isotopically d moiety; a biophysical probe; a phosphorescent group; a chemiluminescent group; an electron dense group; a magnetic group; an intercalating group; a chromophore; an energy transfer agent; a biologically active agent (in which case, the biologically active agent can include an agent with therapeutic ty and the tural amino acid polypeptide or modified non-natural amino acid can serve either as a co-therapeutic agent with the attached eutic agent or as a means for delivery the therapeutic agent to a desired site within an organism); a detectable label; a small molecule; an inhibitory cleic acid; a radionucleotide; a neutron-capture agent; a derivative of biotin; quantum dot(s); a nanotransmitter; a radiotransmitter; 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 f The term “diamine,”as used herein, refers to groups/molecules comprising at least two amine functional groups, including, but not limited to, a ine group, an amidine group, an imine group, a l,l-diamine group, a l,2-diamine group, a 1,3-diamine group, and a l,4-diamine group. In on, such groups may be part of linear, branched, or cyclic molecules.

The term “detectable label,” as used herein, refers to a label which may be observable using analytical techniques including, but not limited to, fluorescence, chemiluminescence, electron-spin resonance, ultraviolet/visible ance spectroscopy, mass spectrometry, nuclear magnetic resonance, magnetic resonance, and electrochemical s.

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

The term “drug,” as used herein, refers to any substance used in the prevention, diagnosis, ation, treatment, or cure of a disease or condition.

The term “dye,” as used herein, refers to a soluble, coloring substance which contains a chromophore.

The term “effective amount,” as used herein, refers to a sufficient amount of an agent or a compound being administered which will relieve to some extent one or more of the symptoms of the disease or condition being treated. The result can be reduction and/or alleviation of the signs, symptoms, or causes of a disease, or any other d alteration of a biological system. By way of example, an agent or a compound being administered includes, but is not limited to, a natural amino acid polypeptide, non- natural amino acid polypeptide, d natural amino acid polypeptide, or modified non-amino acid polypeptide. Compositions containing such natural amino acid polypeptides, non-natural amino acid polypeptides, modified natural amino acid ptides, or modified non-natural amino acid ptides can be administered for lactic, enhancing, and/or therapeutic treatments. An appropriate “effective” amount in any individual case may be determined using techniques, such as a dose escalation study.

The term “electron dense group,” as used herein, refers to a group which scatters 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%, indium trichloride anhydrous, lanthanum nitrate, lead acetate trihydrate, lead citrate trihydrate, lead nitrate, periodic acid, phosphomolybdic acid, otungstic acid, potassium ferricyanide, ium ferrocyanide, ruthenium red, silver nitrate, silver proteinate (Ag Assay: 80-85%) “Strong”, silver tetraphenylporphin (S-TPPS), sodium aurate, sodium tungstate, thallium nitrate, micarbazide (TSC), uranyl acetate, uranyl nitrate, and vanadyl sulfate.

The term “energy transfer agent,” as used herein, refers to a molecule which can either donate or accept energy from another molecule. By way of example only, cence nce energy transfer (FRET) is a dipole-dipole coupling process by which the excited-state energy of a fluorescence donor molecule is non-radiatively transferred to an unexcited acceptor molecule which then fluorescently emits the donated energy at a longer wavelength.

The terms “enhance” or “enhancing” means to increase or prolong either in potency or duration a desired effect. By way of example, “enhancing” the effect of eutic agents refers to the y to increase or prolong, either in potency or duration, the effect of therapeutic agents on during treatment of a disease, disorder or condition. An “enhancing-effective ,” as used , refers to an amount adequate to enhance the effect of a therapeutic agent in the treatment of a disease, disorder or condition.

When used in a patient, amounts effective for this use will depend on the severity and course of the e, disorder or condition, previous therapy, the patient's health status and response to the drugs, and the judgment of the treating physician.

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

The term “fatty acid,” as used herein, refers to carboxylic acids with about C6 or longer hydrocarbon side chain.

The term “fluorophore,” as used herein, refers to a molecule which upon tion emits photons and is thereby fluorescent.

The terms “functional group39 ccactive moiety:9 ccactivating group:9 ccleaVing group:9 ccreactive , , , , site”, cally reactive group” and “chemically reactive moiety,” as used herein, refer to portions or units of a molecule at which chemical reactions occur. The terms are somewhat synonymous in the chemical arts and are used herein to indicate the portions of molecules that perform some function or ty and are reactive with other molecules.

The term “halogen” includes fluorine, ne, iodine, and bromine.

The term “haloacyl,” as used herein, refers to acyl groups which contain halogen es, ing, but not limited to, -C(O)CH3, F3, -C(O)CH20CH3, and the like.

The term “haloalkyl,” as used herein, refers to alkyl groups which contain halogen moieties, including, but not limited to, -CF3 and —CH2CF3 and the like.

The term “heteroalkyl,” as used herein, refers to straight or branched chain, or cyclic hydrocarbon radicals, or combinations thereof, consisting of an alkyl group and at least one heteroatom selected from the group consisting of O, N, Si and S, and wherein the nitrogen and sulfur atoms may optionally be oxidized and the en heteroatom may optionally be quatemized. The heteroatom(s) O, N and S and Si may be placed at any interior position of the heteroalkyl group or at the position at which the alkyl group is attached to the remainder of the molecule. Examples include, but are not limited to, -CH2-CH2-O-CH3, - CHZ-CHZ-NH-CHg, -CH2-CH2-N(CH3)-CH3, -CH2-S-CH2-CH3, -CH2-CH2,-S(O)-CH3, H2-S(O)2- CH3, -CH=CH-O-CH3, -Si(CH3)3, -CH2-CH=N-OCH3, and —CH=CH-N(CH3)-CH3. In on, up to two heteroatoms may be consecutive, such as, by way of example, -CH2-NH-OCH3 and —CH2-O-Si(CH3)3.

The terms “heterocyclic-based linkage” or “heterocycle linkage” refers to a moiety formed from the reaction of a dicarbonyl group with a diamine group. The resulting reaction product is a heterocycle, including a heteroaryl group or a heterocycloalkyl group. The resulting heterocycle group serves as a chemical link between a non-natural amino acid or non-natural amino acid polypeptide and another functional group. In one ment, the heterocycle linkage es a nitrogen-containing heterocycle linkage, ing by way of example only a pyrazole linkage, a pyrrole linkage, an indole linkage, a benzodiazepine linkage, and a pyrazalone linkage. rly, the term “heteroalkylene” refers to a divalent l derived from heteroalkyl, as exemplified, but not limited by, -CH2-CH2-S-CH2-CH2- and —CH2-S-CH2-CH2-NH-CH2-. For heteroalkylene groups, the same or different heteroatoms can also occupy either or both of the chain termini (including but not limited to, alkyleneoxy, alkylenedioxy, alkyleneamino, alkylenediamino, aminooxyalkylene, and the like). Still r, for alkylene and heteroalkylene linking groups, no ation of the linking group is implied by the direction in which the formula of the linking group is written. By way of example, the a —C(O)2R’- represents both —C(O)2R’- and —R’C(O)2-.

The term “heteroaryl” or “heteroaromatic,” as used , refers to aryl groups which contain at least one heteroatom selected from N, O, and S; wherein the nitrogen and sulfur atoms may be optionally oxidized, and the nitrogen atom(s) may be optionally quatemized. Heteroaryl groups may be substituted or unsubstituted. A heteroaryl group may be attached to the remainder of the molecule through a heteroatom. Non-limiting examples of heteroaryl groups include olyl, 2-pyrrolyl, olyl, 3- pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyloxazolyl, 5-oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3- thienyl, 2-pyridyl, 3-pyridyl, dyl, 2-pyrimidyl, 4-pyrimidyl, othiazolyl, purinyl, 2- benzimidazolyl, 5-indolyl, l-isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl, olyl, and 6- quinolyl.

The term “homoalkyl,” as used herein refers to alkyl groups which are hydrocarbon .

The term “identical,” as used herein, refers to two or more sequences or subsequences which are the same. In addition, the term “substantially identical,” as used herein, refers to two or more sequences which have a percentage of sequential units which are the same when compared and aligned for m correspondence over a comparison , or designated region as measured using comparison algorithms or by manual alignment and visual inspection. By way of example only, two or more sequences may be “substantially identical” if the sequential units are about 60% identical, about 65% identical, about 70% identical, about 75% identical, about 80% identical, about 85% identical, about 90% identical, or about 95% cal over a ied region. Such percentages to describe the “percent ty” of two or more sequences. The identity of a sequence can exist over a region that is at least about 75-100 sequential units in length, over a region that is about 50 tial units in length, or, where not specified, across the entire 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 about 60% identical, about 65% cal, about 70% identical, about 75% identical, about 80% identical, about 85% identical, about 90% identical, or about 95% identical over a specified region. The identity can exist over a region that is at least about 75 to about 100 amino acids in length, over a region that is about 50 amino acids in length, or, where not specified, across the entire sequence of a ptide 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 cleotide sequences are “substantially identical” if the nucleic acid residues are about 60% identical, about 65% identical, about 70% cal, about 75% identical, about 80% identical, about 85% identical, about 90% identical, or about 95% identical over a specified region. The identity can exist over a region that is at least about 75 to about 100 nucleic acids in , over a region that is about 50 c acids in length, or, where not specified, across the entire sequence of a polynucleotide sequence.

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

The term “immunogenicity,” as used , refers to an antibody response to administration of a therapeutic drug. The immunogenicity toward therapeutic non-natural amino acid ptides can be obtained using quantitative and qualitative assays for detection of anti-non-natural amino acid ptides antibodies in biological fluids. Such assays include, but are not limited to, Radioimmunoassay (RIA), Enzyme-linked immunosorbent assay ), luminescent assay (LIA), and fluorescent immunoassay (FIA). Analysis of immunogenicity toward therapeutic non-natural amino acid polypeptides involves comparing the antibody response upon administration of therapeutic non-natural amino acid polypeptides to the antibody response upon administration of therapeutic natural amino acid polypeptides.

The term “intercalating agent,” also referred to as “intercalating group,” as used herein, refers to a al that can insert into the intramolecular space of a molecule or the intermolecular space between molecules. By way of example only an intercalating agent or group may be a molecule which inserts into the stacked bases of the DNA double helix.

The term “isolated,” as used herein, refers to separating and removing a component of interest from components not of interest. Isolated nces can be in either a dry or ry state, or in solution, including but not limited to an aqueous solution. The isolated component can be in a homogeneous state or the isolated component can be a part of a pharmaceutical composition that ses additional pharmaceutically acceptable carriers and/or excipients. Purity and homogeneity may be ined using analytical chemistry techniques including, but not limited to, polyacrylamide gel electrophoresis or high performance liquid chromatography. In addition, when a component of interest is isolated and is the predominant species present in a preparation, the ent is described herein as substantially purified. The term “purified,” as used herein, may refer to a component of st which is at least 85% pure, at least 90% pure, at least 95% pure, at least 99% or greater pure. By way of example only, nucleic acids or proteins are “isolated” when such nucleic acids or ns are free of at least some of the cellular components with which it is associated in the natural state, or that the nucleic acid or n has been concentrated to a level greater than the concentration of its in Vivo or in Vitro production.

Also, by way of example, a gene is isolated when separated from open reading frames which flank the gene and encode a protein other than the gene of interest.

The term ,” as used herein, refers to a nce which is orated into a compound and is readily detected, whereby its physical distribution may be detected and/or monitored.

The term “linkage,” as used herein to refer to bonds or chemical moiety formed from a chemical reaction between the functional group of a linker and r molecule. Such bonds may e, but are not limited to, covalent linkages and non-covalent bonds, while such chemical moieties may include, but are not limited to, esters, carbonates, imines phosphate esters, hydrazones, acetals, orthoesters, peptide linkages, and oligonucleotide linkages. Hydrolytically stable linkages means that the es are substantially stable in water and do not react with water at useful pH values, including but not limited to, under physiological conditions for an extended period of time, perhaps even indefinitely. Hydrolytically unstable or degradable linkages mean that the linkages are degradable in water or in aqueous solutions, including for example, blood. Enzymatically unstable or degradable linkages mean that the linkage can be degraded by one or more enzymes. By way of example only, PEG and related polymers may include degradable linkages in the polymer backbone or in the linker group between the polymer backbone and one or more of the al fianctional groups of the r molecule. Such degradable linkages include, but are not limited to ester linkages formed by the on of PEG carboxylic acids or activated PEG carboxylic acids with alcohol groups on a biologically active agent, wherein such ester groups generally yze under physiological conditions to release the biologically active agent. Other hydrolytically degradable linkages e but are not limited to carbonate linkages; imine linkages resulted from reaction of an amine and an aldehyde; phosphate ester linkages formed by reacting an alcohol with a phosphate group; hydrazone linkages which are reaction product of a hydrazide and an aldehyde; acetal linkages that are the reaction product of an aldehyde and an alcohol; orthoester linkages that are the reaction product of a formate and an l; peptide linkages formed by an amine group, including but not limited to, at an end of a polymer such as PEG, and a carboxyl group of a peptide; and oligonucleotide linkages formed by a phosphoramidite group, including but not limited to, at the end of a polymer, and a ' hydroxyl group of an oligonucleotide.

The terms “medium” or “media,” 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 “medium” or ” include, but are not limited to, solution, solid, semi-solid, or rigid supports that may support or contain any host cell, including, by way of example, bacterial host cells, yeast host cells, insect host cells, plant host cells, eukaryotic host cells, mammalian host cells, CHO cells, prokaryotic host cells, E. coli, or Pseudomonas host cells, and cell contents. Such “medium” or “media” includes, but is not limited to, medium or media in which the host cell has been grown into which a polypeptide has been ed, including medium either before or after a proliferation step. Such m” or “media” also includes, but is not limited to, buffers or reagents that contain host cell lysates, by way of example a polypeptide produced intracellularly and the host cells are lysed or ted to release the polypeptide.

The term “metabolite,” as used herein, refers to a derivative of a nd, by way of example natural amino acid polypeptide, a non-natural amino acid ptide, a modified natural amino acid polypeptide, or a modified non-natural amino acid polypeptide, that is formed when the nd, by way of e natural amino acid polypeptide, non-natural amino acid polypeptide, modified natural amino acid polypeptide, or modified non-natural amino acid polypeptide, is lized. The term aceutically active metabolite” or “active metabolite” refers to a biologically active derivative of a compound, by way of example natural amino acid polypeptide, a non-natural amino acid polypeptide, a modified natural amino acid polypeptide, or a modified non-natural amino acid ptide, that is formed when such a nd, by way of example a 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 “metabolized,” as used herein, refers to the sum of the processes by which a ular substance is changed by an organism. Such processes include, but are not limited to, hydrolysis reactions and reactions catalyzed by enzymes. Further information on metabolism may be obtained from The cological Basis of Therapeutics, 9th Edition, McGraw—Hill (1996). By way of example only, metabolites of natural amino acid polypeptides, non-natural amino acid polypeptides, modified natural amino acid polypeptides, or modified tural amino acid polypeptides may be identified either by stration of the natural amino acid polypeptides, non-natural amino acid polypeptides, d natural amino acid polypeptides, or modified non-natural amino acid polypeptides to a host and analysis of tissue samples from the host, or by incubation of natural amino acid polypeptides, non-natural amino acid polypeptides, modified natural amino acid polypeptides, or modified non-natural amino acid polypeptides with hepatic cells in Vitro and analysis of the resulting nds.

The term “metal chelator,” as used herein, refers to a le which forms a metal complex with metal ions. By way of example, such molecules may form two or more coordination bonds with a central metal ion and may form ring structures.

The term “metal-containing moiety,” as used herein, refers to a group which ns a metal ion, atom or particle. Such moieties include, but are not limited to, cisplatin, chelated metals ions (such as nickel, iron, and platinum), and metal nanoparticles (such as nickel, iron, and platinum).

The term “moiety incorporating a heavy atom,” as used herein, refers to a group which incorporates an ion of atom which is usually r than . Such ions or atoms include, but are not limited to, silicon, en, gold, lead, and uranium.

The term “modified,” as used herein refers to the presence of a change to a natural amino acid, a non-natural amino acid, a natural amino acid polypeptide or a non-natural amino acid polypeptide. Such changes, or modifications, may be obtained by post synthesis modifications of natural amino acids, non- natural amino acids, l amino acid polypeptides or non-natural amino acid polypeptides, or by co- translational, or by post-translational modification of natural amino acids, non-natural amino acids, natural amino acid polypeptides or non-natural amino acid polypeptides. The form “modified or unmodified” means that the natural amino acid, non-natural amino acid, natural amino acid polypeptide or non-natural amino acid polypeptide being sed are ally modified, that is, he natural amino acid, non-natural amino acid, natural amino acid polypeptide or non-natural amino acid polypeptide under discussion can be modified or unmodified.

As used herein, the term “modulated serum ife” refers to positive or negative changes in the circulating ife of a modified biologically active molecule relative to its non-modified form. By way of example, the d biologically active molecules include, but are not d to, natural amino acid, non-natural amino acid, natural amino acid polypeptide or non-natural amino acid polypeptide. By way of example, serum half-life is measured by taking blood samples at various time points after administration of the biologically active molecule or modified biologically active molecule, and determining the concentration of that molecule in each sample. Correlation of the serum tration with time allows calculation of the serum half-life. By way of example, modulated serum ife may be an increased in serum ife, which may enable an improved dosing regimens or avoid toxic effects. Such increases in serum may be at least about two fold, at least about three-fold, at least about five-fold, or at least about ten-fold. A non-limiting example of a method to evaluate increases in serum half-life is given in example 33. This method may be used for evaluating the serum half-life of any polypeptide.

The term “modulated therapeutic half-life,” as used , refers to positive or negative change in the half-life of the therapeutically effective amount of a modified biologically active molecule, relative to its non-modified form. By way of example, the modified biologically active molecules include, but are not WO 66559 d to, natural amino acid, non-natural amino acid, natural amino acid polypeptide or non-natural amino acid polypeptide. By way of example, therapeutic half-life is measured by measuring pharmacokinetic and/or pharmacodynamic properties of the molecule at various time points after administration. Increased therapeutic half-life may enable a particular beneficial dosing regimen, a particular beneficial total dose, or avoids an undesired effect. By way of example, the increased therapeutic half-life may result from increased potency, increased or decreased binding of the modified molecule to its target, an increase or decrease in another parameter or ism of action of the non- modified molecule, or an increased or decreased breakdown of the molecules by enzymes such as, by way of example only, proteases. A miting example of a method to evaluate increases in therapeutic half- life is given in example 33. This method may be used for evaluating the therapeutic half-life of any polypeptide.

The term “nanoparticle,” as used herein, refers to a particle which has a particle size between about 500 nm to about 1 nm.

The term “near-stoichiometric,” as used herein, refers to the ratio of the moles of nds participating in a chemical reaction being about 0.75 to about 1.5.

]As used herein, the term “non-eukaryote” refers to non-eukaryotic organisms. By way of example, a non-eukaryotic organism may belong to the Eubacteria, (which includes but is not limited to, Escherichia coli, Thermus thermophilus, or Bacillus stearothermophilus, Pseudomonas fluorescens, monas aeruginosa, Pseudomonas putida), enetic domain, or the Archaea, which includes, but is not limited to, Methanococcus jannaschii, Methanobacterium thermoautotrophicum, Archaeoglobus fulgidus, Pyrococcus furiosus, ccus horikoshii, Aeuropyrum pemix, or Halobacterium such as rax ii and Halobacterium species NRC-l, or phylogenetic domain.

A “non-natural amino acid” refers to an amino acid that is not one of the 20 common amino acids or pyrolysine or selenocysteine. Other terms that may be used synonymously with the term “non-natural amino acid” is “non-naturally encoded amino acid,” “unnatural amino acid,” “non-naturally-occurring amino acid,” and variously hyphenated and non-hyphenated versions thereof The term “non-natural amino acid” includes, but is not limited to, amino acids which occur naturally by modification of a naturally encoded amino acid (including but not limited to, the 20 common amino acids or pyrrolysine and selenocysteine) but are not themselves orated into a growing ptide chain by the translation x. Examples of naturally-occurring amino acids that are not naturally-encoded include, but are not limited to, N—acetylglucosaminyl-L-serine, N—acetylglucosaminyl-L-threonine, and O- phosphotyrosine. Additionally, the term “non-natural amino acid” includes, but is not limited to, amino acids which do not occur naturally and may be obtained tically or may be ed by modification of non-natural amino acids.

The term “nucleic acid,” as used herein, refers to deoxyribonucleotides, deoxyribonucleosides, cleosides or ribonucleotides and polymers thereof in either single- or double-stranded form. By way of example only, such nucleic acids and nucleic acid polymers include, but are not limited to, (i) analogues of natural tides which have similar binding properties as a reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides; (ii) oligonucleotide analogs including, but are not limited to, PNA (peptidonucleic acid), analogs of DNA used in antisense technology (phosphorothioates, phosphoroamidates, and the like); (iii) conservatively modified variants f (including but not limited to, degenerate codon substitutions) and complementary sequences and sequence explicitly indicated. By way of example, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed- base and/or deoxyinosine es (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J.

Biol. Chem. 260:2605-2608 (1985); and Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

The term “oxidizing agent,” as used herein, refers to a compound or material which is e of removing an electron from a compound being oxidized. By way of example oxidizing agents include, but are not limited to, ed glutathione, cystine, cystamine, oxidized dithiothreitol, ed erythreitol, and . A wide variety of oxidizing agents are suitable for use in the s and compositions described herein.

The term “pharmaceutically acceptable”, as used herein, refers to a material, including but not limited, to a salt, carrier or diluent, which does not te the biological actiVity or properties of the compound, and is relatively nontoxic, i.e., the material may be stered to an individual without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.

The term “photoaffinity label,” as used herein, refers to a label with a group, which, upon exposure to light, forms a e with a molecule for which the label has an affinity. By way of example only, such a linkage may be covalent or non-covalent.

The term “photocaged moiety,” as used herein, refers to a group which, upon illumination at certain wavelengths, ntly or non-covalently binds other ions or molecules.

The term “photocleavable group,” as used herein, refers to a group which breaks upon exposure to light.

The term crosslinker,” as used herein, refers to a compound comprising two or more functional groups which, upon exposure to light, are reactive and form a covalent or non-covalent linkage with two or more ric or ric molecules.

The term “photoisomerizable moiety,” as used herein, refers to a group wherein upon illumination with light s from one isomeric form to another.

The term “polyalkylene ,” as used herein, refers to linear or branched polymeric polyether polyols. Such kylene glycols, including, but are not limited to, polyethylene glycol, polypropylene glycol, tylene glycol, and derivatives thereof. Other exemplary embodiments are listed, for example, in commercial supplier catalogs, such as Shearwater Corporation's catalog “Polyethylene Glycol 2012/039468 and Derivatives for Biomedical Applications” . By way of example only, such ric polyether polyols have average molecular weights 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 may be between about 100 Da and about 100,000 Da, including but not limited to, about 100,000 Da, about 95,000 Da, about 90,000 Da, about 85,000 Da, about 80,000 Da, about 75,000 Da, about 70,000 Da, about 65,000 Da, about 60,000 Da, about 55,000 Da, about 50,000 Da, about 45,000 Da, about 40,000 Da, about 35,000 Da, about 30,000 Da, about 25,000 Da, about 20,000 Da, about 15,000 Da, about 10,000 Da, about 9,000 Da, about 8,000 Da, about 7,000 Da, about 6,000 Da, about 5,000 Da, about 4,000 Da, about 3,000 Da, about 2,000 Da, about 1,000 Da, about 900 Da, about 800 Da, about 700 Da, about 600 Da, about 500 Da, 400 Da, about 300 Da, about 200 Da, and about 100 Da. In some embodiments 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 ments, the molecular weight of the polymer is between about 2,000 to about 50,000 Da. In some embodiments, the molecular weight of the polymer is between about ,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 may be between about 1,000 Da and about 100,000 Da, including but not limited to, about 0 Da, about 95,000 Da, about 90,000 Da, about 85,000 Da, about 80,000 Da, about 75,000 Da, about 70,000 Da, about 65,000 Da, about 60,000 Da, about 55,000 Da, about 50,000 Da, about 45,000 Da, about 40,000 Da, about 35,000 Da, about 30,000 Da, about 25,000 Da, about 20,000 Da, about 15,000 Da, about 10,000 Da, about 9,000 Da, about 8,000 Da, about 7,000 Da, about 6,000 Da, about 5,000 Da, about 4,000 Da, about 3,000 Da, about 2,000 Da, and about 1,000 Da. In some ments, the molecular weight of the branched chain PEG is between about 1,000 Da and about 50,000 Da. In some ments, the molecular weight of the branched chain PEG is between about 1,000 Da and about 40,000 Da. In some embodiments, the lar 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 ed chain PEG is between about 5,000 Da and about 20,000 Da. In other embodiments, the molecular weight of the branched chain PEG is between about 2,000 to about 50,000 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 applies equally to a description of a e and a ption of a protein, and Vice versa. The terms apply to naturally WO 66559 occurring amino acid polymers as well as amino acid polymers in which one or more amino acid residues is a non-natural amino acid. Additionally, such “polypeptides,39 66peptides” and “proteins” include amino acid chains of any length, including full length proteins, 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 which occurs after such an amino acid has been translationally incorporated into a polypeptide chain. Such modifications include, but are not limited to, co-translational in vivo modifications, co- translational in vitro modifications (such as in a cell-free translation system), post-translational in vivo modifications, and post-translational in vitro ations.

] The terms “prodrug” or “pharmaceutically acceptable prodrug,” as used herein, refers to an agent that is converted into the parent drug in vivo or in vitro, wherein which does not abrogate the biological activity or properties of the drug, and is relatively nontoxic, i.e., the material may be administered to an individual without causing undesirable biological effects or interacting in a deleterious manner with any of the ents of the composition in which it is contained. Prodrugs are generally drug precursors that, following administration to a subject and subsequent absorption, are converted to an active, or a more active species via some s, such as conversion by a metabolic pathway. Some prodrugs have a chemical group present on the prodrug that renders it less active and/or confers solubility or some other property to the drug. Once the chemical group has been cleaved and/or modified from the g the active drug is generated. Prodrugs are converted into active drug within the body through tic or non-enzymatic reactions. Prodrugs may provide improved physiochemical ties such as better solubility, enhanced delivery characteristics, such as specifically targeting a particular cell, tissue, organ or ligand, and improved therapeutic value of the drug. The benefits of such gs include, but are not limited to, (i) ease of administration compared with the parent drug; (ii) the prodrug may be bioavailable by oral administration s the parent is not; and (iii) the prodrug may also have ed solubility in pharmaceutical compositions compared with the parent drug. A pro-drug es a pharmacologically inactive, or reduced-activity, derivative of an active drug. Prodrugs may be designed to modulate the amount of a drug or biologically active molecule that reaches a desired site of action through the manipulation of the properties of a drug, such as physiochemical, biopharmaceutical, or pharmacokinetic properties. An example, without limitation, of a prodrug would be a non-natural amino acid polypeptide which is administered as an ester (the “prodrug”) to tate transmittal across a cell membrane where water solubility is detrimental to ty but which then is metabolically hydrolyzed to the carboxylic acid, the active entity, once inside the cell where water solubility is beneficial. Prodrugs may be designed as reversible drug derivatives, for use as rs to enhance drug transport to site-specific tissues.

The term ylactically effective amount,” as used herein, refers that amount of a composition ning at least one tural amino acid polypeptide or at least one modified non-natural amino acid polypeptide prophylactically d to a patient which will relieve to some extent one or more of the symptoms of a disease, condition or er being treated. In such prophylactic ations, such amounts may depend on the patient's state of health, weight, and the like. It is considered well within the skill of the art for one to determine such prophylactically effective amounts by routine experimentation, including, but not limited to, a dose escalation clinical trial.

The term “protected,” as used herein, refers to the presence of a “protecting group” or moiety that prevents reaction of the chemically reactive functional group under certain reaction conditions. The protecting group will vary depending on the type of chemically reactive group being protected. By way of example only, (i) if the chemically reactive group is an amine or a hydrazide, the ting group may be ed from tert-butyloxycarbonyl (t-Boc) and 9-fluorenylmethoxycarbonyl (Fmoc); (ii) if the chemically reactive group is a thiol, the ting group may be orthopyridyldisulf1de; and (iii) if the chemically reactive group is a carboxylic acid, such as butanoic or propionic acid, or a yl group, the protecting group may be benzyl or an alkyl group such as methyl, ethyl, or tert-butyl.

By way of example only, blocking/protecting groups may be selected from: H O C2 H2 H H2 \ C\ c ’0 H2c¢C\C/C\ O 0 ”2‘3“ E! Y ”3‘3/ H2 2 allyl Bn Cbz alloc Me H2 H3C\ [CH3 0 (H c c/)3 (H3C)3C’5i\ ( H c 8' H3C 3 3 )3 '\/\0A Et t—butyl TBDMS Teoc H2 OJK c\ o HZC’ (CH3)3C/ \n/ 0/ (C6H5)3C~ H ck ° 3 O.o Boo pMBn trityl acetyl Fmoc Additionally, protecting groups include, but are not d to, including photolabile groups such as Nvoc and MeNvoc and other protecting groups known in the art. Other ting 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 moiety,” as used herein, refers to a group whose nuclei spontaneously give off nuclear ion, such as alpha, beta, or gamma particles; wherein, alpha particles are helium nuclei, beta particles are electrons, and gamma particles are high energy photons.

The term ive compound,” as used herein, refers to a compound which under appropriate conditions is reactive toward another atom, molecule or compound.

The term “recombinant host cell,” also referred to as “host cell,” refers to a cell which includes an exogenous polynucleotide, n the methods used to insert the exogenous polynucleotide into a cell include, but are not limited to, direct uptake, transduction, f-mating, or other methods known in the art to create recombinant host cells. By way of e only, such exogenous polynucleotide may be a nonintegrated vector, including but not limited to a d, or may be integrated into the host genome.

The term “redox-active ” as used , refers to a molecule which oxidizes or s another molecule, whereby the redox active agent becomes reduced or oxidized. Examples of redox active - - - - 2T/3T xes, Co +2 /3T complexes, and agent 1nclude, but are not 11m1ted to, ferrocene, qu1nones, Ru Osw3+ complexes.

The term “reducing agent,” as used herein, refers to a compound or material which is capable of adding an electron to a compound being reduced. By way of e reducing agents include, but are not limited to, dithiothreitol (DTT), 2-mercaptoethanol, dithioerythritol, cysteine, cysteamine (2- aminoethanethiol), and reduced glutathione. Such ng agents may be used, by way of example only, to maintain sulfhydryl groups in the reduced state and to reduce intra- or intermolecular disulfide bonds.

“Refolding,” as used herein describes any process, reaction or method which transforms an improperly folded or unfolded state to a native or ly folded conformation. By way of e only, refolding transforms disulfide bond containing polypeptides from an improperly folded or unfolded state to a native or ly folded conformation with respect to disulfide bonds. Such disulfide bond containing polypeptides may be natural amino acid polypeptides or non-natural amino acid polypeptides.

The term “resin,” as used herein, refers to high lar weight, insoluble r beads. By way of example only, such beads may be used as supports for solid phase peptide synthesis, or sites for attachment of molecules prior to purification.

The term “saccharide,” as used herein, refers to a series of carbohydrates including but not limited to sugars, monosaccharides, oligosaccharides, and polysaccharides.

The term “safety” or “safety profile,” as used herein, refers to side effects that might be related to stration of a drug relative to the number of times the drug has been administered. By way of e, a drug which has been administered many times and produced only mild or no side effects is said to have an ent safety profile. A non-limiting example of a method to evaluate the safety profile is given in example 26. This method may be used for evaluating the safety profile of any ptide.

The phrase “selectively hybridizes to” or “specifically hybridizes to,” as used , refers to the binding, duplexing, or hybridizing of a molecule to a particular nucleotide sequence under stringent hybridization conditions when that sequence is present in a complex mixture including but not limited to, total cellular or library DNA or RNA.

The term “spin label,” as used herein, refers to molecules which contain an atom or a group of atoms exhibiting an unpaired electron spin (i.e. a stable paramagnetic group) that can be detected by electron spin nce spectroscopy and can be attached to another molecule. Such spin-label molecules include, but are not limited to, nitryl radicals and nitroxides, and may be single spin-labels or double spin- labels.

The term “stoichiometric,” as used herein, refers to the ratio of the moles of compounds participating in a al reaction being about 0.9 to about 1.1.

The term hiometric-like,” as used herein, refers to a al reaction which becomes stoichiometric or near-stoichiometric upon 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 e, but are not d to, accelerants.

The phrase “stringent hybridization ions” refers to hybridization of sequences of DNA, RNA, PNA or other nucleic acid mimics, or combinations thereof, under conditions of low ionic strength and high temperature. By way of example, under ent conditions a probe will hybridize to its target subsequence in a complex e of nucleic acid (including but not limited to, total cellular or library DNA or RNA) but does not hybridize to other sequences in the complex mixture. Stringent conditions are sequence-dependent and will be ent in different circumstances. By way of example, longer sequences hybridize specifically at higher temperatures. Stringent hybridization ions include, but are not limited to, (i) about 5-10 0C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH; (ii) the salt concentration is about 0.01 M to about 1.0 M at about pH 7.0 to about pH 8.3 and the temperature is at least about 30 0C for short probes (including but not limited to, about 10 to about 50 nucleotides) and at least about 60 0C for long probes (including but not limited to, greater than 50 nucleotides); (iii) the addition of destabilizing agents including, but not limited to, formamide, (iV) 50% formamide, 5X SSC, and 1% SDS, incubating at 42 0C, or 5X SSC, about 1% SDS, ting at 65 0C, with wash in 0.2X SSC, and about 0.1% SDS at 65 0C for between about 5 minutes to about 120 minutes. By way of example only, detection of selective or specific hybridization, includes, but is not limited to, a positive signal at least two times background. An extensive guide to the hybridization of c acids is found in n, Laboratory Techniques in Biochemistry and Molecular Biology-- Hybridization with Nucleic Probes, “Overview of principles of hybridization and the strategy of nucleic acid assays” (1993).

The term “subject” as used herein, refers to an animal which is the object of ent, observation or experiment. By way of example only, a subject may be, but is not limited to, a mammal including, but not limited to, a human.

The term “substantially purified,” as used herein, refers to a component of interest that may be substantially or essentially free of other components which normally accompany or interact with the ent of interest prior to purification. By way of example only, a component of interest may be “substantially purified” when the ation of the component of interest contains less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% (by dry weight) of contaminating components. Thus, a “substantially purified” component of interest may have a purity level of about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99% or greater. By way of example only, a natural amino acid polypeptide or a non-natural amino acid polypeptide may be purified from a native cell, or host cell in the case of recombinantly produced l amino acid polypeptides or non-natural amino acid polypeptides. By way of example a preparation of a natural amino acid polypeptide or a non-natural amino acid polypeptide may be “substantially purified” when the preparation contains less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% (by dry weight) of contaminating al. By way of example when a l amino acid ptide or a non-natural amino acid ptide is recombinantly produced by host cells, the natural amino acid polypeptide or tural 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 e when a natural amino acid polypeptide or a non-natural amino acid polypeptide is recombinantly produced by host cells, the natural amino acid ptide or non-natural amino acid polypeptide may be present in the culture medium at about 5g/L, about 4g/L, about 3g/L, about 2g/L, about 1g/L, about 750mg/L, about 500mg/L, about L, about 100mg/L, about 50mg/L, about 10mg/L, or about 1mg/L or less of the dry weight of the cells. By way of example, “substantially purified” natural amino acid polypeptides or non-natural amino acid polypeptides may 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 s, including, but not limited to, SDS/PAGE analysis, RP-HPLC, SEC, and capillary electrophoresis.

The term “substituents” also referred to as “non-interfering substituents39 66refers to groups which may be used to replace another group on a molecule. Such groups include, but are not limited to, halo, C1- C10 alkyl, C2-C10 alkenyl, C2-C10 alkynyl, C1-C10 alkoxy, C5-C12 aralkyl, C3-C12 cycloalkyl, C4-C12 cycloalkenyl, phenyl, substituted phenyl, toluolyl, xylenyl, biphenyl, C2-C12 alkoxyalkyl, C5-C12 alkoxyaryl, C5-C12 aryloxyalkyl, C7-C12 oxyaryl, C1-C6 alkylsulfinyl, C1-C10 alkylsulfonyl, -(CH2)m-O-(C1- C10 alkyl) wherein m is from 1 to 8, aryl, substituted aryl, substituted alkoxy, fluoroalkyl, heterocyclic radical, substituted heterocyclic radical, lkyl, -N02, -CN, -NRC(O)-(C1-C10 alkyl), -C(O)-(C1-C10 alkyl), C2-C10 alkthioalkyl, -C(O)O-(C1-C10 alkyl), -OH, -S02, =S, -COOH, -NR2, yl, -C(O)-(C1- C10 alkyl)-CF3, -C(O)-CF3, -C(O)NR2, -(C1-C10 aryl)-S-(C6-C10 aryl), -C(O)-(C6-C10 aryl), -(CH2)m-O- (CH2)m-O-(C1-C10 alkyl) wherein each m is from 1 to 8, -C(O)NR2, -C(S)NR2, -S02NR2, -NRC(O)NR2, - NRC(S)NR2, salts thereof, and the like. Each R group in the preceding list includes, but is not limited to, H, alkyl or substituted alkyl, aryl or substituted aryl, or alkaryl. Where substituent groups are specified by their conventional al formulas, n from left to right, they equally encompass the chemically identical tuents that would result from writing the structure from right to left; for example, -CH20- is lent to —OCH2-.

WO 66559 By way of example only, substituents for alkyl and heteroalkyl radicals ding those groups referred to as alkylene, alkenyl, heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl) includes, but is not limited to: -OR, =0, =NR, =N—OR, -NR2, -SR, - halogen, -SiR3, -OC(O)R, -C(O)R, -C02R, -CONR2, -OC(O)NR2, -NRC(O)R, -NRC(O)NR2, -NR(O)2R, - NR-C(NR2)=NR, -S(O)R, -S(O)2R, -S(O)2NR2, -NRSOZR, -CN and —N02. Each R group in the preceding list includes, but is not limited to, hydrogen, substituted or unsubstituted heteroalkyl, substituted or tituted aryl, including but not d to, aryl substituted with 1-3 halogens, substituted or unsubstituted alkyl, alkoxy or thioalkoxy groups, or aralkyl groups. When two R groups are attached to the same nitrogen atom, they can be combined with the en atom to form a 5-, 6-, or 7-membered ring. For example, -NR2 is meant to include, but not be limited to, l-pyrrolidinyl and 4-morpholinyl.

By way of example, substituents for aryl and heteroaryl groups include, but are not limited to, - OR, =0, =NR, =N-OR, -NR2, -SR, -halogen, -SiR3, -OC(O)R, -C(O)R, -C02R, -CONR2, -OC(O)NR2, - R, -NRC(O)NR2, -NR(O)2R, -NR-C(NR2)=NR, -S(O)R, -S(O)2R, -S(O)2NR2, -NRSOZR, -CN, — N02, -R, -N3, -CH(Ph)2, C1-C4)alkoxy, and fluoro(C1-C4)alkyl, in a number ranging from zero to the total number of open valences on the aromatic ring system; and where each R group in the preceding list 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 containing at least one non-natural amino acid polypeptide and/or at least one modified non-natural amino acid polypeptide administered to a patient already suffering from a disease, condition or disorder, sufficient to cure or at least lly arrest, or relieve to some extent one or more of the symptoms of the disease, er or ion being d. The effectiveness of such compositions depend ions including, but not limited to, the severity and course of the disease, disorder or condition, previous y, the patient's health status and se to the drugs, and the nt of the treating physician.

By way of example only, therapeutically effective amounts may be determined by routine experimentation, including but not limited to a dose escalation clinical trial.

The term “thioalkoxy,” as used herein, refers to sulfur containing alkyl groups linked to molecules Via an oxygen atom.

The term “thermal melting point” or Tm is the temperature (under d ionic strength, pH, and nucleic concentration) at which 50% of probes complementary to a target hybridize to the target sequence at equilibrium.

The term “toxic moiety” or “toxic group” as used herein, refers to a compound which can cause harm, disturbances, or death. Toxic moieties include, but are not limited to, auristatin, DNA minor groove binding agent, DNA minor groove alkylating agent, enediyne, lexitropsin, duocarmycin, , puromycin, dolastatin, maytansinoid, Vinca alkaloid, AFP, MMAF, MMAE, AEB, AEVB, auristatin E, paclitaxel, docetaxel, 5, SN—38, topotecan, morpholino-doxorubicin, rhizoxin, cyanomorpholino- doxorubicin, dolastatin-lO, echinomycin, tatstatin, chalicheamicin, maytansine, DM-l, netropsin, podophyllotoxin (e.g. etoposide, teniposide, etc.), baccatin and its derivatives, anti-tubulin agents, cryptophysin, combretastatin, atin E, vincristine, vinblastine, ine, lbine, VP-l6, camptothecin, epothilone A, epothilone B, nocodazole, colchicines, colcimid, estramustine, cemadotin, ermolide, maytansine, erobin, mechlorethamine, cyclophosphamide, melphalan, carmustine, lomustine, semustine, streptozocin, chlorozotocin, uracil mustard, chlormethine, ifosfamide, chlorambucil, pipobroman, triethylenemelamine, triethylenethiophosphoramine, busulfan, dacarbazine, and temozolomide, ytarabine, cytosine arabinoside, fluorouracil, floxuridine, 6-thioguanine, 6- mercaptopurine, pentostatin, 5-fluorouracil, methotrexate, lO-propargyl-S,8-dideazafolate, 5,8- dideazatetrahydrofolic acid, leucovorin, fludarabine phosphate, pentostatine, gemcitabine, Ara-C, paclitaxel, docetaxel, deoxycoformycin, mitomycin—C, L-asparaginase, azathioprine, brequinar, antibiotics (e.g., anthracycline, gentamicin, cefalotin, vancomycin, telavancin, daptomycin, azithromycin, erythromycin, rocithromycin, furazolidone, amoxicillin, llin, carbenicillin, flucloxacillin, methicillin, penicillin, ciprofloxacin, xacin, ofloxacin, doxycycline, minocycline, oxytetracycline, tetracycline, streptomycin, rifabutin, ethambutol, rifaXimin, etc.), antiviral drugs (e. g., ir, acyclovir, ampligen, cidofovir, delavirdine, didanosine, efavirenz, entecavir, fosfonet, ganciclovir, ibacitabine, imunovir, idine, inosine, lopinavir, methisazone, nexavir, nevirapine, oseltamivir, penciclovir, stavudine, trifluridine, truvada, valaciclovir, zanamivir, etc.), ubicin hydrochloride, daunomycin, rubidomycin, cerubidine, idarubicin, doxorubicin, epirubicin and morpholino derivatives, phenoxizone biscyclopeptides (e.g., dactinomycin), basic glycopeptides (e.g., bleomycin), quinone glycosides (e. g., plicamycin, mithramycin), anthracenediones (e. g., mitoxantrone), azirinopyrrolo indolediones (e. g., mitomycin), macrocyclic immunosuppressants (e.g., cyclosporine, FK-506, tacrolimus, prograf, rapamycin etc.), navelbene, CPT-ll, anastrazole, letrazole, capecitabine, reloxafine, cyclophosphamide, ifosamide, droloxafine, allocolchicine, Halichondrin B, colchicine, colchicine derivatives , maytansine, rhizoxin, paclitaxel, paclitaxel tives, docetaxel, thiocolchicine, trityl cysterin, stine sulfate, stine sulfate, cisplatin, carboplatin, yurea, N—methylhydrazine, epidophyllotoxin, procarbazine, ntrone, leucovorin, and tegafur. “Taxanes” include axel, as well as any active taxane derivative or pro-drug.

The terms “treat,” “treating” or “treatment”, as used herein, include alleviating, abating or ameliorating a disease or condition symptoms, preventing additional symptoms, ameliorating or preventing the underlying metabolic causes of symptoms, ting the disease or condition, e.g., ing the development of the disease or condition, relieving the disease or condition, causing regression of the disease or ion, relieving a condition caused by the disease or condition, or ng the symptoms of the disease or condition. The terms “treat,39 66treating” or “treatment”, include, but are not limited to, prophylactic and/or eutic treatments.

As used , the term “water soluble polymer” refers to any polymer that is soluble in s solvents. Such water soluble rs include, but are not limited to, polyethylene glycol, polyethylene WO 66559 glycol propionaldehyde, mono C1-C10 alkoxy or aryloxy derivatives thereof (described in US. Patent No. ,252,714 which is incorporated by reference herein), monomethoxy-polyethylene glycol, polyvinyl pyrrolidone, polyvinyl alcohol, polyamino acids, lether maleic anhydride, N—(2-Hydroxypropyl)- methacrylamide, dextran, dextran derivatives including dextran e, polypropylene glycol, opylene oxide/ethylene oxide copolymer, polyoxyethylated , heparin, heparin fragments, polysaccharides, oligosaccharides, s, cellulose and cellulose derivatives, including but not limited to methylcellulose and carboxymethyl cellulose, serum albumin, starch and starch derivatives, polypeptides, polyalkylene glycol and derivatives thereof, copolymers of kylene 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 only, coupling of such water soluble polymers to natural amino acid polypeptides or non-natural polypeptides may result in changes including, but not limited to, increased water solubility, increased or modulated serum half-life, increased or modulated therapeutic half-life relative to the unmodified form, increased bioavailability, ted biological activity, extended circulation time, modulated immunogenicity, modulated physical association characteristics including, but not d to, aggregation and multimer formation, altered receptor binding, altered binding to one or more binding partners, and altered receptor dimerization or multimerization. In addition, such water soluble polymers may or may not have their own biological activity.

] Unless otherwise indicated, conventional methods of mass spectroscopy, NMR, HPLC, protein chemistry, mistry, recombinant DNA ques and pharmacology, within the skill of the art are Compounds, (including, but not d to non-natural amino acids, non-natural amino acid polypeptides, modified non-natural amino acid polypeptides, and reagents for producing the aforementioned compounds) ted herein include isotopically-labeled compounds, which are identical to those recited in the various formulas and structures presented herein, but for the fact that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number y found in . Examples of isotopes that can be incorporated into the present compounds e isotopes of hydrogen, carbon, nitrogen, oxygen, e and chlorine, such as 2H, 3H, 13C, 14C, 15N, 18O, 17O, 358, 18F, 36Cl, respectively. Certain isotopically-labeled compounds described herein, for example those into which radioactive isotopes such as 3H and 14C are incorporated, are usefiJl in drug and/or substrate tissue distribution assays. Further, substitution with isotopes such as deuterium, i.e., 2H, can afford certain therapeutic advantages resulting from greater metabolic ity, for example increased in vivo half-life or reduced dosage requirements.

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

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

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, tural 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 may exist as tautomers. All tautomers are included within the scope of the tural amino acids, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides presented herein. In addition, the non-natural amino acids, tural amino acid polypeptides and modified non- natural amino acid polypeptides described herein can exist in unsolvated as well as solvated forms with pharmaceutically acceptable solvents such as water, l, and the like. The ed forms of the non- natural amino acids, non-natural amino acid polypeptides and ed non-natural amino acid polypeptides presented herein are also considered to be disclosed herein.

Some of the compounds herein (including, but not d to non-natural amino acids, non-natural amino acid ptides and modified non-natural amino acid polypeptides and reagents for producing the aforementioned compounds) may exist in several tautomeric forms. All such tautomeric forms are considered as part of the compositions described herein. Also, for example all eto forms of any compounds ding, but not limited to non-natural amino acids, tural amino acid polypeptides and modified non-natural amino acid polypeptides and ts for producing the aforementioned compounds) herein are considered as part of the compositions described herein.

Some of the compounds herein (including, but not limited to non-natural amino acids, non-natural amino acid polypeptides and modified non-natural amino acid polypeptides and reagents for ing either of the aforementioned compounds) are acidic and may form a salt with a pharmaceutically acceptable cation. Some of the compounds herein (including, but not limited to non-natural amino acids, non-natural amino acid polypeptides and d non-natural amino acid ptides and reagents for producing the entioned compounds) can be basic and accordingly, may form a salt with a pharmaceutically able anion. All such salts, including di-salts are within the scope of the itions described herein and they can be prepared by conventional methods. For example, salts can be prepared by contacting the acidic and basic entities, in either an aqueous, non-aqueous or partially aqueous medium. The salts are recovered by using at least one of the following techniques: filtration, precipitation with a non-solvent followed by filtration, evaporation of the solvent, or, in the case of s solutions, lyophilization.

Pharmaceutically acceptable salts of the non-natural amino acid polypeptides disclosed herein may be formed when an acidic proton present in the parent non-natural amino acid polypeptides either is replaced by a metal ion, by way of example an alkali metal ion, an ne earth ion, or an aluminum ion; or coordinates with an organic base. In addition, the salt forms of the disclosed non-natural amino acid polypeptides can be ed using salts of the starting als or intermediates. The non-natural amino acid polypeptides described herein may be prepared as a pharmaceutically acceptable acid addition salt (which is a type of a pharmaceutically able salt) by reacting the free base form of non-natural amino acid polypeptides described herein with a pharmaceutically acceptable inorganic or organic acid.

Alternatively, the non-natural amino acid polypeptides described herein may be prepared as ceutically acceptable base addition salts (which are a type of a pharmaceutically acceptable salt) by reacting the free acid form of non-natural amino acid polypeptides described herein with a ceutically acceptable inorganic or organic base.

The type of pharmaceutical acceptable salts, include, but are not limited to: (1) acid addition salts, formed with inorganic acids such as hydrochloric acid, hydrobromic acid, ic acid, nitric acid, phosphoric acid, and the like; or formed with organic acids such as acetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, ic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, ydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, l,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, 2-naphthalenesulfonic acid, 4-methylbicyclo- [2.2.2]octene-l-carboxylic acid, glucoheptonic acid, 4,4’-methylenebis-(3-hydroxyene-l -carboxylic acid), 3-phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid, lauryl sulfuric acid, gluconic acid, glutamic acid, ynaphthoic acid, salicylic acid, stearic acid, muconic acid, and the like; (2) salts formed when an acidic proton present in the parent compound either is replaced by a metal ion, e. g., an alkali metal ion, an alkaline earth ion, or an aluminum ion; or coordinates with an organic base.

Acceptable organic bases include ethanolamine, diethanolamine, triethanolamine, tromethamine, N- methylglucamine, and the like. Acceptable inorganic bases include aluminum hydroxide, calcium hydroxide, potassium hydroxide, sodium carbonate, sodium hydroxide, and the like.

] The corresponding counterions of the non-natural amino acid polypeptide pharmaceutical acceptable salts may be analyzed and identified using various methods ing, but not limited to, ion exchange chromatography, ion chromatography, capillary electrophoresis, inductively d plasma, atomic absorption oscopy, mass spectrometry, or any combination thereof. In addition, the therapeutic ty of such non-natural amino acid polypeptide pharmaceutical acceptable salts may be tested using the techniques and methods described in examples 87-91.

It should be understood that a reference to a salt includes the solvent addition forms or l forms thereof, particularly solvates or polymorphs. Solvates contain either stoichiometric or non- stoichiometric amounts of a solvent, and are often formed during the process of crystallization with pharmaceutically acceptable solvents such as water, ethanol, and the like. Hydrates are formed when the solvent is water, or alcoholates are formed when the solvent is l. Polymorphs include the different crystal g ements of the same elemental composition of a compound. rphs usually have different X-ray diffraction patterns, infrared spectra, melting points, density, hardness, crystal shape, l and electrical properties, stability, and solubility. Various s such as the recrystallization solvent, rate of crystallization, and storage temperature may cause a single crystal form to dominate.

The screening and characterization of non-natural amino acid polypeptide pharmaceutical acceptable salts polymorphs and/or solvates may be accomplished using a variety of techniques including, but not limited to, thermal analysis, X-ray diffraction, spectroscopy, vapor sorption, and microscopy.

Thermal analysis methods address thermo chemical degradation or thermo physical processes including, but not limited to, polymorphic transitions, and such methods are used to analyze the relationships between polymorphic forms, ine weight loss, to find the glass transition temperature, or for excipient compatibility studies. Such methods include, but are not limited to, Differential scanning calorimetry (DSC), Modulated Differential Scanning Calorimetry (MDCS), gravimetric analysis (TGA), and Thermogravi-metric and Infrared is (TG/IR). X-ray diffraction methods include, but are not limited to, single crystal and powder diffractometers and synchrotron sources. The various oscopic ques used include, but are not d to, Raman, FTIR, UVIS, and NMR (liquid and solid state). The various microscopy techniques include, but are not limited to, polarized light microscopy, Scanning Electron Microscopy (SEM) with Energy Dispersive X-Ray Analysis (EDX), Environmental Scanning Electron Microscopy with EDX (in gas or water vapor atmosphere), IR microscopy, and Raman microscopy.

BRIEF DESCRIPTION OF THE DRAWINGS The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the ples of the invention are utilized, and the accompanying drawings of which: Figure 1 presents a graphical illustration of Her-Tox binding to the Her2 receptor.

Figure 2 presents a graphical illustration of the expression of Anti-Her2 variants determined by ELISA analysis.

Figure 3 presents a graphical illustration of the expression of Anti-Her2 variants determined by ELISA analysis.

Figure 4 presents a graphical illustration of the cell proliferation assay with HCC 1954 breast cancer cell line and dolastatin linker derivatives.

Figure 5 presents a graphical illustration of the analysis of the cell proliferation assay with HCC 1954 breast cancer cell line and trastuzumab-tox conjugates.

Figure 6 ts a graphical illustration of the analysis of the cell proliferation assay with SKOV-3 ovarian cancer cell line and dolastatin linker derivatives.

Figure 7 ts a graphical illustration of the analysis of the cell proliferation assay with SKOV-3 ovarian cancer cell line and trastuzumab-tox conjugates.

Figure 8 presents a graphical ration of the analysis of the cell proliferation assay with MDA— MB-468 breast cancer cell line and dolastatin linker derivatives.

Figure 9 presents a graphical ration of the is of the cell proliferation assay with MDA— MB-468 breast cancer line and zumab-tox conjugates.

Figure 10 presents a graphical illustration of tumor volume measurement (mm3) after a single IC dose (3.3 mg/kg, 10 mg/kg, 20mg/kg) of trastuzumab-linked dolastatin derivatives.

] Figure 11 presents assay formats used to measure trastuzumab-linked dolastatin derivatives concentration in SD rat serum Figure 12 presents graphical illustrations of serum trations (ng/mL) of trastuzumab-linked dolastatin derivatives after single IV injections.

Figure 13 presents a graphical illustration of serum concentrations (ng/mL) of trastuzumab-linked dolastatin tives after single IV injections. This assay detects antibody binding to the ErbB2 receptor.

Figure 14 presents a graphical illustration of of serum concentrations (ng/mL) of trastuzumab- linked dolastatin derivatives after IV injection. The in Vivo stability measurements detect at least two dolastatin derivatives linked to rastuzumab.

Figure 15 presents graphical illustrations of the change in rat body weight and tumor volume after treatment with trastuzumab-linked dolastatin tives.

Figure 16 ts graphical illustrations of anti-tumor efficacy of trastuzumab, Her2-HS122- NCDl and Her2-HS122/LK145-HCD1 against ished tumors of HCC1954 in g mice. Mice were administered a single IV injection on day 1 ). Data points ent group average tumor volume and error bars represent standard error of the mean (SEM).

WO 66559 Figure 17 presents graphical illustrations of anti-tumor y of dolastatin linker derivatives in the MDA361DYT2 Breast (2--) aft model.

Figure 18 ts graphical rations of anti-tumor efficacy of dolastatin linker tives in the MDA361DYT2 Breast (2--) Xenograft model.

DETAILED DESCRIPTION OF THE INVENTION While preferred embodiments of the present ion have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only.

Numerous variations, changes, and substitutions will now occur to those skilled in the art without ing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their lents be covered thereby.

I. Introduction Recently, an ly new technology in the protein sciences has been reported, which promises to overcome many of the limitations associated with site-specific modifications of proteins. Specifically, new components have been added to the protein thetic machinery of the prokaryote Escherichia coli (E. 6011') (e. g., L. Wang, et al., (2001), m 292:498-500) and the eukaryote Sacchromyces cerevisiae (S. cerevisiae) (e. g., J. Chin et al., m 301 :964-7 (2003)), which has enabled the incorporation of non-natural amino acids to proteins in vivo. A number of new amino acids with novel chemical, physical or biological properties, including photoaffinity labels and somerizable amino acids, keto amino acids, and glycosylated amino acids have been incorporated efficiently and with high fidelity into proteins in E. coli and in yeast in response to the amber codon, TAG, using this methodology.

Studies have demonstrated that it is le to selectively and routinely introduce chemical functional groups that are not found in proteins, that are chemically inert to all of the functional groups found in the common, genetically-encoded amino acids and that may be used to react efficiently and selectively to form stable covalent linkages.

II. Overview At one level, described herein are the tools (methods, compositions, techniques) for creating and using dolastatin linker tives or analogs comprising at least one carbonyl, dicarbonyl, oxime, hydroxylamine, aldehyde, protected aldehyde, ketone, protected , thioester, ester, dicarbonyl, hydrazine, azide, amidine, imine, diamine, keto-amine, keto-alkyne, alkyne, cycloalkyne, or ene-dione.

At another level, described herein are the tools (methods, compositions, techniques) for creating and using dolastatin linker derivatives or analogs comprising at least one tural amino acid or d non- natural amino acid with an oxime, aromatic amine, heterocycle (e. g., indole, quinoxaline, phenazine, pyrazole, triazole, etc.).

Such dolastatin linker derivatives comprising tural amino acids may contain further functionality, including but not limited to, a polymer; a water-soluble polymer; a derivative of polyethylene glycol; a second protein or polypeptide or polypeptide analog; an antibody or antibody fragment; and any combination thereof. Note that the various aforementioned functionalities are not meant to imply that the members of one functionality cannot be classified as members of another functionality.

Indeed, there will be overlap depending upon the particular circumstances. By way of example only, a water-soluble polymer overlaps in scope with a derivative of polyethylene glycol, r the overlap is not complete and thus both functionalities are cited above.

Provided herein in some ments, is a toxic group linker tive comprising a carbonyl, onyl, oxime, hydroxylamine, aldehyde, protected aldehyde, ketone, protected ketone, thioester, ester, dicarbonyl, hydrazine, azide, amidine, imine, diamine, keto-amine, keto-alkyne, alkyne, cycloalkyne, or ene-dione. In some embodiments, the toxic group derivative comprises any of the linkers disclosed herein. In other embodiments, bed herein are the tools (methods, compositions, techniques) for creating and using toxic group tives or analogs comprising at least one non-natural amino acid or modified non-natural amino acid with an oxime, aromatic amine, heterocycle (e. g., indole, quinoxaline, ine, pyrazole, triazole, etc.).

In some embodiments, such toxic tives comprising non-natural amino acids may contain further functionality, ing but not limited to, a polymer; a water-soluble polymer; a derivative of polyethylene glycol; a second protein or polypeptide or polypeptide analog; an antibody or antibody fragment; and any combination thereof In specific embodiments, the toxic group is dolastatin or auristatin. In certain specific embodiments, the toxic group is dolastatin-10. Note that the various aforementioned functionalities are not meant to imply that the members of one functionality cannot be classified as members of another onality. , there will be overlap depending upon the ular stances. By way of e only, a soluble r overlaps in scope with a derivative of polyethylene glycol, however the overlap is not complete and thus both functionalities are cited above. n embodiments of the present invention be preparations of certain toxic moieties with linkers that reduce the toxicity of the moiety in Vivo while the toxic moiety retains pharmacological activity. In some embodiments, the toxicity of the linked toxic group, when administered to an animal or human, is reduced or eliminated ed to the free toxic group or toxic group derivatives comprising labile linkages, while retaining cological activity. In some embodiments, increased doses of the linked toxic group (e. g., dolastatin linker derivatives, non-natural amino acid linked dolastatin derivatives) may be administered to animals or humans with greater safety. In certain embodiments, the non-natural amino acid polypeptides linked to a toxic moiety (e. g., dolastatin derivative) provides in Vitro and in Vivo stability. In some embodiments, the non-natural amino acid polypeptides linked to a toxic moiety (e. g., dolastatin-10 derivative) are efficacious and less toxic compared to the free toxic moiety (e. g., dolastatin- ).

III. Dolastatin Linker Derivatives At one level, described herein are the tools (methods, compositions, techniques) for creating and using a dolastatin linker derivatives or analogs sing at least one non-natural amino acid or modified non-natural amino acid with a carbonyl, dicarbonyl, oxime or hydroxylamine group. Such dolastatin linker derivatives comprising non-natural amino acids may contain further functionality, including but not limited to, a polymer; a soluble polymer; a derivative of polyethylene glycol; a second protein or polypeptide or polypeptide analog; an antibody or dy fragment; and any combination thereof. Note that the various aforementioned functionalities are not meant to imply that the members of one functionality cannot be classified as members of another functionality. Indeed, there will be overlap depending upon the particular stances. By way of example only, a soluble polymer overlaps in scope with a derivative of polyethylene glycol, however the overlap is not complete and thus both functionalities are cited above.

In one aspect are methods for selecting and designing a dolastatin linker derivative to be modified using the methods, compositions and techniques described herein. The new dolastatin linker derivative may be designed de novo, including by way of e only, as part of high-throughput screening process (in which case us polypeptides may be designed, synthesized, characterized and/or tested) or based on the interests of the cher. The new dolastatin linker derivative may also be ed based on the structure of a known or partially characterized polypeptide. By way of e only, atin has been the subject of e study by the scientific community; a new compound may be designed based on the structure of dolastatin. The principles for selecting which amino acid(s) to substitute and/or modify are described separately herein. The choice of which modification to employ is also described herein, and can be used to meet the need of the experimenter or end user. Such needs may include, but are not limited to, manipulating the therapeutic effectiveness of the ptide, improving the safety profile of the polypeptide, adjusting the pharmacokinetics, pharmacologics and/or pharmacodynamics of the polypeptide, such as, by way of example only, increasing water solubility, bioavailability, increasing serum half-life, increasing therapeutic half-life, ting immunogenicity, modulating biological activity, or extending the circulation time. In addition, such modifications include, by way of example only, providing additional onality to the polypeptide, incorporating an antibody, and any combination of the aforementioned modifications.

Also described herein are dolastatin linker derivatives that have or can be modified to contain an oxime, carbonyl, dicarbonyl, or ylamine group. Included with this aspect are methods for producing, purifying, characterizing and using such atin linker derivatives The dolastatin linker derivative 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 of a carbonyl or dicarbonyl group, oxime group, hydroxylamine group, or protected forms thereof. The atin linker derivative can be the same or different, for example, there can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, ll, 12, l3, 14, , 16, 17, 18, 19, 20, or more different sites in the derivative that comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more different reactive groups.

A. Structure and Synthesis of Dolastatin Linker Derivatives: Electrophilic and Nucleophilic Groups Dolastatin derivatives with linkers containing a hydroxylamine (also called an xy) group allow for reaction with a variety of electrophilic groups to form conjugates (including but not limited to, with PEG or other water soluble polymers). Like hydrazines, hydrazides and rbazides, the enhanced nucleophilicity of the aminooxy group permits it to react efficiently and selectively with a variety of molecules that contain carbonyl- or dicarbonyl-groups, ing but not d to, ketones, aldehydes or other functional groups with similar chemical reactivity. Whereas the result of reaction with a hydrazine group is the corresponding hydrazone, however, an oxime results generally from the reaction of an aminooxy group with a carbonyl- or dicarbonyl-containing group such as, by way of example, a ketones, aldehydes or other functional groups with similar chemical reactivity. In some embodiments, dolastatin derivatives with linkers comprising an azide, alkyne or cycloalkyne allow for linking of molecules Via cycloaddition reactions (e. g., 1,3-dipolar cycloadditions, azide-alkyne Huisgen cycloaddition, etc.) Thus, in n embodiments described herein are dolastatin derivatives with linkers comprising a hydroxylamine, aldehyde, protected aldehyde, , ted , thioester, ester, dicarbonyl, hydrazine, amidine, imine, diamine, keto-amine, keto-alkyne, and ene-dione hydroxylamine group, a hydroxylamine-like group (which has reactivity similar to a hydroxylamine group and is structurally similar to a hydroxylamine group), a masked hydroxylamine group (which can be readily ted into a ylamine , or a ted hydroxylamine group (which has reactiVity similar to a hydroxylamine group upon deprotection). In some embodiments, the dolastatin derivatives with linkers comprise azides, alkynes or lkynes. Such dolastatin linker derivatives include compounds haVing the structure of Formula (1): Wm,R70 /\ Me OMeO wherein: Z has the structure of: €Yé\(KAr R5 is H, CORg, C1-C6alkyl, or thiazole; R8 is OH; R6 is OH or H; Ar is phenyl or pyridine; R7 is C1-C6alkyl or hydrogen; Y is ylamine, methyl, aldehyde, protected aldehyde, ketone, protected ketone, thioester, ester, dicarbonyl, hydrazine, azide, amidine, imine, e, keto-amine, keto-alkyne, alkyne, lkyne, or ene-dione; L is—alkylene— or —(alkylene—O)n—alkylene—; and n is an integer greater than or equal to one.

Such dolastatin linker tives may be in the form of a salt, or may be incorporated into a non-natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post translationally modified.

In certain ments of nds of Formula (I), R5 is thiazole or carboxylic acid. In certain embodiments of compounds of Formula (I), R5 is hydrogen. In certain embodiments of compounds of Formula (I), R5 is methyl, ethyl, propyl, iso-propyl, butyl, iso-butyl, sec-butyl, tert-butyl, , or hexyl.

In some embodiments, Y is azide. In other embodiments, Y is cycloalkyne. In specific embodiments, the cyclooctyne has a structure of: |©—§ (R19)q each R19 is independently selected from the group ting of C1-C6 alkyl, C1-C6 alkoxy, ester, ether, thioether, aminoalkyl, n, alkyl ester, aryl ester, amide, aryl amide, alkyl halide, alkyl amine, alkyl ic acid, alkyl nitro, thioester, sulfonyl ester, halosulfonyl, nitrile, alkyl nitrile, and nitro; and qis 0,1, 2, 3, 4, 5, 6, 7, 8, 9,10 or 11.

In n embodiments of compounds of Formula (I), R6 is H. In some embodiments of compounds of Formula (1), R6 is hydroxy.

] In certain embodiments of compounds of Formula (1), Ar is phenyl.

In certain embodiments of compounds of Formula (I), R7 is methyl, ethyl, propyl, iso-propyl, butyl, sec-butyl iso-butyl, tert-butyl, , or hexyl. In certain embodiments of compounds of Formula (1), R7 is hydrogen.

In certain embodiments of compounds of Formula (I), Y is hydroxylamine, aldehyde, protected aldehyde, ketone, protected ketone, thioester, ester, dicarbonyl, hydrazine, amidine, imine, diamine, keto- amine, keto-alkyne, or ene-dione.

In certain embodiments of compounds of Formula (I), each alkylene independently is —CH2—, — CHZCH2—, —CH2CH2CH2—, —CH2CH2CH2CH2—, —CH2CH2CH2CH2CH2—, —CH2CH2CH2CH2CH2CH2—, — CH2CH2CH2CH2CH2CH2CH2—, —CH2CH2CHZCHZCHZCHZCHZCH2—, — CH2CH2CHZCHZCHQCHZCHZCHZCH2—, 2CHQCHQCHQCH2CH2CH2CH2CH2—, — CH2CH2CH2CH2CHZCHQCHZCHZCHZCHZCH2—, or —CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2—.

In certain embodiments of compounds ofFormula(1), n is O, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100.

B. Structure and Synthesis statin Linker Derivatives: Hydroxylamine Groups Thus, in certain embodiments described herein are dolastatin tives with linkers comprising a hydroxylamine group, a hydroxylamine-like group (Which has reactivity similar to a hydroxylamine group and is structurally similar to a hydroxylamine group), a masked hydroxylamine group (Which can be readily ted into a hydroxylamine group), or a protected hydroxylamine group (which has reactivity similar to a hydroxylamine group upon deprotection). Such dolastatin linker derivatives include compounds having the structure of Formula (1): mm,R70 /\ Me OMeO wherein: Z has the structure of: ‘7;\KKAr R5 ; R5 is H, CORg, C1-C6alkyl, or thiazole; R8 is OH; R6 is OH or H; Ar is phenyl or pyridine; R7 is C1-C6alkyl or hydrogen; Y is NHg—O— or methyl; L is—alkylene— or —(alkylene—O)n—alkylene—; and n is an integer greater than or equal to one.

Such dolastatin linker tives may be in the form of a salt, or may be incorporated into a non-natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post ationally modified.

In certain embodiments of compounds of Formula (I), R5 is thiazole. In certain embodiments of compounds of Formula (I), R5 is hydrogen. In certain embodiments of compounds of Formula (I), R5 is methyl, ethyl, propyl, iso-propyl, butyl, iso-butyl, sec-butyl, tert-butyl, pentyl, or hexyl.

In certain embodiments of compounds of Formula (I), R6 is H. In some ments of compounds of Formula (I), R6 is hydroxy.

In certain embodiments of compounds of Formula (I), Ar is phenyl.

In n embodiments of compounds of Formula (I), R7 is methyl, ethyl, propyl, iso-propyl, butyl, sec-butyl iso-butyl, tert-butyl, pentyl, or hexyl. In certain embodiments of compounds of Formula (I), R7 is hydrogen.

In certain ments of compounds of Formula (I), Y is hydroxylamine, de, protected aldehyde, ketone, protected ketone, thioester, ester, onyl, hydrazine, amidine, imine, diamine, keto- amine, keto-alkyne, or ene-dione.

In certain embodiments of compounds of Formula (I), alkylene is —CH2—, -—CH2CH2—, -— CH2CH2CH2—, —CH2CH2CH2CH2—, 2CH2CH2CH2—, —CH2CH2CH2CH2CH2CH2—, — CH2CH2CH2CH2CH2—, —CH2CH2CH2CH2CH2CH2CH2CH2—, — CH2CH2CH2CH2CH2CH2CH2CH2CH2—, —CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2—, — CH2CHZCH2CH2CHZCHZCHZCHZCHZCHZCH2—, or —CH2CH2CH2CHZCH2CHZCHZCHZCHZCHZCHZCH2—.

In certain embodiments of compounds ofFormula (I), n is O, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100.

In certain embodiments, dolastatin linker derivatives include compounds having the ure of Formula (II): H2N\O/L\NMBNJL“AnimNo R7 0 Me/\MeMe OMe OM OK/8 In some ments of compounds of Formula (II), L is —(alkylene—O)n—alkylene—. In some embodiments, each alkylene is —CH2CH2—, n is equal to 3, and R7 is methyl. In some embodiments, L is — alkylene—. In some embodiments of compounds of Formula (II), each alkylene is —CH2CH2— and R7 is methyl or hydrogen. Such atin linker derivatives may be in the form of a salt, or may be incorporated into a non-natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post translationally modified.

In certain ments of compounds of Formula (II), R7 is methyl, ethyl, propyl, iso-propyl, butyl, sec-butyl iso-butyl, tert-butyl, , or hexyl. In certain embodiments of compounds of Formula (II), R7 is hydrogen.

In certain ments of compounds of Formula (II), alkylene is —CH2—, -—CH2CH2—, -— CH2CH2CH2—, —CH2CH2CH2CH2—, —CH2CH2CH2CH2CH2—, —CH2CH2CH2CH2CH2CH2—, — CH2CH2CH2CH2CH2CH2CH2—, 2CH2CH2CH2CH2CH2CH2—, — CH2CH2CH2CH2CH2CH2CH2CH2CH2—, —CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2—, — CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2—, or —CH2CHZCHZCHZCH2CHZCHgCHgCHZCHZCHgCH2—.

In certain embodiments of compounds of Formula (II), n is O, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100.

In certain embodiments, compounds of Formula (I) are stable in aqueous on for at least 1 month under mildly acidic conditions. In certain embodiments, compounds of Formula (I) are stable for at least 2 weeks under mildly acidic conditions. In certain embodiments, compound of Formula (I) are stable for at least 5 days under mildly acidic conditions. In certain embodiments, such acidic conditions are pH 2 to 8.

The s and compositions provided and described herein include ptides comprising dolastatin linker derivative ning at least one carbonyl or dicarbonyl group, oxime group, hydroxylamine group, or protected or masked forms f. Introduction of at least one reactive group into a dolastatin linker derivative can allow for the application of conjugation chemistries that involve specific chemical reactions, including, but not limited to, With one or more dolastatin linker derivative(s) While not reacting With the commonly occurring amino acids. Once incorporated, the dolastatin linker derivative side chains can also be d by utilizing chemistry methodologies described herein or suitable for the particular functional groups or substituents t in the dolastatin linker derivative.

The atin linker derivative methods and compositions described herein provide conjugates of substances having a Wide variety of functional groups, substituents or moieties, With other substances including but not limited to a polymer; a water-soluble polymer; a tive of polyethylene glycol; a second protein or polypeptide or polypeptide analog; an dy or antibody fragment; and any combination thereof.

In certain embodiments, the dolastatin linker derivatives, linkers and reagents described herein, including compounds of Formulas (I) are stable in aqueous solution under mildly acidic conditions (including but not limited to pH 2 to 8). In other embodiments, such compounds are stable for at least one month under mildly acidic ions. In other embodiments, such compounds are stable for at least 2 weeks under mildly acidic conditions. In other embodiments, such compounds are stable for at least 5 days under mildly acidic conditions.

]In another aspect of the compositions, methods, techniques and strategies described herein are methods for studying or using any of the aforementioned “modified or unmodified” non-natural amino acid atin linker derivatives. Included within this aspect, by way of example only, are therapeutic, diagnostic, assay-based, industrial, cosmetic, plant biology, nmental, energy-production, consumer- products, and/or military uses which would benefit from a dolastatin linker derivative sing a “modified or unmodified” non-natural amino acid polypeptide or protein.

Non—limiting examples of dolastatin linker derivatives include: IV. Non-Natural Amino Acid tives The non-natural amino acids used in the methods and compositions described herein have at least one of the following four properties: (1) at least one onal group on the sidechain of the non- l amino acid has at least one characteristics and/or activity and/or reactivity orthogonal to the chemical reactivity of the 20 common, genetically-encoded amino acids (i.e., alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, , threonine, tryptophan, tyrosine, and ), or at least orthogonal to the chemical reactivity of the naturally ing amino acids present in the polypeptide that includes the non-natural amino acid; (2) the introduced non-natural amino acids are substantially chemically inert toward the 20 , genetically-encoded amino acids; (3) the non-natural amino acid can be stably incorporated into a polypeptide, preferably with the stability commensurate with the naturally-occurring amino acids or under typical logical conditions, and further preferably such incorporation can occur via an in vivo system; and (4) the non-natural amino acid includes an oxime functional group or a functional group that can be transformed into an oxime group by reacting with a reagent, preferably under conditions that do not destroy the biological properties of the polypeptide that includes the non-natural amino acid (unless of course such a destruction of biological ties is the purpose of the modif1cation/transformation), or where the transformation can occur under aqueous conditions at a pH between about 4 and about 8, or where the reactive site on the non-natural amino acid is an electrophilic site. Any number of tural amino acids can be introduced into the polypeptide.

Non-natural amino acids may also include protected or masked oximes or protected or masked groups that can be transformed into an oxime group after deprotection of the protected group or unmasking of the masked group. Non-natural amino acids may also e protected or masked carbonyl or onyl groups, which can be transformed into a carbonyl or dicarbonyl group after deprotection of the protected group or unmasking of the masked group and thereby are available to react with hydroxylamines or oximes to form oxime groups.

Non-natural amino acids that may be used in the methods and compositions described herein include, but are not limited to, amino acids comprising a amino acids with novel onal groups, amino acids that covalently or noncovalently interact with other molecules, glycosylated amino acids such as a sugar substituted serine, other carbohydrate modified amino acids, keto-containing amino acids, de- containing amino acids, amino acids sing polyethylene glycol or other polyethers, heavy atom substituted amino acids, chemically cleavable and/or photocleavable amino acids, amino acids with an ted side chains as compared to natural amino acids, including but not limited to, polyethers or long chain hydrocarbons, including but not limited to, greater than about 5 or greater than about 10 carbons, carbon-linked sugar-containing amino acids, redox-active amino acids, amino thioacid containing amino acids, and amino acids comprising one or more toxic moiety.

In some embodiments, non-natural amino acids comprise a saccharide moiety. es of such amino acids include N—acetyl-L-glucosaminyl-L-serine, N—acetyl-L-galactosaminyl-L-serine, N- acetyl-L-glucosaminyl-L-threonine, N—acetyl-L-glucosaminyl-L-asparagine and O-mannosaminyl-L- serine. Examples of such amino acids also include examples where the naturally-occurring N— or O- linkage between the amino acid and the saccharide is ed by a covalent linkage not commonly found in nature — including but not limited to, an alkene, an oxime, a thioether, an amide and the like. Examples of such amino acids also include saccharides that are not commonly found in naturally-occurring proteins such as 2-deoxy-glucose, ygalactose and the like.

The chemical es incorporated into polypeptides Via oration of non-natural amino acids into such polypeptides offer a variety of advantages and manipulations of polypeptides. For example, the unique reactiVity of a carbonyl or dicarbonyl functional group (including a keto- or aldehyde- functional group) allows selective modification of proteins with any of a number of hydrazine- or hydroxylamine-containing ts in vivo and in vitro. A heavy atom non-natural amino acid, for e, can be useful for phasing x-ray structure data. The pecific introduction of heavy atoms using tural amino acids also provides selectiVity and flexibility in choosing positions for heavy atoms. Photoreactive non-natural amino acids (including but not d to, amino acids with benzophenone and arylazides (including but not limited to, phenylazide) side chains), for example, allow for efficient in vivo and in vitro photocrosslinking of polypeptides. Examples of photoreactive non-natural amino acids include, but are not limited to, p-azido-phenylalanine and p-benzoyl—phenylalanine. The polypeptide with the photoreactive non-natural amino acids may then be crosslinked at will by excitation of the photoreactive group-providing temporal control. In a non-limiting example, the methyl group of a non-natural amino can be substituted with an ically labeled, including but not limited to, with a methyl group, as a probe of local structure and dynamics, including but not limited to, with the use of nuclear magnetic nce and Vibrational spectroscopy.

A. ure and Synthesis Natural Amino Acid Derivatives: Carbonyl, Carbonyl like, Masked Carbonyl, and Protected Carbonyl Groups Amino acids with an electrophilic reactive group allow for a variety of reactions to link molecules Via various chemical reactions, including, but not limited to, nucleophilic addition reactions. Such electrophilic reactive groups include a carbonyl- or dicarbonyl-group (including a keto- or aldehyde group), a carbonyl-like- or dicarbonyl-like-group (which has reactivity similar to a yl- or dicarbonyl-group and is structurally similar to a carbonyl- or dicarbonyl-group), a masked carbonyl- or masked dicarbonyl-group (which can be readily converted into a carbonyl- or dicarbonyl-group), or a protected carbonyl- or protected dicarbonyl-group (which has vity similar to a carbonyl- or dicarbonyl-group upon deprotection). Such amino acids include amino acids having the ure of Formula (XXXVII): R3 A\ / K \ R3 B R (XXXVII) R1\ R2 wherein: A is optional, and when present is lower alkylene, substituted lower ne, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower lene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, arylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene; B is optional, and when t 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-, kylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O)k- where k is l, 2, or 3, -S(O)k(alkylene or substituted alkylene)-, -C(O)-, -NS(O)2-, -OS(O)2-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -, -NR’-(alkylene or substituted alkylene)-, -C(O)N(R’)-, -CON(R’)-(alkylene or tuted alkylene)-, -CSN(R’)-, -CSN(R’)-(alkylene or substituted alkylene)-, -N(R’)CO-(alkylene or substituted alkylene)-, -N(R’)C(O)O-, -S(O)kN(R’)-, -N(R’)C(O)N(R’)-, -N(R’)C(S)N(R’)-, S(O)kN(R’)-, -N(R’)-N=, -C(R’)=N—, -C(R’)=N—N(R’)-, -C(R’)=N—N=, -C(R’)2-N=N-, and -C(R’)2-N(R’)-N(R’)-, where each R’ is independently H, alkyl, or substituted alkyl; O \ / 12$!12%gadgfiffigHAfl/“QfiorakgO S Bu fi SR" R" ”'2, o 0" I | R is H, alkyl, tuted alkyl, cycloalkyl, or substituted cycloalkyl; each R” is independently H, alkyl, substituted alkyl, or a protecting group, or when more than one R” group is t, two R” optionally form a heterocycloalkyl; R1 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 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; or the -R groups together form a bicyclic or tricyclic cycloalkyl or heterocycloalkyl comprising at least one carbonyl group, including a dicarbonyl group, protected carbonyl group, including a protected dicarbonyl group, or masked carbonyl group, including a masked dicarbonyl group; or the —K-R group together forms a monocyclic or bicyclic cycloalkyl or heterocycloalkyl sing at least one carbonyl group, including a dicarbonyl group, protected carbonyl group, including a protected dicarbonyl group, or masked carbonyl group, including a masked dicarbonyl group; with a o that when A is phenylene and each R3 is H, B is present; and that when A is —(CH2)4- and each R3 is H, B is not —NHC(O)(CH2CH2)-; and that when A and B are absent and each R3 is H, R is not methyl. Such non-natural amino acids may be in the form of a salt, or may be incorporated into a non- natural amino acid polypeptide, polymer, polysaccharide, or a cleotide and optionally post translationally modified.

In certain embodiments, compounds of Formula (XXXVII) are stable in aqueous on for at least 1 month under mildly acidic conditions. In certain embodiments, compounds of a (XXXVII) are stable for at least 2 weeks under mildly acidic ions. In certain embodiments, compound of a (XXXVII) are stable for at least 5 days under mildly acidic ions. In certain embodiments, such acidic conditions are pH 2 to 8.

In certain embodiments of nds of a (XXXVII), B is lower alkylene, tuted lower alkylene, -O-(alkylene or substituted alkylene)-, -C(R’)=N—N(R’)-, -N(R’)CO-, -C(O)-, -C(R’)=N—, -C(O)-(alkylene or substituted alkylene)-, -CON(R’)-(alkylene or substituted alkylene)-, -S(alkylene or substituted alkylene)-, -S(O)(alkylene or substituted alkylene)-, or -S(O)2(alkylene or substituted alkylene)-. In certain embodiments of compounds of Formula (XXXVII), B is —O(CH2)-, -CH=N—, -CH=N—NH-, -NHCH2-, -NHCO-, -C(O)-, -C(O)-(CH2)-, -CONH-(CH2)-, -SCH2-, -S(=O)CH2-, or - H2-. In certain embodiments of compounds of Formula (XXXVII), R is C1_6 alkyl or cycloalkyl. In n embodiments of compounds of Formula (XXXVII) R is —CH3, -CH(CH3)2, or cyclopropyl. In certain embodiments of compounds of Formula (XXXVII), R1 is H, tert-butyloxycarbonyl (Boc), 9- Fluorenylmethoxycarbonyl , N—acetyl, tetrafluoroacetyl (TFA), or benzyloxycarbonyl (Cbz). In certain embodiments of compounds of a (XXXVII), R1 is a resin, amino acid, polypeptide, dy, or polynucleotide. In certain embodiments of compounds of Formula (XXXVII), R2 is OH, 0- methyl, O-ethyl, or O-t—butyl. In certain embodiments of compounds of Formula (XXXVII), R2 is a resin, amino acid, polypeptide, antibody, or polynucleotide. In certain embodiments of compounds of a (XXXVII), R2 is a polynucleotide. In certain embodiments of compounds of Formula (XXXVII), R2 is ribonucleic acid (RNA).

A\E/m In certain embodiments of compounds of Formula (XXXVII), 13/ is selected from the group consisting of: (i) A is substituted lower alkylene, lene, substituted e, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene; B is optional, and when present is a divalent linker selected from the group consisting of lower ne, substituted lower alkylene, lower alkenylene, substituted lower lene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S(O)-, -S(O)2-, -NS(O)2-, - OS(O)2-, , -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -N(R’)-, -C(O)N(R’)-, -CON(R’)-(alkylene or substituted alkylene)-, -CSN(R’)-, CO-(alkylene or substituted alkylene)-, -N(R’)C(O)O-, -N(R’)C(S)-, -S(O)N(R’), -S(O)2N(R’), -N(R’)C(O)N(R’)-, _N(R’)C(S)N(R’)_a S(O)N(R’)-, -N(R’)S(0)2N(R’)-, - N=, -C(R’)=N—N(R’)-, -C(R’)=N—N=, -C(R’)2-N=N-, and -C(R’)2-N(R’)-N(R’)-; (ii) A is optional, and when present is substituted lower alkylene, C4-arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted lene; B is a divalent linker selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S(O)-, -S(O)2-, -NS(O)2-, -OS(O)2-, -C(O)-, -C(O)-(alkylene or tuted alkylene)-, -C(S)-, -N(R’)-, (R’)-, -CON(R’)-(alkylene or substituted alkylene)-, -CSN(R’)-, -N(R’)CO-(alkylene or substituted alkylene)-, _N(R’)C(O)O_a -N(R’)C(S)-, -S(0)N(R’), -S(0)2N(R’), -N(R’)C(O)N(R’)-, C(S)N(R’)-, -N(R’)S(O)N(R’)-, -N(R’)S(O)2N(R’)-, -N(R’)-N=, -C(R’)=N—N(R’)-, -C(R’)=N—N=, -C(R’)2-N=N-, and -C(R’)2-N(R’)-N(R’)-; (iii) A is lower alkylene; B is optional, and when present is a divalent linker ed from the group consisting of lower ne, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, -O-, kylene or substituted alkylene)-, -S-, -S(O)-, -S(O)2-, -NS(O)2-, - OS(O)2-, -C(O)-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -N(R’)-, -C(O)N(R’)-, -CSN(R’)-, -CON(R’)-(alkylene or tuted alkylene)-, -N(R’)C(O)O-, -N(R’)C(S)-, -S(O)N(R’), -S(0)2N(R’), -N(R’)C(O)N(R’)-, _N(R’)C(S)N(R’)_a -N(R’)S(O)N(R’)-, - N(R’)S(O)2N(R’)-, -N(R’)-N=, -C(R’)=N—N(R’)-, -C(R’)=N—N=, -C(R’)2-N=N-, and -C(R’)2-N(R’)-N(R’)-; and (M A is phenylene; B is a divalent linker selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S(O)-, -S(O)2-, -NS(O)2-, -OS(O)2-, -C(O)-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -N(R’)-, -C(O)N(R’)-, ’)-(alkylene or substituted alkylene)-, -CSN(R’)-, -N(R’)CO-(alkylene or substituted alkylene)-, -N(R’)C(0)0-, -N(R’)C(S)-, -S(0)N(R’), -S(0)2N(R’), -N(R’)C(0)N(R’)-, -N(R’)C(S)N(R’)-, S(O)N(R’)-, -N(R’)S(O)2N(R’)-, -N(R’)-N=, -C(R’)=N—N(R’)-n -C(R’)=N—N=, -C(R’)2-N=N-, and 2-N(R’)-N(R’)-; m s /—\0 OR' R' A: W“ A; figawtfloI *1, I‘ 7 7 7 each R’ is independently H, alkyl, or substituted alkyl, R1 is optional, and when present, is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and R2 is optional, and when present, is OH, an ester protecting group, resin, amino acid, polypeptide, or cleotide; and each R3 and R4 is independently H, n, lower alkyl, or tuted lower alkyl, R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl, In addition, amino acids haVing the ure of Formula (XXXVHI) are included: 0 (XXXVHI), A is optional, and when present is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower cycloalkylene, e, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene, B is optional, and when present is a linker selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O)k- where k is l, 2, or 3, -S(O)k(alkylene or substituted alkylene)-, -C(O)-, -NS(O)2-, -OS(O)2-, -C(O)-(alkylene or substituted alkylene)-, , -C(S)-(alkylene or substituted alkylene)-, -N(R’)-, -NR’-(alkylene or substituted alkylene)-, -C(O)N(R’)-, ’)-(alkylene or tuted alkylene)-, ’)-, -CSN(R’)-(alkylene or substituted alkylene)-, -N(R’)CO-(alkylene or substituted alkylene)-, -N(R’)C(O)O-, -S(O)kN(R’)-, C(O)N(R’)-, C(S)N(R’)-, -N(R’)S(O)kN(R’)-, -N(R’)-N=, -C(R’)=N—, -C(R’)=N—N(R’)-, -C(R’)=N—N=, -C(R’)2-N=N-, and 2-N(R’)-N(R’)-, where each R’ is independently H, alkyl, or substituted alkyl; R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; R1 is H, an amino protecting group, resin, at least one amino acid, polypeptide, or polynucleotide; and R2 is OH, an ester ting group, resin, at least one amino acid, polypeptide, or polynucleotide; with a proviso that when A is phenylene, B is present; and that when A is —(CH2)4-, B is not — NHC(O)(CH2CH2)-; and that when A and B are absent, R is not methyl. Such non-natural amino acids may be in the form of a salt, or may be orated into a non-natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post translationally modified.

In addition, amino acids haVing the structure of Formula (XXXIX) are included: Ra BTR R1 \ R2 O (XXXIX), wherein: B is a linker selected from the group consisting of lower alkylene, substituted lower alkylene, lower lene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O)k- where k is l, 2, or 3, -S(O)k(alkylene or substituted alkylene)-, -C(O)-, -NS(O)2-, -OS(O)2-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -, -NR’-(alkylene or substituted alkylene)-, (R’)-, -CON(R’)-(alkylene or substituted alkylene)-, -CSN(R’)-, -CSN(R’)-(alkylene or substituted alkylene)-, -N(R’)CO-(alkylene or substituted alkylene)-, -N(R’)C(0)0-, -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’)=N—N(R’)-, -C(R’)=N—N=, -C(R’)2-N=N-, and -C(R’)2-N(R’)-N(R’)-, where each R’ is independently H, alkyl, or substituted alkyl; R is H, alkyl, substituted alkyl, cycloalkyl, or substituted lkyl; R1 is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and R2 is OH, an ester protecting group, resin, amino acid, ptide, or polynucleotide; each R21 is ndently selected from the group consisting of H, halogen, alkyl, substituted alkyl, - N(R’)2, -C(O)kR’ where k is 1, 2, or 3, -C(O)N(R’)2, -OR’, and -S(O)kR’, where each R’ is independently H, alkyl, or substituted alkyl. Such non-natural amino acids may be in the form of a salt, or may be incorporated into a non-natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post translationally modified.

In addition, the following amino acids are included: 0 O OQK H 3k \N/NY \N OH 0 H OH H2N H N H2N O HgN COOH 0 O 7 7 7 7 O O O O SQK N\)J\H OH OH H H2N H2N H2N H2N COOH 0 0 and 0 , , , .

Such non-natural amino acids may be are optionally amino protected group, carboxyl protected and/or in the form of a salt, or may be incorporated into a tural amino acid polypeptide, polymer, polysaccharide, or a cleotide and optionally post translationally modified.

In addition, the following amino acids haVing the structure of Formula (XXXX) are included: (CR8)\BJ\Rn R1\N R2 (XXXX) wherein -NS(O)2-, -OS(O)2-, optional, and when present is a linker selected from the group consisting of lower alkylene, substituted lower alkylene, lower lene, substituted lower alkenylene, lower alkylene, substituted lower heteroalkylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S- (alkylene or substituted alkylene)-, -S(O)k- where k is l, 2, or 3, -S(O)k(alkylene or substituted alkylene)-, -C(O)-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R’)-, -NR’-(alkylene or substituted alkylene)-, -C(O)N(R’)-, -CON(R’)-(alkylene or substituted alkylene)-, -CSN(R’)-, -CSN(R’)-(alkylene or substituted ne)-, -N(R’)CO-(alkylene or substituted alkylene)-, -N(R’)C(O)O-, -S(O)kN(R’)-, -N(R’)C(O)N(R’)-, -N(R’)C(S)N(R’)-, S(O)kN(R’)-, -N(R’)-N=, -C(R’)=N—, -C(R’)=N—N(R’)-, -C(R’)=N—N=, -C(R’)2-N=N-, and -C(R’)2-N(R’)-N(R’)-, where each R’ is independently H, alkyl, or tuted alkyl, R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl, R1 is H, an amino ting group, resin, amino acid, polypeptide, or polynucleotide; and R2 is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide; each R5 is independently ed from the group consisting of H, halogen, alkyl, substituted alkyl, - N(R’)2, -C(O)kR’ where k is l, 2, or 3, -C(O)N(R’)2, -OR’, and -S(O)kR’, where each R’ is independently H, alkyl, or substituted alkyl; and n is O to 8; with a proviso that when A is —(CH2)4-, B is not —NHC(O)(CH2CH2)-. Such non-natural amino acids may be in the form of a salt, or may be incorporated into a non-natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post translationally modified.

In addition, the following amino acids are included: 5 5 050 85, 5f Y 55555 w,herein such compounds are optionally amino protected, optionally carboxyl protected, optionally amino protected and carboxyl ted, or a salt thereof, or may be incorporated into a non-natural amino acid polypeptide, polymer, ccharide, or a polynucleotide and optionally post translationally modified.

In addition, the following amino acids haVing the structure of Formula (XXXXI) are included: 0 (XXXXI), wherein, A is optional, and when present is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower lkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted arylene, alkarylene, substituted alkarylene, aralkylene, or tuted aralkylene, B is optional, and when present is a linker selected from the group consisting of lower alkylene, tuted lower alkylene, lower alkenylene, tuted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O)k- where k is l, 2, or 3, -S(O)k(alkylene or substituted alkylene)-, -C(O)-, -NS(O)2-, -OS(O)2-, -C(O)-(alkylene or substituted alkylene)-, , -C(S)-(alkylene or substituted alkylene)-, -N(R’)-, -NR’-(alkylene or substituted alkylene)-, -C(O)N(R’)-, -CON(R’)-(alkylene or substituted alkylene)-, -CSN(R’)-, -CSN(R’)-(alkylene or substituted alkylene)-, -N(R’)CO-(alkylene or substituted alkylene)-, -N(R’)C(O)O-, -S(O)kN(R’)-, -N(R’)C(O)N(R’)-, -N(R’)C(S)N(R’)-, -N(R’)S(O)kN(R’)-, -N(R’)-N=, =N—, -C(R’)=N—N(R’)-, -C(R’)=N—N=, -C(R’)2-N=N-, and -C(R’)2-N(R’)-N(R’)-, where each R’ is independently H, alkyl, or substituted alkyl; R1 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 cleotide.

Such non-natural amino acids may be in the form of a salt, or may be incorporated into a non-natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post translationally modified.

In addition, the following amino acids haVing the structure of Formula (XXXXH) are Ra BYOOJ R1 \ R2 0 (XXXXII), wherein, B is optional, and when present is a linker selected from the group consisting of lower alkylene, tuted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, tuted lower heteroalkylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O)k- where k is l, 2, or 3, -S(O)k(alkylene or substituted alkylene)-, -C(O)-, -NS(O)2-, -OS(O)2-, -C(O)-(alkylene or substituted ne)-, -C(S)-, -C(S)-(alkylene or substituted ne)-, -N(R’)-, -NR’-(alkylene or substituted alkylene)-, -C(O)N(R’)-, -CON(R’)-(alkylene or substituted ne)-, -CSN(R’)-, -CSN(R’)-(alkylene or substituted alkylene)-, -N(R’)CO-(alkylene or substituted alkylene)-, -N(R’)C(O)O-, -S(O)kN(R’)-, -N(R’)C(O)N(R’)-, -N(R’)C(S)N(R’)-, -N(R’)S(O)kN(R’)-, -N=, -C(R’)=N—, -C(R’)=N—N(R’)-, -C(R’)=N—N=, 2-N=N-, and -C(R’)2-N(R’)-N(R’)-, where each R’ is independently H, alkyl, or tuted alkyl; R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; R1 is H, an amino ting group, resin, amino acid, polypeptide, or polynucleotide; and R2 is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide; wherein each R9 is independently selected from the group consisting of H, halogen, alkyl, substituted alkyl, -N(R’)2, -C(O)kR’ where k is l, 2, or 3, -C(O)N(R’)2, -OR’, and -S(O)kR’, where each R’ is independently H, alkyl, or substituted alkyl.

Such non-natural amino acids may be in the form of a salt, or may be incorporated into a non-natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post translationally modified.

In addition, the following amino acids are included: 9999939” ”<9<9,9” wherein such compounds are optionally amino protected, optionally yl protected, optionally amino protected and carboxyl protected, or a salt thereof, or may be incorporated into a tural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post translationally modified.

In addition, the following amino acids haVing the structure of Formula (XXXXIV) are included: (CR9)9\B/K/O>0 R1\ R2 0 (XXXXIV), wherein, B is al, and when present is a linker selected from the group consisting of lower alkylene, tuted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O)k- where k is l, 2, or 3, -S(O)k(alkylene or substituted alkylene)-, -C(O)-, -NS(O)2-, -OS(O)2-, (alkylene or substituted ne)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -, -NR’-(alkylene or substituted alkylene)-, -C(O)N(R’)-, -CON(R’)-(alkylene or tuted alkylene)-, -CSN(R’)-, -CSN(R’)-(alkylene or substituted alkylene)-, -N(R’)CO-(alkylene or tuted ne)-, -N(R’)C(O)O-, -S(O)kN(R’)-, -N(R’)C(O)N(R’)-, -N(R’)C(S)N(R’)-, -N(R’)S(O)kN(R’)-, -N(R’)-N=, -C(R’)=N—, -C(R’)=N—N(R’)-, -C(R’)=N—N=, 2-N=N-, and -C(R’)2-N(R’)-N(R’)-, where each R’ is independently H, alkyl, or substituted alkyl; R is H, alkyl, substituted alkyl, lkyl, or substituted cycloalkyl; R1 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 R21 is independently selected from the group consisting of H, n, alkyl, substituted alkyl, - , R’ where k is l, 2, or 3, -C(O)N(R’)2, -OR’, and -S(O)kR’, where each R’ is independently H, alkyl, or substituted alkyl; and n is O to 8.

Such non-natural amino acids may be in the form of a salt, or may be incorporated into a non-natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post translationally modified.

] In on, the following amino acids are included: o o o o OH OH OH OH OH OH OH H2N H2N H2N HZN HZN H2N H2N O O O O and 7 7 7 7 7 wherein such compounds are optionally amino ted, optionally carboxyl protected, optionally amino protected and carboxyl protected, or a salt thereof, or may be orated into a non-natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post translationally modified.

In addition to monocarbonyl structures, the non-natural amino acids described herein may include groups such as dicarbonyl, dicarbonyl like, masked dicarbonyl and protected dicarbonyl groups.

For example, the following amino acids haVing the ure of Formula (XXXXV) are included: A\8% R1 \ R2 0 (XXXXV), wherein, A is optional, and when present is lower alkylene, substituted lower alkylene, lower cycloalkylene, tuted lower cycloalkylene, lower alkenylene, tuted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene; B is optional, and when present is a linker selected from the group consisting of lower alkylene, substituted lower ne, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O)k- where k is l, 2, or 3, -S(O)k(alkylene or substituted alkylene)-, -C(O)-, -NS(O)2-, -OS(O)2-, -C(O)-(alkylene or tuted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -, -NR’-(alkylene or substituted alkylene)-, (R’)-, -CON(R’)-(alkylene or substituted ne)-, -CSN(R’)-, -CSN(R’)-(alkylene or tuted alkylene)-, -N(R’)CO-(alkylene or substituted alkylene)-, -N(R’)C(O)O-, -S(O)kN(R’)-, -N(R’)C(O)N(R’)-, -N(R’)C(S)N(R’)-, -N(R’)S(O)kN(R’)-, -N(R’)-N=, -C(R’)=N—, -C(R’)=N—N(R’)-, -C(R’)=N—N=, -C(R’)2-N=N-, and -C(R’)2-N(R’)-N(R’)-, where each R’ is independently H, alkyl, or substituted alkyl; R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; R1 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.

Such non-natural amino acids may be in the form of a salt, or may be incorporated into a non-natural amino acid polypeptide, r, polysaccharide, or a cleotide and optionally post translationally modified.

In addition, the following amino acids haVing the structure of Formula (XXXXVI) are included: Ra B R1 \ R2 0 (XXXXVI), wherein, B is optional, and when present is a linker selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene or substituted ne)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O)k- where k is l, 2, or 3, -S(O)k(alkylene or substituted alkylene)-, , -NS(O)2-, -OS(O)2-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R’)-, -NR’-(alkylene or substituted alkylene)-, -C(O)N(R’)-, ’)-(alkylene or substituted alkylene)-, -CSN(R’)-, -CSN(R’)-(alkylene or substituted ne)-, -N(R’)CO-(alkylene or substituted alkylene)-, -N(R’)C(O)O-, -S(O)kN(R’)-, C(O)N(R’)-, -N(R’)C(S)N(R’)-, -N(R’)S(O)kN(R’)-, -N(R’)-N=, -C(R’)=N—, -C(R’)=N—N(R’)-, -C(R’)=N—N=, 2-N=N-, and -C(R’)2-N(R’)-N(R’)-, where each R’ is independently H, alkyl, or substituted alkyl; R is H, alkyl, substituted alkyl, cycloalkyl, or substituted lkyl; R1 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; 2012/039468 wherein each Ra is independently selected from the group consisting of H, halogen, alkyl, substituted alkyl, -N(R’)2, -C(O)kR’ where k is l, 2, or 3, -C(O)N(R’)2, -OR’, and -S(O)kR’, where each R’ is independently H, alkyl, or substituted alkyl.

Such non-natural amino acids may be in the form of a salt, or may be incorporated into a non-natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post translationally modified.

In addition, the following amino acids are included: O O O W m (gH”wk0 0 +H3N 000' +H3N 000' +H3N 000' and+H3N 000' 7 7 7 wherein such compounds are optionally amino ted and carboxyl ted, or a salt f. Such non-natural amino acids may be in the form of a salt, or may be incorporated into a non-natural amino acid ptide, polymer, polysaccharide, or a polynucleotide and optionally post translationally modified.

In addition, the following amino acids haVing the structure of Formula (XXXXVH) are included: 'rRz(CRa>n\ JWVRB O O (XXXXVII), wherein, B is optional, and when t is a linker selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, tuted lower alkenylene, lower alkylene, substituted lower heteroalkylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O)k- where k is l, 2, or 3, -S(O)k(alkylene or substituted alkylene)-, -C(O)-, -NS(O)2-, -OS(O)2-, -C(O)-(alkylene or tuted 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 ne)-, -CSN(R’)-, -CSN(R’)-(alkylene or substituted alkylene)-, -N(R’)CO-(alkylene or substituted alkylene)-, -N(R’)C(O)O-, -S(O)kN(R’)-, -N(R’)C(O)N(R’)-, -N(R’)C(S)N(R’)-, -N(R’)S(O)kN(R’)-, -N(R’)-N=, -C(R’)=N—, -C(R’)=N—N(R’)-, -C(R’)=N—N=, -C(R’)2-N=N-, and -C(R’)2-N(R’)-N(R’)-, where each R’ is independently H, alkyl, or substituted alkyl; R is H, alkyl, tuted alkyl, cycloalkyl, or substituted cycloalkyl; R1 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 R21 is independently selected from the group consisting of H, halogen, alkyl, tuted alkyl, -N(R’)2, -C(O)kR’ where k is l, 2, or 3, -C(O)N(R’)2, -OR’, and -S(O)kR’, where each R’ is independently H, alkyl, or substituted alkyl; and n is O to 8.

Such non-natural amino acids may be in the form of a salt, or may be orated into a non-natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post translationally modified.

In addition, the following amino acids are included: 0% 0% O (3ng (3ng (3ng O O 0%O O 8 O NH \ O 3 NH HZNLWOH HZNLWOH HZNLWOH OH H H H OH H2N H2N HZN HZN HZN O O O O O O O O , 7 7 7 7 7 7 7 OYAO OYAO OYAO o i; 1; mi: O 3 NH OH OH OH OH OH OH OH H2N H2N H2N H2N H2N H2N H2N O O O O O O O 7 7 7 7 7 7 7 and 0 wherein such compounds are optionally amino protected and carboxyl protected, or a salt thereof, or may be incorporated into a non-natural amino acid polypeptide, polymer, polysaccharide, or a cleotide and optionally post ationally modified.

In addition, the following amino acids haVing the ure of Formula (XXXXVIII) are included: 0 O A/ 1\L)KR R1HNXClOlR2 III); wherein: A is optional, and when present is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower lene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, lene, or substituted aralkylene, R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl, R1 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; X1 is C, S, or S(O); and L is alkylene, tuted alkylene, N(R’)(alkylene) or N(R’)(substituted alkylene), where R’ is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl.

Such non-natural amino acids may be in the form of a salt, or may be orated into a non-natural amino acid polypeptide, r, polysaccharide, or a polynucleotide and optionally post translationally modified.

In addition, the following amino acids haVing the structure of Formula (XXXXIX) are included: 0 O é' A A/ \L R R1HNXClolR2 (XXXXIX) wherein: A is optional, and when present is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, lene, substituted alkarylene, aralkylene, or substituted lene, R is H, alkyl, tuted alkyl, cycloalkyl, or substituted lkyl, R1 is H, an amino protecting group, resin, amino acid, ptide, or polynucleotide; and R2 is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide; L is alkylene, substituted alkylene, N(R’)(alkylene) or N(R’)(substituted alkylene), where R’ is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl.

Such non-natural amino acids may be in the form of a salt, or may be incorporated into a non-natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post translationally modified.

] In on, the following amino acids haVing the structure of Formula ) are included: 0 0 A/ \L R (XXXXX) A is optional, and when present is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower lkylene, lower lene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower cycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene, R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl, R1 is H, an amino ting group, resin, amino acid, polypeptide, or polynucleotide; and R2 is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide; L is alkylene, substituted alkylene, alkylene) or N(R’)(substituted alkylene), where R’ is H, alkyl, substituted alkyl, lkyl, or substituted cycloalkyl.

Such non-natural amino acids may be in the form of a salt, or may be incorporated into a non-natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post translationally modified.

In addition, the following amino acids haVing the structure of Formula (XXXXXI) are included: 0 o l' A1 A/ \ R X (c R 8 R 9)“ MN 0(0le (XXXXXI) wherein: A is optional, and when present is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, lene, lower heteroalkylene, substituted alkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene, R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl, R1 is H, an amino protecting group, resin, amino acid, polypeptide, or cleotide; and R2 is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide; X1 is C, S, or S(O); and n is O, l, 2, 3, 4, or 5; and each R8 and R9 on each CRgR9 group is independently selected from the group consisting of H, alkoxy, alkylamine, halogen, alkyl, aryl, or any R8 and R9 can together form =0 or a cycloalkyl, or any to adjacent R8 groups can together form a cycloalkyl, Such non-natural amino acids may be in the form of a salt, or may be incorporated into a non-natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post translationally modified. 2012/039468 ] In addition, the ing amino acids having the structure of Formula (XXXXXH) are included: 0 0 A/C \ R X (c R ER 9)“ MN 0(0le (XXXXXII) wherein: A is optional, and when present is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene, R is H, alkyl, substituted alkyl, lkyl, or substituted cycloalkyl, R1 is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and R2 is OH, an ester protecting group, resin, amino acid, ptide, or polynucleotide; n is O, 1, 2, 3, 4, or 5; and each R8 and R9 on each CRgR9 group is independently selected from the group ting of H, , alkylamine, halogen, alkyl, aryl, or any R8 and R9 can together form =0 or a cycloalkyl, or any to adjacent R8 groups can together form a cycloalkyl, Such non-natural amino acids may be in the form of a salt, or may be incorporated into a non-natural amino acid ptide, polymer, polysaccharide, or a cleotide and optionally post translationally modified.

In addition, the following amino acids haVing the structure of Formula (XXXXXHI) are 0 O 0 \S/ A A/ \ R X (c R ER 9)“ MN 0(0le (XXXXXIII) wherein: A is optional, and when present is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene, R is H, alkyl, substituted alkyl, lkyl, or substituted cycloalkyl, R1 is H, an amino protecting group, resin, amino acid, polypeptide, or cleotide; and R2 is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide; n is O, l, 2, 3, 4, or 5; and each R8 and R9 on each CRgR9 group is independently ed from the group consisting of H, alkoxy, alkylamine, halogen, alkyl, aryl, or any R8 and R9 can together form =0 or a cycloalkyl, or any to adjacent R8 groups can together form a cycloalkyl.

Such non-natural amino acids may be in the form of a salt, or may be incorporated into a non-natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post translationally modified.

In addition, the following amino acids haVing the structure of Formula (XXXXXIV) are included: MIN 0(0le (XXXXXIV); wherein: A is optional, and when present is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted alkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, arylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted lene, R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl, R1 is H, an amino protecting group, resin, amino acid, ptide, or polynucleotide; and R2 is OH, an ester ting group, resin, amino acid, polypeptide, or polynucleotide; X1 is C, S, or S(O); and L is ne, substituted alkylene, N(R’)(alkylene) or N(R’)(substituted alkylene), where R’ is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl.

Such non-natural amino acids may be in the form of a salt, or may be incorporated into a non-natural amino acid polypeptide, r, polysaccharide, or a polynucleotide and optionally post translationally modified.

In addition, the following amino acids haVing the ure of Formula (XXXXXV) are included: R1HN 0(0le (XXXXXV) wherein: A is al, and when present is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted e, heteroarylene, substituted heteroarylene, alkarylene, tuted alkarylene, aralkylene, or substituted aralkylene, R is H, alkyl, substituted alkyl, lkyl, or substituted cycloalkyl, R1 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; L is alkylene, substituted alkylene, N(R’)(alkylene) or N(R’)(substituted alkylene), where R’ is H, alkyl, tuted alkyl, cycloalkyl, or substituted cycloalkyl.

Such non-natural amino acids may be in the form of a salt, or may be incorporated into a non-natural amino acid ptide, polymer, polysaccharide, or a polynucleotide and optionally post translationally modified.

In addition, the following amino acids haVing the structure of Formula (XXXXXVI) are included: 0\\S// A0 o A/ \N —L R X RI R1HN 0(0le (XXXXXVI) wherein: A is optional, and when t is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower lene, tuted lower alkenylene, lene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower cycloalkylene, arylene, substituted arylene, heteroarylene, substituted arylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene, R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl, R1 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; L is alkylene, substituted alkylene, N(R’)(alkylene) or N(R’)(substituted alkylene), where R’ is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl.

Such tural amino acids may be in the form of a salt, or may be incorporated into a non-natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post translationally modified.

In addition, amino acids haVing the structure of Formula (XXXXXVII) are included: O (XXXXXVH), wherein: A is optional, and when present is lower alkylene, substituted lower ne, lower cycloalkylene, substituted lower cycloalkylene, lower lene, substituted lower lene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene; (b) (b) m R3 m WV‘ m I l /C\—C\C—§/ E/C=C—§ l (b) /C\—C—§<b> M is -C(R3)-, (a) R4 R4 (3) R4 (3) R4 E/CrS—EG» ’ ’(a) , (b) (b) (b) m R3 m R3 I .5“: /R3 J‘Pr / c=c—§ (b) o—c—E (b) s—C—g (b) R/Cx—Cri C / | I I R4 JJJ R4 m m m (a) (a) (a) (a) or where (a) indicates a , , , bonding to the A group and (b) indicates bonding to respective carbonyl groups, R3 and R4 are independently chosen from H, halogen, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl, or R3 and R4 or two R3 groups or two R4 groups optionally form a lkyl or a heterocycloalkyl; R is H, halogen, alkyl, substituted alkyl, lkyl, or substituted cycloalkyl, T3 is a bond, C(R)(R), O, or S, and R is H, halogen, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl, R1 is H, an amino protecting group, resin, amino acid, ptide, or polynucleotide; and R2 is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide.

Such non-natural amino acids may be in the form of a salt, or may be incorporated into a non-natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post ationally modified.

In addition, amino acids haVing the structure of Formula (XXXXXVIH) are included: Ra T3\ I{1\g R2 0 VIII), wherein: (b) (b) (b) WV‘ R3 m m m / \(|:—C/—§ (b) (|:=C—§ (b) (|:—o—§ é—s—é (b) \ \ / \ Mis -C(R3)-, (3) R4 R4 (a) 16/ R4 (3) R4 (80 as/ \R4 , , ’ , (b) (b) (b) (b) m R3 “l“ 1“? /R3 "I /3 | / s—C—g (b) (b) (b) (b) /c=c—§ o—c—g R3/c\—c—§ >9; R4 I I I R4 m m WV‘ (a) (a) (a) or (a) Where (a) indicates a , , , g to the A group and (b) indicates bonding to respective carbonyl groups, R3 and R4 are independently chosen from H, halogen, alkyl, substituted alkyl, lkyl, or substituted lkyl, or R3 and R4 or two R3 groups or two R4 groups optionally form a cycloalkyl or a heterocycloalkyl; R is H, halogen, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl, T3 is a bond, C(R)(R), O, or S, and R is H, halogen, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl, R1 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 R2, is independently ed from the group consisting of H, halogen, alkyl, substituted alkyl, 2, -C(O)kR’ Where k is l, 2, or 3, -C(O)N(R’)2, -OR’, and -S(O)kR’, Where each R’ is independently H, alkyl, or substituted alkyl.

Such non-natural amino acids may be in the form of a salt, or may be incorporated into a non-natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post translationally modified.

In addition, amino acids having the structure of Formula (XXXXXIX) are included: R O wherein: R is H, halogen, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; and T3 is O, or S.

Such non-natural amino acids may be in the form of a salt, or may be incorporated into a tural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post translationally modified.

] In addition, amino acids having the structure of Formula (XXXXXX) are included: R O O (XXXXXX), wherein: R is H, n, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl.

In addition, the following amino acids having structures of Formula (XXXXXX) are included: 0 O O O RPE R2 R1% R2 0 and 0 , .

Such non-natural amino acids may be in the form of a salt, or may be incorporated into a non-natural amino acid polypeptide, polymer, polysaccharide, or a cleotide and optionally post ationally modified.

The carbonyl or dicarbonyl functionality can be reacted selectively With a hydroxylamine- containing reagent under mild ions in aqueous solution to form the corresponding oxime linkage that is stable under physiological conditions. See, e.g., Jencks, W. P., J. Am. Chem Soc. 81, 475-481 (1959); Shao, J. and Tam, J. P., J. Am. Chem. Soc. 117(14):3893-3899 (1995). er, the unique reactivity of the carbonyl or dicarbonyl group allows for ive cation in the presence of the other amino acid side chains. See, e.g., Cornish, V. W., et al., J. Am. Chem. Soc. 118:8150-8151 (1996); Geoghegan, K. F. & Stroh, J. G., Bioconjug. Chem. 3:138-146 (1992); Mahal, L. K., et al., Science 276:1125-1128 (1997).

The synthesis of p-acetyl-(+/-)-phenylalanine and m—acetyl-(+/-)-phenylalanine is described in Zhang, Z., et al., Biochemistry 42: 6735-6746 (2003), incorporated by nce. Other carbonyl- or dicarbonyl-containing amino acids can be similarly prepared.

WO 66559 In some embodiments, a polypeptide sing a non-natural amino acid is chemically ed to generate a reactive carbonyl or dicarbonyl functional group. For instance, an aldehyde functionality useful for conjugation reactions can be generated from a functionality haVing adjacent amino and hydroxyl groups. Where the biologically active molecule is a polypeptide, for example, an inal serine or threonine (which may be normally present or may be exposed Via chemical or enzymatic digestion) can be used to te an aldehyde onality under mild oxidative cleavage ions using periodate. See, e.g., Gaertner, et. al., jug. Chem. 3: 262-268 (1992); Geoghegan, K. & Stroh, J., Bioconjug. Chem. 3:138-146 (1992); Gaertner et al., J. Biol. Chem. 269:7224-7230 (1994). However, methods known in the art are restricted to the amino acid at the N—terminus of the peptide or protein.

Additionally, by way of example a non-natural amino acid bearing adjacent hydroxyl and amino groups can be incorporated into a polypeptide as a “masked” aldehyde functionality. For example, -hydroxylysine bears a hydroxyl group adjacent to the epsilon amine. Reaction conditions for generating the aldehyde typically involve addition of molar excess of sodium metaperiodate under mild conditions to avoid oxidation at other sites within the polypeptide. The pH of the oxidation reaction is typically about 7.0. A typical reaction es the addition of about 1.5 molar excess of sodium meta periodate to a buffered solution of the polypeptide, followed by incubation for about 10 minutes in the dark. See, e. g.

US. Patent No. 6,423,685.

B. Structure and Synthesis 0fN0n-Natural Amino Acids: Dicarbonyl, Dicarbonyl-like, Masked Dicarbonyl, and Protected onyl Groups Amino acids with an ophilic ve group allow for a variety of reactions to link molecules Via philic addition reactions among others. Such electrophilic reactive groups include a dicarbonyl group (including a diketone group, a ketoaldehyde group, a ketoacid group, a ketoester group, and a ketothioester group), a dicarbonyl-like group (which has reactiVity similar to a dicarbonyl group and is structurally r to a dicarbonyl group), a masked dicarbonyl group (which can be readily converted into a dicarbonyl group), or a protected dicarbonyl group (which has reactiVity similar to a onyl group upon deprotection). Such amino acids include amino acids having the structure of Formula (xxxvn): 0 (xxxvn), wherein: A is optional, and when present is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene, B is optional, and when t is a linker linked at one end to a diamine containing moiety, the linker selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-(alkylene or substituted alkylene)-, -S-(alkylene or substituted alkylene)-, ”-, - S(O)k(alkylene or substituted alkylene)-, where k is l, 2, or 3, -C(O)-(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 ne)-, and CO-(alkylene or substituted alkylene)-, where each R” is independently H, alkyl, or substituted alkyl, 0 O k 0 O T2 A k at“ KZ"? R / 5:; T1 T2 \55: E)k A /‘71; T 1 T2 \T; \ T1 as, T2 Y KlS O VV‘VV O 3—75\T3_T3_T2}?a T3 T fry—T3_T2A / \ / 3\ A R T ‘1 T2 0 fiT/T1\TIT2\SSS- 3 3 or “N” , , where, T1 is a bond, optionally substituted C1-C4 alkylene, optionally substituted C1-C4 alkenylene, or ally substituted heteroalkyl; wherein each optional substituents is independently selected from lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, tuted lower lene, alkynylene, lower alkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower cycloalkylene, arylene, tuted arylene, heteroarylene, tuted arylene, lene, substituted lene, aralkylene, or substituted aralkylene, T2, is selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, tuted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O)k- where k is l, 2, or 3, -S(O)k(alkylene or substituted alkylene)-, -C(O)-, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N(R’)-, -NR’-(alkylene or substituted alkylene)-, -C(O)N(R’)-, -CON(R’)-(alkylene or substituted alkylene)-, ’)-, -CSN(R’)-(alkylene or substituted alkylene)-, -N(R’)CO-(alkylene or substituted alkylene)-, _N(R’)C(O)O_a 'S(O)kN(R’)'a _N(R’)C(O)N(R’)_a -N(R’)C(S)N(R’)-, 'N(R’)S(O)kN(R’)'a -N(R’)- N=, -C(R’)=N—, =N—N(R’)-, -C(R’)=N—N=, -C(R’)2-N=N-, and -C(R’)2-N(R’)-N(R’)-, Where each R’ is independently H, alkyl, or tuted alkyl; 95,57, X?“ 5%?“1 1"” “31‘” X2 X2 T3 is R'O ORV \1—/ v ‘7?“ 921' ; ; or Where each X1 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 Where each R’ is independently H, alkyl, or substituted alkyl; R is H, halogen, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; R1 is H, an amino protecting group, resin, amino acid, polypeptide, or cleotide; and R2 is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide; or the —A—B-K-R groups together form a bicyclic or lic cycloalkyl or heterocycloalkyl comprising at least one carbonyl group, including a dicarbonyl group, protected carbonyl group, including a protected dicarbonyl group, or masked carbonyl group, including a masked dicarbonyl group; or the —K-R group together forms a monocyclic or bicyclic cycloalkyl or heterocycloalkyl comprising at least one carbonyl group, including a onyl group, protected yl group, including a ted dicarbonyl group, or masked carbonyl group, including a masked dicarbonyl group.

Non-limiting example of dicarbonyl amino acids haVing the structure of Formula (X X XVII) include: O O O O OH OH HZN H2N HZN O O 7 7 7 O O O O OH OH HzN HZN WNW O O O 7 7 7 \)OJ\/\n/ O O O O O / o o OH OH OH HZN E I HZN OH H2N HzN O O O o o o o o o o O O CF3 CHF2 OH OH OH OH HZN HZN HZN HzN o 0 o o 7 7 7 7 o o o o 0 o o o OH OH OH OH HZN HZN HzN HZN 0 o O 0 7 7 7 7 o o o o o o o o 0/ s/ 0/ s/ OH OH OH OH HZN HZN HZN HZN 0 0 0 and 0 , , , .

The following amino acids having structures of Formula (XXXVII) are also ed: O OYLO O S NH HZNLKOH HZNL'l/OH OH OH HZN HzN OH O O O O O 7 7 7 7 7 1/ o o o 0 O O O 51; HN‘Z; o OH OH OH OH OH HZN HZN HZN HZN HZN o o o 0 o gr 0? HZNLKOH s HzN HZN O O o o OH OH HZN HZN O O Such non-natural amino acids may be in the form of a salt, or may be incorporated into a non-natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post translationally modified.

C. Structure and Synthesis of Non-Natural Amino Acids: Ketoalkyne, Ketoalkyne - like, Masked Ketoalkyne, Protected Ketoalkyne , Alkyne, and Cycloalkyne Groups Amino acids containing reactive groups with dicarbonyl-like reactivity allow for the linking of molecules via nucleophilic addition reactions. Such ophilic ve groups include a ketoalkyne group, a ketoalkyne-like group (which has reactivity r to a ketoalkyne group and is structurally similar to a ketoalkyne group), a masked ketoalkyne group (which can be y converted into a ketoalkyne , or a protected ketoalkyne group (which has reactivity similar to a ketoalkyne group upon deprotection). In some embodiments, amino acids containing ve groups with a terminal alkyne, internal alkyne or cycloalkyne allow for linking of molecules via cycloaddition reactions (e.g., 1,3-dipolar cycloadditions, azide-alkyne n cycloaddition, etc.) Such amino acids include amino acids having the structure of Formula (XXXXXXI-A) or (XXXXXXl-B): R A G—CEC—‘R | H 3 \B/ R3 A\ Q R3 B R R (R19)q 4 H 0 R4 (XXXXXXI-A), o (XXXXXXI-B), wherein: A is optional, and when present is lower alkylene, substituted lower alkylene, lower cycloalkylene, tuted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower alkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted e, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene, B is optional, and when present is a linker linked at one end to a e containing moiety, the 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 tuted alkylene)-, -S-(alkylene or substituted alkylene)-, -C(O)R”-, - S(O)k(alkylene or substituted alkylene)-, where k is l, 2, or 3, -C(O)-(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)-, and -N(R”)CO-(alkylene or substituted alkylene)-, where each R” is independently H, alkyl, or tuted alkyl; “37* 5'S) gsj/T‘K 5“5" G is optional, and when present is or , ; as “a ’35?“ $9 T4 is a carbonyl protecting group including, but not limited to, R'O OR' \_/ fyk “w X X1 92?-11: v 09-239 ; ; or where each X1 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, - c), -N(R)(OMe), or N3, and where each R’ is independently H, alkyl, or substituted alkyl; R is H, halogen, alkyl, substituted alkyl, lkyl, or substituted cycloalkyl; R1 is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; R2 is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide; 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 R19 is independently selected from the group consisting of C1-C6 alkyl, C1-C6 alkoxy, ester, ether, thioether, aminoalkyl, halogen, alkyl ester, aryl ester, amide, aryl amide, alkyl halide, alkyl amine, alkyl ic acid, alkyl nitro, ter, sulfonyl ester, halosulfonyl, nitrile, alkyl e, and nitro; and qis 0,1, 2, 3, 4, 5, 6, 7, 8, 9,10 or 11.

D. Structure and Synthesis ofNon-Natural Amino Acids: ine, Ketoamine-like, Masked Ketoamine, and ted Ketoamine Groups Amino acids containing reactive groups with dicarbonyl-like reactivity allow for the linking of molecules via nucleophilic addition reactions. Such reactive groups e a ketoamine group, a ketoamine-like group (which has reactivity similar to a ketoamine group and is structurally similar to a ketoamine group), a masked ine group (which can be readily converted into a ketoamine group), or a ted ketoamine group (which has reactivity similar to a ine group upon deprotection). Such amino acids include amino acids having the structure of Formula (XXXXXXH): O (XXXXXXII) wherein: A is optional, and when present is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted alkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, lene, or substituted aralkylene; B is optional, and when present is a linker linked at one end to a diamine containing moiety, the linker selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, tuted lower alkenylene, lower alkylene, substituted lower heteroalkylene, -O-(alkylene or substituted alkylene)-, -S-(alkylene or substituted alkylene)-, -C(O)R”-, - S(O)k(alkylene or substituted alkylene)-, where k is l, 2, or 3, -C(O)-(alkylene or tuted alkylene)-, -C(S)-(alkylene or substituted alkylene)-, -NR”-(alkylene or substituted ne)-, -CON(R”)-(alkylene or substituted alkylene)-, -CSN(R”)-(alkylene or substituted alkylene)-, and -N(R”)CO-(alkylene or substituted alkylene)-, where each R” is independently H, alkyl, or substituted alkyl, “57* 5' T S) “37/ 4\ 5“50 G is or , Q T1 is an optionally substituted C1-C4 alkylene, an optionally substituted C1-C4 lene, or an optionally substituted heteroalkyl; 555><9LL )?(X1 T4 is a carbonyl protecting group including, but not limited to, R'O OR' \_/ fyk “w X X1 afLXZ at11: v ; ; or where each X1 is ndently selected from the group consisting of -O-, -S-, -N(H)-, -N(R’)-, -N(Ac)-, and )-; X2 is —OR, -OAc, -SR’, -N(R’)2, -N(R’)(Ac), -N(R’)(OMe), or N3, and where each R’ is independently H, alkyl, or substituted alkyl; R is H, halogen, alkyl, tuted alkyl, cycloalkyl, or substituted cycloalkyl; R1 is H, an amino ting group, resin, amino acid, polypeptide, or polynucleotide; and R2 is OH, an ester protecting group, resin, amino acid, ptide, or polynucleotide; 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 lkyl or a heterocycloalkyl.

Amino acids haVing the ure of a (XXXXXXII) include amino acids haVing the ure of Formula (XXXXXXIII) and Formula (XXXXXXIV): N’R' XIII), 0 (XXXXXXIV) wherein each Ra is independently selected from the group consisting of H, halogen, alkyl, tuted alkyl, -N(R’)2, -C(O)kR’ where k is 1, 2, or 3, -C(O)N(R’)2, -OR’, and -S(O)kR’, where each R’ is independently H, alkyl, or substituted alkyl.

E. ure and Synthesis ofNon-Natural Amino Acids: Diamine, Diamine-like, Masked Diamine, Protected Amines and Azides Amino acids with a nucleophilic reactive group allow for a variety of reactions to link molecules Via electrophilic addition reactions among . Such nucleophilic reactive groups include a diamine group (including a hydrazine group, an e group, an imine group, a l,l-diamine group, a 1,2-diamine group, a 1,3-diamine group, and a l,4-diamine group), a diamine-like group (which has reactiVity similar to a diamine group and is structurally similar to a diamine group), a masked diamine group (which can be readily converted into a diamine group), or a protected diamine group (which has reactiVity similar to a diamine group upon deprotection). In some embodiments, amino acids containing reactive groups with azides allow for linking of molecules Via cycloaddition reactions (e.g., 1,3-dipolar cycloadditions, azide-alkyne Huisgen cycloaddition, etc.).

In another aspect are methods for the chemical synthesis of hydrazine-substituted molecules for the derivatization of carbonyl-substituted dolastatin derivatives. In one embodiment, the hydrazine- substituted molecule can dolastatin linked derivatives. In one embodiment are methods for the preparation of hydrazine-substituted molecules suitable for the derivatization of yl-containing non-natural amino acid polypeptides, including by way of e only, ketone-, or aldehyde-containing non-natural amino acid polypeptides. In a further or additional embodiment, the non-natural amino acids are incorporated site-specifically during the in Vivo translation of proteins. In a further or onal embodiment, the hydrazine-substituted dolastatin derivatives allow for the site-specific derivatization of carbonyl-containing non-natural amino acids via nucleophilic attack of each carbonyl group to form a heterocycle-derivatized ptide, including a nitrogen-containing heterocycle-derivatized polypeptide in a site-specific fashion. In a further or additional embodiment, the method for the preparation of ine-substituted dolastatin derivatives provides access to a wide variety of site-specifically derivatized polypeptides. In a further or additional ment are methods for sizing hydrazine- functionalized polyethyleneglycol (PEG) linked dolastatin derivatives.

Such amino acids include amino acids having the ure of Formula (XXXVH-A) or (XXXVH-B): R3 A\ R3 /K\ A\ /N 3 R3 B R R3 B Rhm R R2 Fm” R R2 40 (XXXVII), 4o (XXXVII-B), wherein: A is optional, and when present is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, tuted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene, B is optional, and when present is a linker linked at one end to a diamine containing moiety, the 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)-, -C(O)R”-, -C(O)R”- or tuted ne)-, where k is , -S(O)k(alkylene l, 2, or 3, -C(O)-(alkylene or substituted alkylene)-, -C(S)-(alkylene or tuted alkylene)-, -NR”-(alkylene or substituted alkylene)-, -CON(R”)-(alkylene or substituted alkylene)-, -CSN(R”)-(alkylene or substituted ne)-, and CO-(alkylene or substituted alkylene)-, where each R” is independently H, alkyl, or substituted alkyl, R8 R9 [RB H H ,Rg $8 $9 l l W I WA \ / \ /N \R N\ /N “'11/ 1 H T2 \ T2 9 / T1 \ \ / / \ 5f K ls I'LLL yr? JJJ “Ll-L “211 5‘55 9 ’ 9 9 H\N I \\T2/N R8 and R9 are independently selected from H, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, or amine protecting group; T1 is a bond, optionally substituted C1-C4 alkylene, optionally substituted C1-C4 alkenylene, or optionally substituted heteroalkyl; T2 is optionally substituted C1-C4 alkylene, ally substituted C1-C4 alkenylene, optionally substituted heteroalkyl, optionally tuted aryl, or ally substituted heteroaryl; wherein each optional substituents is independently selected from lower alkyl, substituted lower alkyl, lower cycloalkyl, substituted lower cycloalkyl, lower alkenyl, substituted lower alkenyl, alkynyl, lower heteroalkyl, substituted heteroalkyl, lower heterocycloalkyl, substituted lower heterocycloalkyl, aryl, substituted aryl, heteroaryl, substituted aryl, alkaryl, substituted alkaryl, aralkyl, or substituted aralkyl; R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; R1 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 cleotide; each of R3 and R4 is independently H, halogen, lower alkyl, or substituted lower alkyl, or R3 and R4 or two R3 groups ally form a cycloalkyl or a cycloalkyl; or the —A—B-K-R groups er form a bicyclic or tricyclic cycloalkyl or heterocycloalkyl comprising at least one diamine group, ted diamine group or masked e group; or the —B-K-R groups er form a bicyclic or tricyclic cycloalkyl or cycloaryl or heterocycloalkyl comprising at least one diamine group, protected diamine group or masked diamine group; or the —K-R group together forms a monocyclic or bicyclic cycloalkyl or heterocycloalkyl comprising at least one diamine group, protected diamine group or masked diamine group; wherein at least one amine group on —A—B-K-R is optionally a protected amine.

In one aspect are compounds comprising the structures 1 or 2: R3 R3A, 1% NH B R 3A l \T; 2 3 \B/ ZNHZ Rl-N R2 RlN R2 H R4O H R40 1 2 wherein: A is optional, and when t is lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, arylene, tuted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene, B is optional, and when present is a linker linked at one end to a diamine containing moiety, the linker selected from the group consisting of lower ne, substituted lower alkylene, lower lene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-(alkylene or substituted ne)-, -S-(alkylene or substituted alkylene)-, -C(O)R”-,-S(O)k(alkylene or tuted alkylene)-, where k is l, 2, or 3, -C(O)-(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)-, and -N(R”)CO-(alkylene or substituted ne)-, where each R” is independently H, alkyl, or substituted alkyl; T1 is a bond or CH2; and T2 is CH; wherein each optional substituents is independently selected from lower alkyl, substituted lower alkyl, lower cycloalkyl, substituted lower cycloalkyl, lower alkenyl, substituted lower alkenyl, alkynyl, lower heteroalkyl, substituted heteroalkyl, lower heterocycloalkyl, substituted lower heterocycloalkyl, aryl, tuted aryl, heteroaryl, substituted heteroaryl, alkaryl, substituted alkaryl, aralkyl, or tuted aralkyl; R1 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 cleotide; 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; or the iamine containing moiety together form a bicyclic cycloalkyl or cycloalkyl comprising at least one diamine group, protected diamine group or masked diamine group; or the —B-diamine containing moiety groups together form a ic or tricyclic cycloalkyl or cycloaryl or heterocycloalkyl comprising at least one diamine group, protected diamine group or masked diamine group; wherein at least one amine group on iamine ning moiety is optionally a protected amine; or an active metabolite, salt, or a pharmaceutically acceptable prodrug or solvate thereof.

The following non-limiting examples of amino acids haVing the structure of Formula (XXXVII) are included: H2N\ HZN HZN \ HZN NHz NH2 NHz NHz NH2 OH OH OH OH OH H2N HZN HZN HZN HZN O O O O O 7 7 7 7 7 NH2 NH2 HZN HZN OH OH OH HZN HZN HZN HzN O O O O 7 7 7 7 NH NH O\/“\ HZN NH HN NH2 NH2 OH OH OH OH HZN HZN HZN HZN O O O O NH2 NH2 OH OH OH HzN H2N HZN O O and 0 Such non-natural amino acids may also be in the form of a salt or may be incorporated into a non-natural amino acid polypeptide, polymer, polysaccharide, or a cleotide and/or optionally post translationally modified.

In certain ments, compounds of Formula (XXXVII) are stable in aqueous solution for at least 1 month under mildly acidic conditions. In certain embodiments, compounds of Formula (XXXVII) are stable for at least 2 weeks under mildly acidic conditions. In certain embodiments, compound of Formula (XXXVII) are stable for at least 5 days under mildly acidic ions. In certain embodiments, such acidic conditions are pH about 2 to about 8.

In certain embodiments of compounds of Formula (XXXVII), B is lower alkylene, substituted lower alkylene, O-(alkylene or substituted alkylene)-, C(R’)=NN(R’)-, -N(R’)CO-, C(O)-, -C(R’)=N-, C(O)-(alkylene or substituted alkylene)-, CON(R’)(alkylene or substituted alkylene)-, -S(alkylene or substituted alkylene)-, -S(O)(alkylene or substituted alkylene)-, or -S(O)2(alkylene or substituted ne)-. In n embodiments of compounds of Formula (XXXVII), B is —O(CH2)-, -CH=N-, CH=NNH-, -NHCH2-, -NHCO-, C(O)-, C(O)(CH2)-, CONH(CH2)-, -SCH2-, -S(=O)CH2-, or -S(O)2CH2-.

In n embodiments of compounds of a I), R is C1-6 alkyl or cycloalkyl. In certain embodiments of compounds of Formula (XXXVII) R is —CH3, -CH(CH3)2, or cyclopropyl. In certain embodiments of compounds of Formula (XXXVII), R1 is H, tert-butyloxycarbonyl (Boc), 9- nylmethoxycarbonyl (Fmoc), N-acetyl, tetrafluoroacetyl (TFA), or benzyloxycarbonyl (Cbz). In n embodiments of compounds of Formula I), R1 is a resin, amino acid, polypeptide, or cleotide. In certain embodiments of compounds of Formula (XXXVII), R1 is an antibody, dy fragment or monoclonal antibody. In certain embodiments of nds of Formula (XXXVII), R2 is OH, O-methyl, O-ethyl, or O-t-butyl. In certain embodiments of compounds of Formula (XXXVII), R2 is a resin, at least one amino acid, polypeptide, or polynucleotide. In certain embodiments of compounds of Formula (XXXVII), R2 is an antibody, antibody fragment or monoclonal antibody.

The following non-limiting examples of amino acids having the structure of Formula (XXXVII) are also included: ‘NHZ 0 gH OH HZN HZN o 0 / NHz Non—limiting examples of protected amino acids having the structure of Formula (XXXVII) include: N’N= \ g /CH3 E CH3 H CH3 0 ‘N=C\ V \N=C: CH3 CH3 OH OH OH H2N HZN HzN 0 0 0 g , ,and N\ [CH3 N-C\_ F. Structure and Synthesis 0fN0n-Natural Amino Acids: Aromatic Amines Non-natural amino acids with nucleophilic reactive , such as, by way of example only, an aromatic amine group (including secondary and tertiary amine groups), a masked ic amine group (Which can be readily converted into a aromatic amine , or a protected aromatic amine group (Which has vity similar to an aromatic amine group upon deprotection) allow for a variety of reactions to link molecules via s reactions, including but not limited to, reductive alkylation reactions With aldehyde containing dolastatin linker derivatives. Such aromatic amine containing non- natural amino acids include amino acids having the ure of Formula (XXXXXXV): (Ra)n\/A NH R3 B R1\ R2 H R4 0 (XXXXXXV) wherein: (Ron\/Aan?NH is selected from the group consisting of a monocyclic aryl ring, a bicyclic aryl ring, a multicyclic aryl ring, a monocyclic heteroaryl ring, a ic heteroaryl ring, and a multicyclic heteroaryl ring; A is independently CR,, or N; B is independently CRa, N, O, or S; each R2, is independently selected from the group consisting of H, halogen, alkyl, -N02, -CN, substituted alkyl, -N(R’)2, -C(O)kR’, -C(O)N(R’)2, -OR’, and -S(O)kR’, Where k is l, 2, or 3; andn is O, l, 2, 3, 4, , or 6; R1 is H, an amino protecting group, resin, at least one amino acid, ptide, or polynucleotide; and R2 is OH, an ester protecting group, resin, at least one amino acid, polypeptide, or polynucleotide; 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; M is H or —CH2R5; or the M-N—C(R5) moiety may form a 4 to 7 membered ring structure; R5 is alkyl, tuted alkyl, alkenyl, substituted l, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, alkylalkoxy, substituted alkylalkoxy, polyalkylene oxide, substituted polyalkylene oxide, cycloalkyl, tuted cycloalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocycle, substituted heterocycle, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl, -C(O)R”, -C(O)OR”, -C(O)N(R”)2, -C(O)NHCH(R”)2, -(alkylene or substituted alkylene)-N(R”)2, -(alkenylene or substituted alkenylene)-N(R”)2, lene 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 ne)-S-S-(aryl or substituted aryl), wherein each R” is ndently hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, aryl, tuted aryl, heteroaryl, substituted heteroaryl, heterocycle, tuted heterocycle, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl, or R’; or two R5 groups optionally form a cycloalkyl or a 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 may be incorporated into a non-natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally reductively ted.

(Ron\/ARx}NH The structure (as presented in all examples herein) does not present the relative orientations of “A,” “B,” “NH-M” and “Ra”; rather these four features of this structure may be oriented in any ally-sound manner (along with other features of this structure), as illustrated by example herein.

Non-natural amino acids containing an aromatic amine moiety haVing the ure of Formula (A) include non-natural amino acids haVing the ures: /A B-A1 1:11”?V A Aé B\ I , \A,IA' R3 \ 7A R3 A R R 1\N 2 R4 H R4 0 and , , A‘=A\‘ B ,,A' R3 A R‘N R2 R4 “'4 0 ; wherein, each A’ is ndently ed from CRa, N, or C_NH, and 1‘14 up to two A’ may be C_NH with the remaining A’ selected from CR,, or N.

Such non-natural amino acids may also be in the form of a salt, or may be incorporated into a non-natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally ively alkylated.

Non-limiting examples of non-natural amino acids containing an aromatic amine moiety haVing the structure of Formula (XXXXXXV) include non-natural amino acids haVing the structure of Formula (XXXXXXVI), and Formula (XXXXXXVII), if if (XXXXXXVI), (XXXXXXVH), wherein; G is an amine protecting group, including, but not limited to, A, “are; , on flog , ,5,%are, H: orw Such non-natural amino acids may be in the form of a salt, or may be incorporated into a non-natural amino acid polypeptide, r, polysaccharide, or a polynucleotide and ally reductively alkylated.

Non-natural amino acids containing an aromatic amine moiety 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(O)kR’, -C(O)N(R’)2, -OR’, and -S(O)kR’, Where k is l, 2, or 3; M is H or ; or the M-N—C(R5) moiety may form a 4 to 7 membered ring structure; R1 is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; R2 is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide; R5 is alkyl, substituted alkyl, alkenyl, tuted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, alkylalkoxy, substituted alkylalkoxy, polyalkylene oxide, tuted polyalkylene oxide, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocycle, substituted heterocycle, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl, ”, -C(O)OR”, -C(O)N(R”)2, -C(O)NHCH(R”)2, lene or substituted 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)-S-S-(aryl or substituted aryl), Wherein each R” is independently hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, , tuted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocycle, substituted heterocycle, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl, or -C(O)OR’; or R5 and any Ra optionally form a cycloalkyl or a cycloalkyl; 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 may be incorporated into a non-natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide.

] Such non-natural amino acids of Formula (XXXXXXV) may be formed by reduction of protected or masked amine moieties on the aromatic moiety of a non-natural amino acid. Such ted or masked amine moieties e, but are not limited to, imines, hydrazines, nitro, or azide substituents.

The ng agents used to reduce such protected or masked amine moieties include, but are not limited to, TCEP, NagS, 4, LlAlH4, NaBH4 01‘ NaBCNH3.

V. Non-Natural Amino Acid Linked Dolastatin Derivatives In another aspect described herein are methods, strategies and ques for incorporating at least one such atin linker derivatives into a non-natural amino acid. Also ed With this aspect are methods for producing, purifying, characterizing and using such atin linker derivatives containing 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 (including DNA and RNA) that can be used to produce, at least in part, a dolastatin linker derivative containing at least one 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 dolastatin linker derivative containing at least one non-natural amino acid.

Thus, dolastatin linker derivatives comprising at least one non-natural amino acid or modified non-natural amino acid With a carbonyl, dicarbonyl, alkyne, cycloalkyne, azide, oxime or hydroxylamine group are provided and described herein. In n embodiments, dolastatin linker derivatives with at least one non-natural amino acid or d non-natural amino acid With a carbonyl, dicarbonyl, alkyne, cycloalkyne, azide, oxime or hydroxylamine group include at least one post-translational modification at some position on the ptide. In some embodiments the co-translational or post-translational modification occurs via the cellular machinery (e. g., glycosylation, acetylation, acylation, lipid- modification, palmitoylation, palmitate addition, phosphorylation, glycolipid-linkage modification, and the like), in many instances, such cellular-machinery-based co-translational or ranslational modifications occur at the naturally occurring amino acid sites on the polypeptide, however, in certain embodiments, the cellular-machinery-based co-translational or post-translational modifications occur on the non-natural amino acid ) on the ptide.

In other embodiments, the ranslational modification does not utilize the cellular machinery, but the onality is instead provided by attachment of a molecule (a polymer; a water-soluble polymer; a derivative of polyethylene glycol; a second protein or polypeptide or polypeptide analog; an antibody or antibody fragment; and any ation thereof) comprising a second reactive group to the at least one non-natural amino acid comprising a first reactive group ding but not limited to, non-natural amino acid containing a ketone, aldehyde, acetal, hemiacetal, alkyne, cycloalkyne, azide, oxime, or hydroxylamine functional group) utilizing chemistry methodology described herein, or others le for the particular reactive groups. In n embodiments, the co-translational or post-translational modification is made in vivo in a eukaryotic cell or in a non-eukaryotic cell. In certain embodiments, the ranslational modification is made in vitro not utilizing the cellular machinery. Also ed With this aspect are methods for producing, purifying, characterizing and using such dolastatin linker derivatives containing at least one such co-translationally or post-translationally d non-natural amino acids.

Also included Within the scope of the methods, compositions, strategies and techniques described herein are reagents capable of reacting With a dolastatin linker derivative (containing a carbonyl or dicarbonyl group, oxime group, alkyne, cycloalkyne, azide, hydroxylamine group, or masked or protected forms thereof) that is part of a polypeptide so as to produce any of the aforementioned ranslational modifications. In certain ments, the resulting post-translationally modified dolastatin linker derivative Will contain at least one oxime group; the resulting modified oxime-containing dolastatin linker derivative may undergo subsequent modification reactions. Also included With this aspect are methods for producing, purifying, characterizing and using such reagents that are capable of any such post- translational modifications of such dolastatin linker derivative(s).

In certain embodiments, the polypeptide or non-natural amino acid linked dolastatin tive includes at least one co-translational or post-translational modification that is made in vivo by one host cell, Where the post-translational modification is not ly made by another host cell type. In certain embodiments, the ptide es at least one co-translational or post-translational modification that is made in vivo by a eukaryotic cell, Where the co-translational or post-translational ation is not normally made by a non-eukaryotic cell. Examples of such co-translational or post-translational modifications include, but are not limited to, glycosylation, acetylation, ion, lipid-modification, palmitoylation, ate addition, phosphorylation, glycolipid-linkage modification, and the like. In one embodiment, the co-translational or post-translational modification comprises attachment of an oligosaccharide to an asparagine by a GlcNAc-asparagine linkage ding but not limited to, Where the oligosaccharide comprises c-Man)Z-Man-GlcNAc-GlcNAc, and the like). In another ment, the co-translational or post-translational modification comprises attachment of an oligosaccharide (including but not limited to, Gal-GalNAc, Gal-GlcNAc, etc.) to a serine or threonine by a GalNAc- serine, a GalNAc-threonine, a GlcNAc-serine, or a GlcNAc-threonine linkage. In certain embodiments, a protein or polypeptide can comprise a secretion or zation ce, an epitope tag, a FLAG tag, a polyhistidine tag, a GST fusion, and/or the like. Also included With this aspect are methods for ing, ing, characterizing and using such polypeptides containing at least one such co-translational or post- translational modification. In other embodiments, the glycosylated non-natural amino acid polypeptide is produced in a non-glycosylated form. Such a non-glycosylated form of a glycosylated non-natural amino acid may be produced by methods that include chemical or enzymatic removal of accharide groups from an isolated or substantially purified or unpurified glycosylated non-natural amino acid polypeptide; production of the non-natural amino acid in a host that does not glycosylate such a non-natural amino acid polypeptide (such a host including, yotes or eukaryotes engineered or mutated to not glycosylate such a polypeptide), the introduction of a glycosylation inhibitor into the cell culture medium in Which such a tural amino acid polypeptide is being produced by a eukaryote that normally would glycosylate such a polypeptide, or a combination of any such methods. Also described herein are such non-glycosylated forms of normally-glycosylated non-natural amino acid polypeptides (by normally- glycosylated is meant a ptide that would be glycosylated when produced under conditions in which naturally-occurring polypeptides are glycosylated). Of , such non-glycosylated forms of normallyglycosylated non-natural amino acid polypeptides (or indeed any ptide described herein) may be in an unpurified form, a substantially purified form, or in an isolated form.

] In certain embodiments, the non-natural amino acid polypeptide includes at least one post- translational modification that is made in the ce of an accelerant, wherein the post-translational modification is stoichiometric, stoichiometric-like, or near-stoichiometric. In other embodiments the polypeptide is contacted with a reagent of Formula (XIX) in the presence of an accelerant. In other embodiments the rant is selected from the group consisting of: Cfifljjcr0“CENHZ’ M1: (N1: fir“:NH2 HZN/\/NH2 H2N/\/OH /\/SH 2 HOO;OHCE[5/3 El:/> H2N ,and A. Chemical Synthesis 0fN0n-Nataral Amino Acid Linked Dolastatin Derivatives: Oxime- Containing Linked Dolastatin Derivatives Non—natural amino acid dolastatin linked derivatives containing an oxime group allow for on with a variety of reagents that contain certain reactive carbonyl- or dicarbonyl- groups (including but not limited to, ketones, des, or other groups with r reactivity) to form new non-natural amino acids comprising a new oxime group. Such an oxime exchange reaction allows for the further functionalization of dolastatin linked derivatives. Further, the original atin linked derivative containing an oxime group may be useful in their own right as long as the oxime linkage is stable under conditions necessary to incorporate the amino acid into a polypeptide (e.g., the in vivo, in vitro and chemical synthetic methods described herein).

Thus, in n embodiments described herein are non-natural amino acid dolastatin linked derivatives with sidechains comprising an oxime group, an oxime-like group (which has reactivity similar to an oxime group and is structurally similar to an oxime group), a masked oxime group (which can be readily converted into an oxime group), or a protected oxime group (which has reactivity similar to an oxime group upon deprotection).

] Such non-natural amino acid dolastatin linked derivatives include dolastatin linked derivatives having the structure of Formula (VIII): , Italifigr R/N R7 0 /\ Me OMeO wherein: A is optional, and when present is lower alkylene, substituted lower ne, lower cycloalkylene, substituted lower lkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted alkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene; B is optional, and when present is a linker selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene or substituted alkylene)-, - S-, kylene or tuted alkylene)-, -S(O)k- where k is l, 2, or 3, -S(O)k(alkylene or substituted alkylene)-, , -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)- (alkylene or substituted ne)-, -N(R’)-, -NR’-(alkylene or substituted alkylene)-, -C(O)N(R’)-, -CON(R’)-(alkylene or substituted alkylene)-, -CSN(R’)-, -CSN(R’)-(alkylene or substituted alkylene)-, -N(R’)CO-(alkylene or substituted alkylene)-, -N(R’)C(O)O-, 'S(O)kN(R’)'a -N(R’)C(0)N(R’)-, _N(R’)C(S)N(R’)_a -N(R’)S(O)kN(R’)-, -N=, - C(R’)=N—, -C(R’)=N—N(R’)-, -C(R’)=N—N=, 2-N=N-, and -C(R’)2-N(R’)-N(R’)-, where each R’ is independently H, alkyl, or substituted alkyl, R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; R1 is H, an amino protecting group, resin, at least one amino acid, polypeptide, or cleotide; R2 is OH, an ester protecting group, resin, at least one amino acid, polypeptide, or polynucleotide; R3 and R4 are each independently H, halogen, lower alkyl, or substituted lower alkyl, or R3 and R4 or two R3 groups optionally form a cycloalkyl or a heterocycloalkyl; Z has the structure of: ‘7;\KKAr R5 ; R5 is H, CORg, C1-C6alkyl, or thiazole; R8 is OH R6 is OH or H; Ar is phenyl or pyridine; R7 is lkyl or hydrogen; L is a linker selected from the group consisting of —alkylene—, —(alkylene—O)n—alkylene—; and n is an integer greater than or equal to one; or an active metabolite, or a pharmaceutically acceptable prodrug or solvate thereof.

In certain embodiments of compounds of Formula (VIII), R5 is le. In certain embodiments of compounds of Formula (VIII), R6 is H. In certain embodiments of compounds of Formula (VIII), Ar is phenyl. In certain embodiments of compounds of Formula , R7 is methyl. In certain embodiments of compounds of Formula (VIII), n is an integer from O to 20. In certain embodiments of nds of Formula (VIII), n is an integer from O to 10. In certain embodiments of compounds of Formula (VIII), n is an r from O to 5.

In certain embodiments of compounds of Formula (VIII), R5 is thiazole. In certain embodiments of compounds of a (VIII), R5 is hydrogen. In certain ments of compounds of Formula (VIII), R5 is methyl, ethyl, propyl, iso-propyl, butyl, iso-butyl, sec-butyl, tert-butyl, pentyl, or hexyl.

In certain ments of compounds of Formula (VIII), R6 is H.

In n embodiments of compounds of Formula (VIII), Ar is phenyl.

In certain embodiments of compounds of Formula (VIII), R7 is methyl, ethyl, propyl, iso-propyl, butyl, sec-butyl iso-butyl, utyl, pentyl, or hexyl. In certain embodiments of compounds of Formula (VIII) and (IX), R7 is hydrogen.

In certain embodiments of compounds of Formula (VIII), each alkylene independently is —CH2—, —CH2CH2—, —CH2CH2CH2—, —CH2CH2CH2CH2—, —CH2CH2CH2CH2CH2—, —CH2CH2CH2CH2CH2CH2—, — CH2CH2CH2CH2CH2CH2CH2—, —CH2CH2CH2CH2CH2CH2CH2CH2—, — CH2CH2CH2CH2CH2CH2CH2CH2CH2—, —CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2—, — CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2—, or —CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2—.

In n embodiments of compounds of Formula (VIII), alkylene is methylene, ethylene, ene, nes, pentylene, ne, or heptylene.

In certain embodiments of compounds of Formula (VIII), each n independently is O, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, , 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100.

In certain embodiments of compounds of Formula (VIII), R1 is a polypeptide. In certain embodiments of compounds of Formula (VIII), R2 is a polypeptide. In certain embodiments of compounds of Formula , the polypeptide is an antibody. In certain embodiments of compounds of a (VIII), the antibody is herceptin.

In n embodiments, compounds of Formula (VIII) are stable in aqueous solution for at least 1 month under mildly acidic conditions. In certain embodiments, compounds of Formula (VIII) are stable for at least 2 weeks under mildly acidic conditions. In certain embodiments, compound of Formula (VIII) are stable for at least 5 days under mildly acidic conditions. In certain embodiments, such acidic conditions are pH 2 to 8. Such non-natural amino acids may be in the form of a salt, or may be incorporated into a non-natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post translationally d.

Oxime-based non-natural amino acids may be sized by methods already described in the art, or by methods described herein, including: (a) reaction of a hydroxylamine-containing non-natural amino acid with a carbonyl- or dicarbonyl-containing reagent; (b) reaction of a carbonyl- or dicarbonyl- containing non—natural amino acid with a hydroxylamine-containing reagent; or (c) reaction of an oximecontaining tural amino acid with certain carbonyl- or dicarbonyl-containing ts.

B. Chemical ure and Synthesis of Non-Natural Amino Acid Linked Dolastatin Derivatives: Alkylatea' Aromatic Amine Linked Dolastatin Derivatives In one aspect are dolastatin linker derivatives for the al derivatization of non-natural amino acids based upon the reactivity of an aromatic amine group. In further or additional embodiments, at least one of the aforementioned non-natural amino acids is incorporated into a dolastatin linker derivative, that is, such embodiments are non-natural amino acid linked dolastatin derivatives. In further or additional ments, the dolastatin linker derivatives are functionalized on their ains such that their reaction with a derivatizing non-natural amino acid generates an amine linkage. In further or additional embodiments, the dolastatin linker derivatives are selected from dolastatin linker derivatives having aromatic amine sidechains. In further or additional embodiments, the dolastatin linker derivatives comprise a masked ain, ing a masked aromatic amine group. In further or additional embodiments, the non-natural amino acids are selected from amino acids having aromatic amine sidechains. In r or additional embodiments, the non-natural amino acids se a masked ain, including a masked aromatic amine group.

In another aspect are carbonyl-substituted dolastatin linker derivatives such as, by way of example, aldehydes, and ketones, for the production of derivatized non-natural amino acid polypeptides based upon an amine linkage. In a further embodiment are aldehyde-substituted dolastatin linker tives used to derivatize aromatic amine-containing non-natural amino acid polypeptides via the formation of an amine linkage between the derivatizing dolastatin linker and the aromatic amine- containing non-natural amino acid ptide.

In further or additional embodiments, the tural amino acids comprise aromatic amine sidechains where the aromatic amine is selected from an aryl amine or a heteroaryl amine. In a r or additional embodiment, the tural amino acids resemble a natural amino acid in structure but contain aromatic amine groups. In another or further embodiment the non-natural amino acids resemble WO 66559 phenylalanine or tyrosine tic amino acids). In one embodiment, the non-natural amino acids have properties that are distinct from those of the natural amino acids. In one embodiment, such distinct properties are the chemical reactivity of the sidechain; in a further embodiment this ct chemical reactiVity permits the sidechain of the non-natural amino acid to undergo a reaction while being a unit of a polypeptide even though the sidechains of the naturally-occurring amino acid units in the same polypeptide do not undergo the aforementioned reaction. In a further ment, the sidechain of the non-natural amino acid has a chemistry orthogonal to those of the naturally-occurring amino acids. In a r embodiment, the sidechain of the tural amino acid comprises a nucleophile-containing moiety; in a further embodiment, the nucleophile-containing moiety on the sidechain of the non-natural amino acid can undergo a reaction to generate an amine-linked derivatized dolastatin. In a further embodiment, the sidechain of the non-natural amino acid comprises an electrophile-containing moiety; in a further ment, the electrophile-containing moiety on the sidechain of the non-natural amino acid can undergo nucleophilic attack to generate an amine-linked derivatized dolastatin. In any of the aforementioned embodiments in this paragraph, the non-natural amino acid may exist as a separate molecule or may be incorporated into a polypeptide of any length; if the latter, then the polypeptide may further incorporate naturally-occurring or non-natural amino acids.

Modification of non-natural amino acids described herein using reductive alkylation or reductive amination reactions have any or all of the following advantages. First, aromatic amines can be reductively alkylated with carbonyl-containing compounds, including aldehydes, and ketones, 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 te substituted amine, including secondary and tertiary amine, linkages. Second, under these reaction conditions the try is selective for non-natural amino acids as the sidechains of naturally occurring amino acids are unreactive. This allows for site-specific derivatization of polypeptides which have orated non-natural amino acids containing aromatic amine moieties or protected aldehyde moieties, including, by way of example, recombinant proteins. Such derivatized ptides and proteins can thereby be prepared as defined homogeneous products. Third, the mild ions needed to effect the reaction of an aromatic amine moiety on an amino acid, which has been incorporated into a polypeptide, with an aldehyde-containing t generally do not irreversibly destroy the tertiary structure of the polypeptide (excepting, of course, where the purpose of the reaction is to destroy such tertiary structure). rly, the mild conditions needed to effect the reaction of an aldehyde moiety on an amino acid, which has been incorporated into a polypeptide and ected, with an aromatic amine-containing reagent generally do not rsibly destroy the tertiary structure of the polypeptide ting, of , where the purpose of the reaction is to destroy such tertiary structure). Fourth, the reaction occurs rapidly at room ature, which allows the use of many types of polypeptides or reagents that would otherwise be unstable at higher atures. Fifth, the reaction occurs y is aqueous conditions, again allowing use of polypeptides and reagents incompatible (to any extent) with non-aqueous solutions. Six, the reaction occurs readily even when the ratio of polypeptide or amino acid to reagent is stoichiometric, iometric-like, or near-stoichiometric, so that it is unnecessary to add excess reagent or polypeptide to obtain a useful amount of reaction t. Seventh, the resulting amine can be produced regioselectively and/or regiospecifically, depending upon the design of the amine and carbonyl portions of the reactants. Finally, the reductive alkylation of aromatic amines with aldehyde-containing reagents, and the reductive amination of aldehydes with aromatic amine containing reagents, generates amine, including secondary and tertiary amine, linkages which are stable under biological conditions. tural amino acids with nucleophilic reactive groups, such as, by way of example only, an ic amine group (including secondary and tertiary amine ), a masked aromatic amine group (which can be readily converted into a aromatic amine group), or a protected aromatic amine group (which has reactiVity similar to a aromatic amine group upon deprotection) allow for a variety of reactions to link molecules Via various ons, ing but not limited to, reductive alkylation reactions with aldehyde containing dolastatin linked derivatives. Such alkylated non-natural amino acid linked dolastatin tives include amino acids haVingMthe ure of Formula (XXV): (“fink/gR — H I R 0Me/\MeMe OMeOM (XXV) 0 R2 wherein: Z has the structure of: ‘7;\KKAr R5 ; R5 is H, COZH, C1-C6alkyl, or thiazole; R6 is OH or H; Ar is phenyl or pyridine; R1 is H, an amino protecting group, resin, at least one amino acid, polypeptide, or polynucleotide; R2 is OH, an ester protecting group, resin, at least one amino acid, ptide, or polynucleotide; R4 is H, halogen, lower alkyl, or substituted lower alkyl; R7 is C1-C6alkyl or hydrogen; L is—alkylene— or —(alkylene—O)n—alkylene—; n is an integer greater than or equal to one; and each R16 is independently selected from the group consisting of hydrogen, halogen, alkyl, N02, CN, and substituted alkyl.

Such alkylated non-natural amino acid linked dolastatin derivatives may also be in the form of a salt, or may be incorporated into a non-natural amino acid ptide, polymer, polysaccharide, or a polynucleotide and optionally reductively alkylated.

In certain embodiments of compounds of a (XXV), R5 is thiazole or ylic acid. In certain embodiments of compounds of Formula (XXV), R6 is H. In certain embodiments of compounds of Formula (XXV), Ar is phenyl. In certain ments of compounds of Formula (XXV), R7 is methyl.

In certain embodiments of compounds of Formula (XXV), n is an integer from O to 20. In certain embodiments of compounds of Formula (XXV), n is an integer from O to 10. In certain embodiments of compounds of Formula (XXV), n is an integer from O to 5.

In n embodiments of compounds of Formula (XXV), R5 is thiazole or carboxylic acid. In certain ments of compounds of Formula (XXV), R5 is hydrogen. In certain embodiments of compounds of Formula (XXV), R5 is methyl, ethyl, propyl, iso-propyl, butyl, iso-butyl, sec-butyl, tert- butyl, pentyl, or hexyl.

In certain embodiments of compounds of Formula (XXV), R6 is H. In some embodiments of compounds of Formula (XXV), R6 is hydroxy.

In certain embodiments of compounds of Formula (XXV), Ar is phenyl.

In certain embodiments of compounds of Formula (XXV), R7 is methyl, ethyl, propyl, iso-propyl, butyl, tyl iso-butyl, utyl, pentyl, or hexyl. In certain embodiments of compounds of Formula (XXV), R7 is hydrogen.

In certain embodiments of compounds of Formula (XXV), each alkylene independently is —CH2—, H2—, -—CH2CH2CH2—, —CH2CH2CH2CH2—, 2CH2CH2CH2—, —CH2CH2CH2CH2CH2CH2—, —CH2CH2CH2CH2CH2CH2CH2—, —CH2CH2CH2CH2CH2CH2CH2CH2—, — CH2CH2CH2CH2CH2CH2CH2CH2CH2—, —CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2—, — CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2—, or —CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2—.

In certain embodiments of compounds of Formula (XXV), alkylene is methylene, ethylene, propylene, nes, ene, hexylene, or heptylene.

In certain embodiments of compounds of Formula (XXV), n is O, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100.

In certain ments of compounds of Formula (XXV), R1 is a polypeptide. In certain ments of compounds of Formula (XXV), R2 is a polypeptide. In certain embodiments of nds of Formula (XXV), the polypeptide is an antibody. In certain embodiments of compounds of Formula (XXV), the antibody is herceptin.

Compounds of Formula (XXV) are formed by the reductive alkylation of aromatic amine compounds with carbonyl containing reagents such as, by way of e, ketones, esters, thioesters, and aldehydes.

In some embodiments, the masked amine moieties of non-natural amino acids contained in polypeptides are initially reduced to give non-natural amino acids containing aromatic amine moieties incorporated into non-natural amino acid polypeptides. Such aromatic amine moieties are then reductive alkylated with carbonyl-containing reagents described above to give polypeptides containing non-natural amino acids of Formula (XXV). Such reactions may also be applied to non-natural amino acids orated into tic polymers, polysaccharides, or polynucleotides. Additionally, such reactions may be applied to non-incorporated tural amino acids. By way of example the reducing agent used to reduce masked amine moieties includes, but is not limited to, TCEP, NaZS, Na2S204, LiAlH4, B2H6, and NaBH4. By way of example only, reductive alkylation may occur in aqueous s with a pH of about 4 to about 7 and using a mild reducing agent, such as, by way of example only, sodium cyanoborohydride (NaBCNHg). In addition, other reducing agents may be used for reductive alkylation including, but not limited to, TCEP, NaZS, Na2S204, LiAlH4, B2H6, and NaBH4.

A non-limiting exemplary syntheses of non-natural amino acid polypeptides containing amino acids of a (XXV) by reductive alkylation of ary aromatic amine moieties, contained in non- natural amino acids, 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 tural amino acids incorporated into synthetic polymers, ccharides, or polynucleotides. Additionally, such reactions may be applied to non-incorporated non-natural amino acids. By way of example only, ive alkylation may occur in aqueous buffers with a pH of about 4 to about 7 and using a mild reducing agent, such as, by way of example only, sodium cyanoborohydride (NaBCNHg). In on, other reducing agents may be used for reductive alkylation including, but not limited to, TCEP, NaZS, Na2S204, LiAlH4, B2H6, and NaBH4.

C. Chemical Synthesis of Non-Natural Amino Acid Linked Dolastatin Derivatives: Heteroaryl-Containing Linked Dolastatin Derivatives In one aspect are non-natural amino acids for the chemical derivatization of dolastatin linked derivatives based upon the reactivity of a onyl group, including a group ning at least one ketone group, and/or at least one aldehyde , and/or at least one ester group, and/or at least one carboxylic acid, and/or at least one thioester group, and wherein the dicarbonyl group can be a 1,2- dicarbonyl group, a l,3-dicarbonyl group, or a l,4-dicarbonyl group. In further or additional aspects are non-natural amino acids for the chemical derivatization of dolastatin linked derivatives based upon the reactivity of a e group, including a hydrazine group, an amidine group, an imine group, a 1,1- diamine group, a l,2—diamine group, a l,3-diamine group, and a l,4-diamine group. In r or additional embodiments, at least one of the aforementioned non-natural amino acids is incorporated into a dolastatin linked derivative, that is, such embodiments are non-natural amino acid linked dolastatin derivatives. In further or additional ments, the non-natural amino acids are functionalized on their sidechains such that their reaction With a derivatizing le generates a linkage, including a heterocyclic-based linkage, including a nitrogen-containing heterocycle, and/or an aldol-based e. In further or additional ments are non-natural amino acid polypeptides that can react With a derivatizing dolastatin linker to generate a non-natural amino acid linked dolastatin derivatives containing a linkage, including a heterocyclic-based linkage, including a nitrogen-containing heterocycle, and/or an aldol-based linkage. In further or additional embodiments, the non-natural amino acids are selected from amino acids having dicarbonyl and/or diamine sidechains. In further or additional embodiments, the non- natural amino acids comprise a masked sidechain, including a masked diamine group and/or a masked dicarbonyl group. In further or additional embodiments, the tural amino acids comprise a group selected from: keto-amine (i.e., a group containing both a ketone and an amine); keto-alkyne (i.e., a group containing both a ketone and an alkyne); and an ene-dione (i.e., a group containing a dicarbonyl group and an alkene).

In r or additional embodiments, the non-natural amino acids comprise dicarbonyl sidechains Where the carbonyl is ed from a ketone, an aldehyde, a carboxylic acid, or an ester, including a thioester. In another embodiment are non-natural amino acids containing a functional group that is e of forming a heterocycle, including a nitrogen-containing heterocycle, upon treatment With an appropriately functionalized reagent. In a further or additional ment, the non-natural amino acids resemble a natural amino acid in ure but contain one of the aforementioned functional groups.

In another or further ment the non-natural amino acids resemble phenylalanine or tyrosine (aromatic amino acids); While in a separate embodiment, the tural amino acids resemble alanine and leucine (hydrophobic amino acids). In one ment, the non-natural amino acids have properties that are distinct from those of the natural amino acids. In one embodiment, such distinct properties are the chemical reactivity of the sidechain, in a further embodiment this distinct chemical reactivity s the sidechain of the non-natural amino acid to undergo a reaction While being a unit of a polypeptide even though the ains of the naturally-occurring amino acid units in the same polypeptide do not undergo the aforementioned reaction. In a further embodiment, the sidechain of the non-natural amino acid has a chemistry onal to those of the naturally-occurring amino acids. In a further embodiment, the sidechain of the non-natural amino acid comprises an ophile-containing moiety; in a further embodiment, the electrophile-containing moiety on the sidechain of the non-natural amino acid can undergo nucleophilic attack to generate a cycle-derivatized protein, including a nitrogen-containing heterocycle-derivatized protein. In any of the aforementioned embodiments in this paragraph, the non- natural amino acid may exist as a separate molecule or may be incorporated into a polypeptide of any ; if the latter, then the ptide may further incorporate naturally-occurring or non-natural amino acids.

In another aspect are e-substituted molecules, wherein the diamine group is selected from a hydrazine, an amidine, an imine, a l,l-diamine, a l,2-diamine, a 1,3-diamine and a l,4-diamine group, for the production of derivatized non-natural amino acid linked dolastatin derivatives based upon a heterocycle, ing a en-containing heterocycle, linkage. In a further embodiment are diamine- substituted dolastatin derivatives used to derivatize dicarbonyl-containing non-natural amino acid polypeptides via the formation of a cycle, including a nitrogen-containing heterocycle, linkage between the derivatizing molecule and the dicarbonyl-containing non-natural amino acid polypeptide. In further embodiments the aforementioned dicarbonyl-containing tural amino acid polypeptides are diketone-containing non-natural amino acid polypeptides. In further or additional embodiments, the dicarbonyl-containing non-natural amino acids comprise sidechains where the yl is selected from a ketone, an aldehyde, a carboxylic acid, or an ester, including a thioester. In further or additional embodiments, the diamine-substituted molecules comprise a group ed from a desired functionality.

In a further embodiment, the sidechain of the non-natural amino acid has a chemistry orthogonal to those of the naturally-occurring amino acids that allows the non-natural amino acid to react selectively with the diamine-substituted molecules. In a r embodiment, the sidechain of the non-natural amino acid comprises an electrophile-containing moiety that reacts selectively with the diamine-containing molecule; in a further embodiment, the electrophile-containing moiety on the sidechain of the tural amino acid can undergo philic attack to te a heterocycle-derivatized protein, including a nitrogen- containing heterocycle-derivatized protein. In a further aspect related to the embodiments bed in this paragraph are the ed non-natural amino acid polypeptides that result from the reaction of the derivatizing le with the non-natural amino acid polypeptides. Further embodiments include any further modifications of the already modified non-natural amino acid polypeptides.

] In another aspect are dicarbonyl-substituted molecules for the production of derivatized non- natural amino acid polypeptides based upon a heterocycle, including a nitrogen-containing heterocycle, linkage. In a further embodiment are dicarbonyl-substituted molecules used to derivatize diamine- containing non-natural amino acid polypeptides Via the formation of a heterocycle, including a nitrogencontaining heterocycle group. In a further embodiment are dicarbonyl-substituted molecules that can form such heterocycle, ing a nitrogen-containing heterocycle groups with a diamine-containing non- natural amino acid polypeptide in a pH range n about 4 and about 8. In a further embodiment are dicarbonyl-substituted molecules used to derivatize diamine-containing non-natural amino acid polypeptides Via the formation of a heterocycle, including a nitrogen-containing heterocycle, linkage between the derivatizing molecule and the diamine-containing non-natural amino acid polypeptides. In a r embodiment the dicarbonyl-substituted molecules are diketone-substitued molecules, in other aspects ketoaldehyde-substituted molecules, in other aspects id—substituted molecules, in other aspects ketoester-substituted molecules, including ketothioester-substituted molecules. In further embodiments, the dicarbonyl-substituted les comprise a group selected from a desired functionality. In further or additional embodiments, the aldehyde-substituted molecules are aldehyde- substituted polyethylene glycol (PEG) molecules. In a further embodiment, the sidechain of the non- natural amino acid has a chemistry orthogonal to those of the naturally-occurring amino acids that allows the non-natural amino acid to react ively With the carbonyl-substituted molecules. In a further embodiment, the sidechain of the non-natural amino acid comprises a moiety (e.g., diamine group) that reacts selectively With the dicarbonyl-containing molecule; in a further embodiment, the nucleophilic moiety on the sidechain of the non-natural amino acid can undergo electrophilic attack to generate a heterocyclic-derivatized protein, including a en-containing heterocycle-derivatized protein. In a further aspect related to the embodiments described in this paragraph are the modified non-natural amino acid polypeptides that result from the reaction of the tizing molecule With the non-natural amino acid polypeptides. Further ments include any r modifications of the y modified non- natural amino acid polypeptides.

] In one aspect are methods to derivatize proteins via the reaction of carbonyl and ine reactants to generate a heterocycle-derivatized n, including a nitrogen-containing heterocycle-derivatized dolastatin. Included Within this aspect are methods for the derivatization of dolastatin linker derivatives based upon the condensation of carbonyl- and hydrazine-containing reactants to generate a cycle-derivatized dolastatin, including a nitrogen-containing cycle-derivatized dolastatin. In additional or further embodiments are methods to derivatize ketone-containing dolastatin derivatives or aldehyde-containing dolastatin derivatives With hydrazine-fianctionalized non-natural amino acids. In yet additional or further aspects, the hydrazine-substituted molecule can include ns, other polymers, and small molecules.

In another aspect are methods for the chemical synthesis of hydrazine-substituted molecules for the derivatization of carbonyl-substituted dolastatin derivatives. In one embodiment, the hydrazine-substituted molecule is a dolastatin linked tive suitable for the derivatization of carbonylcontaining non-natural amino acid polypeptides, including by way of example only, ketone-, or aldehyde- containing non-natural amino acid polypeptides.

In one aspect are non-natural amino acids for the chemical derivatization of dolastatin analogs based upon a quinoxaline or phenazine linkage. In further or onal embodiments, the non-natural amino acids are functionalized on their ains such that their reaction With a derivatizing dolastatin linker generates a quinoxaline or phenazine linkage. In further or additional embodiments, the non-natural amino acids are selected from amino acids having l,2-dicarbonyl or l,2-aryldiamine sidechains. In further or additional embodiments, the tural amino acids are selected from amino acids having protected or masked l,2-dicarbonyl or l,2-aryldiamine ains. Further included are equivalents to carbonyl sidechains, or protected or masked equivalents to l,2-dicarbonyl sidechains.

In another aspect are derivatizing molecules for the production of derivatized non-natural amino acid polypeptides based upon quinoxaline or phenazine linkages. In one embodiment are 1,2- WO 66559 dicarbonyl substituted dolastatin linker derivatives used to derivatize 1,2-aryldiamine containing non- natural amino acid polypeptides to form quinoxaline or phenazine linkages. In another embodiment are 1,2-aryldiamine substituted dolastatin linker derivatives used to tize 1,2-dicarbonyl containing non- natural amino acid polypeptides to form quinoxaline or phenazine linkages. In a further aspect related to the above embodiments are the modified non-natural amino acid polypeptides that result from the on of the derivatizing dolastatin linker with the non-natural amino acid polypeptides. In one embodiment are 1,2-aryldiamine containing non-natural amino acid polypeptides derivatized with 1,2-dicarbonyl substituted dolastatin linker derivative to form quinoxaline or phenazine linkages. In another embodiment are 1,2-dicarbonyl containing non-natural amino acid polypeptides derivatized with yldiamine substituted dolastatin linker derivatives to form quinoxaline or phenazine linkages.

Provided herein in certain embodiments are derivatizing molecules for the production of toxic compounds comprising tural amino acid polypeptides based upon triazole linkages. In some embodiments, the reaction between the first and second reactive groups can proceed Via a dipolarophile reaction. In n embodiments, the first reactive group can be an azide and the second reactive group can be an alkyne. In further or alternative embodiments, the first reactive group can be an alkyne and the second ve group can be an azide. In some embodiments, the Huisgen cycloaddition reaction (see, e.g., Huisgen, in 1,3-DIPOLAR CYCLOADDITION CHEMISTRY, (ed. Padwa, A., 1984), p. 1-176) provides for the incorporation of turally d amino acids bearing azide and alkyne-containing side chains permits the resultant polypeptides to be ed with extremely high selectiVity. In certain embodiments, both the azide and the alkyne functional groups are inert toward the twenty common amino acids found in naturally-occurring polypeptides. When brought into close proximity, however, the ”spring-loaded” nature of the azide and alkyne groups is revealed and they react selectively and ently Via Huisgen [3 2] ddition reaction to generate the corresponding triazole. See, e.g., Chin et al., Science 301:964-7 (2003); Wang et al., J. Am. Chem. Soc., 125, 3192-3193 (2003); Chin et al., J. Am.

Chem. Soc., 124:9026-9027 (2002). Cycloaddition reaction ing azide or alkyne-containing polypeptides can be d out at room temperature under aqueous conditions by the addition of Cu(II) (e. g., in the form of a catalytic amount of CuSO4) in the presence of a ng agent for reducing Cu(II) to Cu(I), in situ, in catalytic amount. See, e.g., Wang et al., J. Am. Chem. Soc. 125, 3192-3193 (2003); Tomoe et al., J. Org. Chem. 67:3057-3064 (2002); tseV, Angew. Chem. Int. Ed. 41:2596-2599 (2002). Preferred reducing agents include ascorbate, metallic copper, e, hydroquinone, Vitamin K, glutathione, cysteine, Fez, C02, and an applied electric potential.

Such non-natural amino acid heteroaryl-linked dolastatin tives include amino acids haVing the structure of Formula (XXXI): 2012/039468 E;$1NJ0 e/\MeMe Me 7 OMeO MeO NH (XXXI) wherein: Z has the structure of: ‘7;\KKAr R5 ; R5 is H, COZH, C1-C6alkyl, or thiazole; R6 is OH or H; Ar is phenyl or pyridine; R1 is H, an amino protecting group, resin, at least one amino acid, polypeptide, or polynucleotide; R2 is OH, an ester ting group, resin, at least one amino acid, polypeptide, or polynucleotide; R4 is H, halogen, lower alkyl, or substituted lower alkyl; R7 is C1-C6alkyl or hydrogen; L is—alkylene— or —(alkylene—O)n—alkylene—; n is an integer greater than or equal to one; D has the structure of: ’ R17 ’ R17 R17 R N\ R N\ ‘zL WY 17 / I1 ;; R18\ a: M4f N \s *2: M2? N g—W/ X»; R17/T3 ’ R17/T3 ’ N N a— \_5 s1 I \—§ 3 E- / K / N N H H ’ each R17 is independently selected from the group consisting of H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, , substituted alkoxy, alkylalkoxy, substituted alkylalkoxy, polyalkylene oxide, substituted polyalkylene oxide, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl, -(alkylene or substituted alkylene)-ON(R”)2, -(alkylene or substituted alkylene)-C(O)SR”, -(alkylene or substituted alkylene)-S-S-(aryl or substituted aryl), -C(O)R”, R”, or (R”)2, wherein each R” is ndently hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, alkaryl, tuted alkaryl, aralkyl, or substituted aralkyl; each 21 is a bond, CR17R17, O, S, NR’, CR17R17-CR17R17, CR17R17-O, O-CR17R17, CR17R17-S, S-CR17R17, CR17R17-NR’, or NR’-CR17R17; each R’ is H, alkyl, or substituted alkyl; each 22 is ed from the group consisting of a bond, -C(O)-, -C(S)-, optionally substituted C1-C3 alkylene, optionally tuted C1-C3 lene, and optionally substituted heteroalkyl; each 23 are independently selected from the group consisting of a bond, optionally substituted C1-C4 alkylene, optionally substituted C1-C4 alkenylene, optionally substituted heteroalkyl, -O-, -S-, -C(O)-, -C(S)-, and -N(R’)-; each T3 is a bond, C(R”)(R”), O, or S; With the proviso that When T3 is O or S, R” cannot be halogen; each R” is H, halogen, alkyl, tuted alkyl, cycloalkyl, or substituted cycloalkyl; m and p are 0, l, 2, or 3, provided that at least one ofm or p is not 0; (b) (b) ‘ ”I“ it "ii “T" “I“ /C (b) \R—g E/C—R—E (b) [ls/C=C\—§ 03) /c—o—E @E/CrS—g (b) M2 is (a) 3 (a) R4 R4 (a) R4 (a) R4 (a) , , , (b) as) (b) (b) aw R3 R3 “’W‘ JJJ‘ R3 | \ / \ / I / 020—3 (b) 0—C—E (b) s—c—g (b) /C\—C—E <b> / I I I R4 >55 R4 m m m (a) (a) (a) or (a) Where (a) indicates a , , , bonding to the B group and (b) indicates bonding to tive positions Within the heterocycle group; If: a” (b) ,3: Wllw /R3 /E_ §(b) /|C|__C/R3‘3“) /i|:_O_E(b /C|_S_§(b) E—ngw) M3 is (8051’ ,(afiv CR\4 (:02 @577 4 Where (a) indicates bonding to the B group and (b) indicates bonding to respective positions Within the heterocycle group; (b) (b) (b) (b) m m aw m I | 3(1)) /(|:\R_(%R=§ (b) R3 E4 (Eli-g 0—3:; (b) s—CSE (b) M4 1s. ( 19/]:a) (a) 3 3 (a) (a) or (a) 7 7 g g j where (a) indicates bonding to the B group and (b) indicates bonding to tive ons Within the heterocycle group; each R19 is independently selected from the group consisting of C1-C6 alkyl, C1-C6 alkoxy, ester, ether, thioether, aminoalkyl, halogen, alkyl ester, aryl ester, amide, aryl amide, alkyl halide, alkyl amine, alkyl sulfonic acid, alkyl nitro, thioester, sulfonyl ester, halosulfonyl, nitrile, alkyl nitrile, and nitro; qis 0,1, 2, 3, 4, 5, 6, 7, 8, 9,10 or 11; and each R16 is ndently selected from the group consisting of hydrogen, halogen, alkyl, N02, CN, and substituted alkyl.

In some embodiments, the compound of a (XXXI) include compounds having the ure of Formula (XXXI-A): QR N Me =N R79/1,“OM eMe OMeO MeO NH (XXXI—A) R 0 40 Z R1 R2 In n embodiments of compounds of Formula (XXXI), R5 is thiazole or carboxylic acid. In certain embodiments of compounds of Formula (XXXI), R6 is H. In certain embodiments of compounds of Formula (XXXI), Ar is phenyl. In certain embodiments of compounds of Formula (XXXI), R7 is methyl. In certain embodiments of compounds of Formula (XXXI), n is an r from O to 20. In certain embodiments of compounds of Formula (XXXI), n is an integer from O to 10. In certain ments of compounds of Formula (XXXI), n is an integer from O to 5.

In certain ments of compounds of Formula (XXXI), R5 is thiazole or carboxylic acid. In certain embodiments of compounds of Formula (XXXI), R5 is hydrogen. In certain embodiments of compounds of Formula (XXXI), R5 is methyl, ethyl, propyl, iso-propyl, butyl, iso-butyl, sec-butyl, tertbutyl , pentyl, or hexyl.

In certain embodiments of nds of Formula (XXXI), R6 is H. In some embodiments of compounds of Formula (XXXI), R6 is hydroxy.

] In certain embodiments of compounds of Formula (XXXI), Ar is phenyl.

In certain embodiments of nds of Formula (XXXI), R7 is methyl, ethyl, propyl, iso- propyl, butyl, tyl iso-butyl, tert-butyl, pentyl, or hexyl. In certain embodiments of nds of Formula (XXXI), R7 is hydrogen.

In certain embodiments of compounds of Formula (XXXI), each alkylene independently is —CH2— , -—CH2CH2—, -—CH2CH2CH2—, —CH2CH2CH2CH2—, —CH2CH2CH2CH2CH2—, —CH2CH2CH2CH2CH2CH2—, —CH2CH2CH2CH2CH2CH2CH2—, —CH2CH2CH2CH2CH2CH2CH2CH2—, — CH2CH2CH2CH2CH2CH2CH2CH2CH2—, —CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2—, — CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2—, or —CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2—.

In certain embodiments of compounds of Formula (XXXI), alkylene is methylene, ethylene, propylene, butylenes, pentylene, hexylene, or heptylene.

] In certain embodiments of compounds of Formula (XXXI), n is O, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100.

In certain embodiments of compounds of Formula (XXXI), R1 is a polypeptide. In certain ments of nds of Formula (XXXI), R2 is a polypeptide. In certain embodiments of compounds of Formula (XXXI), the ptide is an antibody. In certain embodiments of compounds of Formula (XXXI), the antibody is herceptin.

The formation of such non-natural amino acid heterocycle-linked dolastatin derivatives haVing the structure of Formula (XXXI) includes, but is not limited to, (i) reactions of diamine-containing non- natural amino acids With dicarbonyl-containing dolastatin linked derivatives or reactions of e- containing non—natural amino acids With ketoalkyne-containing atin linked derivatives, (ii) reactions of dicarbonyl-containing non-natural amino acids With either diamine-containing dolastatin linked derivatives or reactions of dicarbonyl-containing non-natural amino acids With ketoamine-containing dolastatin linked derivatives, (iii) reactions of ketoalkyne-containing non-natural amino acids With diamine-containing dolastatin linked derivatives, or (iV) reactions of ketoamine-containing non-natural amino acids With dicarbonyl-containing V.

Modification of dolastatin linked derivatives described herein With such ons have any or all of the ing advantages. First, diamines undergo sation With dicarbonyl-containing compounds in a pH range of about 5 to about 8 (and in further embodiments in a pH range of about 4 to about 10, in other embodiments in a pH range of about 3 to about 8, in other embodiments in a pH range of about 4 to about 9, and in further embodiments a pH range of about 4 to about 9, in other embodiments a pH of about 4, and in yet another ment a pH of about 8) to generate cycle, including a en- containing heterocycle, linkages. Under these conditions, the sidechains of the naturally occurring amino acids are unreactive. Second, such selective chemistry makes possible the pecific derivatization of recombinant ns: derivatized proteins can now be prepared as defined homogeneous products. Third, the mild conditions needed to effect the reaction of the diamines described herein with the dicarbonyl- containing polypeptides bed herein generally do not irreversibly destroy the tertiary structure of the polypeptide (excepting, of course, where the purpose of the reaction is to y such tertiary structure). , the reaction occurs y at room temperature, which allows the use of many types of ptides or reagents that would be unstable at higher temperatures. Fifth, the reaction occurs readily is s conditions, again allowing use of polypeptides and reagents atible (to any ) with non-aqueous solutions. Six, the reaction occurs readily even when the ratio of polypeptide or amino acid to reagent is stoichiometric, near stoichiometric, or stoichiometric-like, so that it is unnecessary to add excess t or ptide to obtain a useful amount of on product. Seventh, the resulting heterocycle can be produced regioselectively and/or regiospecifically, depending upon the design of the diamine and dicarbonyl portions of the reactants. Finally, the condensation of diamines with dicarbonyl- containing molecules generates heterocycle, including a nitrogen-containing heterocycle, linkages which are stable under biological conditions.

V1 Location 0fn0n-nataral amino acids in atin linker derivatives The methods and compositions described herein include incorporation of one or more non-natural amino acids into a dolastatin linker derivative. One or more tural amino acids may be incorporated at one or more particular positions which do not disrupt activity of the dolastatin linker derivative. This can be achieved by making “conservative” substitutions, including but not limited to, tuting hydrophobic amino acids with non-natural or natural hydrophobic amino acids, bulky amino acids with non-natural or natural bulky amino acids, hydrophilic amino acids with non-natural or l hydrophilic amino acids) and/or inserting the non-natural amino acid in a location that is not required for activity.

A variety of biochemical and structural approaches can be employed to select the desired sites for tution with a non-natural amino acid within the dolastatin linker derivative. In some embodiments, the non-natural amino acid is linked at the C-terminus of the dolastatin derivative. In other embodiments, the non-natural amino acid is linked at the N—terminus of the atin derivative. Any position of the dolastatin linker derivative is suitable for selection to incorporate a non-natural amino acid, and selection may be based on rational design or by random selection for any or no particular desired purpose. Selection of desired sites may be based on producing a non-natural amino acid polypeptide (which may be further modified or remain unmodified) having any desired property or activity, including but not limited to a receptor binding modulators, receptor activity modulators, modulators of binding to binder partners, binding partner activity modulators, g partner conformation modulators, dimer or multimer formation, no change to activity or property compared to the native molecule, or manipulating any physical or chemical property of the polypeptide such as solubility, aggregation, or stability.

Alternatively, the sites fied as critical to biological activity may also be good candidates for substitution with a non-natural amino acid, again depending on the desired activity sought for the polypeptide. r alternative would be to simply make serial substitutions in each on on the ptide chain with a non-natural amino acid and observe the effect on the actiVities of the polypeptide. Any means, technique, or method for selecting a on for tution with a non-natural amino acid into any polypeptide is suitable for use in the methods, techniques and compositions described herein.

The structure and actiVity of naturally-occurring mutants of a polypeptide that contain deletions can also be examined to ine regions of the protein that are likely to be tolerant of substitution with a non-natural amino acid. Once residues that are likely to be intolerant to substitution with tural amino acids have been eliminated, the impact of proposed substitutions at each of the remaining positions can be examined using methods including, but not limited to, the three-dimensional structure of the nt polypeptide, and any associated ligands or binding proteins. X-ray crystallographic and NMR structures of many polypeptides are available in the Protein Data Bank (PDB, W‘vsr\i~x.>‘"<:s§:s :53), a centralized database containing three-dimensional structural data of large molecules of proteins and nucleic acids, one can be used to identify amino acid positions that can be substituted with non-natural amino acids. . In addition, models may be made investigating the secondary and tertiary structure of polypeptides, if three-dimensional structural data is not available. Thus, the identity of amino acid positions that can be substituted with non-natural amino acids can be readily obtained.

Exemplary sites of incorporation of a non-natural amino acid e, but are not limited to, those that are excluded from potential or binding regions, or regions for binding to binding proteins or ligands may be fully or lly t exposed, have minimal or no hydrogen-bonding interactions with nearby residues, may be minimally exposed to nearby reactive residues, and/or may be in regions that are highly flexible as predicted by the three-dimensional crystal structure of a particular polypeptide with its associated receptor, ligand or binding proteins.

A wide variety of non-natural amino acids can be substituted for, or incorporated into, a given position in a polypeptide. By way of example, a particular non-natural amino acid may be selected for incorporation based on an examination of the three ional crystal structure of a polypeptide with its associated ligand, receptor and/or binding proteins, a preference for conservative substitutions In one embodiment, the methods described herein include incorporating into the dolastatin linker tive, where the dolastatin linker derivative ses a first reactive group; and contacting the dolastatin linker derivative with a molecule (including but not limited to a second n or polypeptide or polypeptide analog; an antibody or antibody fragment; and any combination f) that comprises a second reactive group. In certain ments, the first reactive group is a hydroxylamine moiety and the second reactive group is a yl or dicarbonyl moiety, whereby an oxime linkage is formed. In certain embodiments, the first reactive group is a carbonyl or dicarbonyl moiety and the second reactive group is a hydroxylamine moiety, whereby an oxime linkage is formed. In certain embodiments, the first reactive group is a yl or dicarbonyl moiety and the second reactive group is an oxime moiety, whereby an oxime exchange reaction occurs. In certain embodiments, the first reactive group is an oxime moiety and the second reactive group is carbonyl or dicarbonyl moiety, whereby an oxime exchange reaction occurs.

In some cases, the dolastatin linker derivative incorporation(s) will be ed with other additions, substitutions, or deletions within the polypeptide to affect other chemical, physical, pharmacologic and/or biological . In some cases, the other additions, substitutions or deletions may increase the stability (including but not limited to, resistance to proteolytic degradation) of the polypeptide or increase y of the ptide for its appropriate receptor, ligand and/or binding proteins. In some cases, the other additions, substitutions or deletions may increase the solubility (including but not limited to, when expressed in i M or other host cells) of the polypeptide. In some embodiments sites are selected for substitution with a lly encoded or tural amino acid in addition to another site for incorporation of a non-natural amino acid for the e of increasing the polypeptide solubility following sion in E coli, or other inant host cells. In some embodiments, the polypeptides se another addition, substitution, or deletion that modulates affinity for the associated ligand, binding proteins, and/or receptor, tes (including but not limited to, increases or decreases) receptor dimerization, stabilizes receptor dimers, tes circulating half-life, modulates release or bio- availability, facilitates purification, or improves or alters a ular route of administration. Similarly, the non-natural amino acid polypeptide can comprise chemical or enzyme ge sequences, protease cleavage sequences, reactive groups, antibody-binding domains (including but not limited to, FLAG or poly-His) or other affinity based sequences (including but not limited to, FLAG, poly-His, GST, etc.) or linked molecules ding but not limited to, biotin) that improve detection (including but not limited to, GFP), purification, transport thru tissues or cell membranes, prodrug release or activation, size reduction, or other traits of the polypeptide.

VII. HER2 Gene as Exemplar ] The methods, compositions, strategies and techniques described herein are not limited to a particular type, class or family of polypeptides or proteins. Indeed, virtually any ptides may be designed or modified to include at least one “modified or unmodified” non-natural amino acids containing dolastatin linker derivative described herein. By way of example only, the ptide can be homologous to a therapeutic protein selected from the group consisting of: alpha-l ypsin, angiostatin, antihemolytic factor, antibody, antibody fragment, monoclonal antibody (e. g., bevacizumab, mab, panitumumab, infliximab, adalimumab, basiliximab, daclizumab, omalizumab, ustekinumab, etanercept, gemtuzumab, alemtuzumab, rituximab, trastuzumab, nimotuzumab, palivizumab, and abciximab), apolipoprotein, apoprotein, atrial natriuretic , atrial natriuretic polypeptide, atrial peptide, C-X-C chemokine, T39765, NAP-2, ENA—78, gro-a, gro-b, gro-c, IP-lO, GCP-2, NAP-4, SDF-l, PF4, MIG, calcitonin, c-kit ligand, cytokine, CC ine, te chemoattractant protein-l, monocyte chemoattractant protein-2, monocyte chemoattractant protein-3, monocyte inflammatory protein-l alpha, te inflammatory protein-i beta, RANTES, 1309, R83915, R91733, HCCl, T58847, D31065, T64262, CD40, CD40 ligand, c-kit ligand, collagen, colony stimulating factor (CSF), complement factor 5a, complement inhibitor, complement receptor 1, cytokine, epithelial neutrophil activating peptide-78, MIP-l6, MCP-l, epidermal growth factor (EGF), epithelial neutrophil activating peptide, erythropoietin (EPO), exfoliating toxin, Factor IX, Factor VII, Factor VIII, Factor X, fibroblast grth factor (FGF), fibrinogen, fibronectin, four-helical bundle protein, G-CSF, glp-l, GM-CSF, glucocerebrosidase, tropin, grth factor, grth factor receptor, grf, og protein, obin, hepatocyte grth factor (hGF), hirudin, human grth hormone (hGH), human serum albumin, ICAM-l, ICAM-l receptor, LFA—l, LFA—l receptor, insulin, insulin-like grth factor (IGF), IGF-I, IGF-II, interferon (IFN), IFN—alpha, IFN—beta, IFN—gamma, interleukin (IL), IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, keratinocyte grth factor (KGF), lactoferrin, leukemia inhibitory factor, luciferase, neurturin, neutrophil inhibitory factor (NIF), atin M, osteogenic protein, oncogene t, paracitonin, parathyroid hormone, PD-ECSF, PDGF, peptide hormone, pleiotropin, protein A, protein G, pth, nic exotoxin A, pyrogenic exotoxin B, pyrogenic exotoxin C, pyy, relaxin, renin, SCF, small biosynthetic protein, soluble ment or I, soluble I-CAM l, soluble interleukin receptor, soluble TNF or, somatomedin, somatostatin, somatotropin, streptokinase, superantigens, staphylococcal enterotoxin, SEA, SEB, SECl, SEC2, SEC3, SED, SEE, steroid e receptor, superoxide ase, toxic shock syndrome toxin, thymosin alpha 1, tissue plasminogen activator, tumor grth factor (TGF), tumor necrosis factor, tumor necrosis factor alpha, tumor necrosis factor beta, tumor necrosis factor or (TNFR), VLA—4 protein, VCAM-l n, vascular endothelial grth factor (VEGF), urokinase, mos, ras, raf, met, p53, tat, fos, myc, jun, myb, rel, estrogen receptor, progesterone receptor, testosterone receptor, aldosterone receptor, LDL receptor, and corticosterone.

In one embodiment is a method for treating solid tumor which overexpresses HER-2 selected from the group consisting of breast cancer, small cell lung carcinoma, ovarian cancer, prostate cancer, gastric carcinoma, cervical cancer, esophageal carcinoma, and colon cancer. In another embodiment, the solid tumor is breast cancer. In a further embodiment the solid tumor is ovarian cancer.

] Thus, the following description of trastuzumab is provided for illustrative purposes and by way of e only, and not as a limit on the scope of the methods, itions, strategies and techniques described herein. Further, reference to trastuzumab in this application is intended to use the generic term as an e of any antibody. Thus, it is understood that the modifications and chemistries described herein with reference to trastuzumab can be y applied to any antibody or monoclonal antibody, including those specifically listed herein.

Trastuzumab is a humanized monoclonal antibody that binds to the domain IV of the ellular segment of the HER2/neu receptor. The HER2 gene (also known as HER2/neu and ErbB2 gene) is amplified in 20-30% of early-stage breast cancers, which makes it overexpressed. Also, in , HER2 may send signals without mitogens arriving and binding to any receptor, making it overactive.

HER2 extends through the cell membrane, and carries s from outside the cell to the inside.

In healthy people, signaling compounds called mitogens arrive at the cell membrane, and bind to the outside part of other members of the HER family of receptors. Those bound receptors then link (dimerize) with HER2, activating it. HER2 then sends a signal to the inside of the cell. The signal passes through different mical pathways. This includes the Pl3K/Akt pathway and the MAPK pathway. These signals promote invasion, survival and growth of blood s (angiogenesis) of cells.

Cells treated with trastuzumab undergo arrest during the G1 phase of the cell cycle so there is d proliferation. It has been suggested that trastuzumab induces some of its effect by downregulation of HER2/neu leading to disruption of receptor dimerization and signaling through the downstream PI3K cascade. P27Kip1 is then not phosphorylated and is able to enter the nucleus and inhibit cdk2 actiVity, causing cell cycle . Also, trastuzumab suppresses angiogenesis by both induction of antiangiogenic factors and repression of proangiogenic factors. It is thought that a contribution to the unregulated grth observed in cancer could be due to proteolytic cleavage of HER2/neu that results in the release of the extracellular domain. zumab has been shown to inhibit HER2/neu main cleavage in breast cancer cells.

VIII. Cellular uptake nataral amino acids Non-natural amino acid uptake by a eukaryotic cell is one issue that is typically considered when designing and selecting non-natural amino acids, including but not limited to, for incorporation into a protein. For example, the high charge density of oc-amino acids suggests that these nds are unlikely to be cell permeable. Natural amino acids are taken up into the eukaryotic cell Via a collection of protein-based transport systems. A rapid screen can be done which assesses which non-natural amino acids, if any, are taken up by cells (examples 15 & 16 herein rate non-limiting examples of tests which can be done on non-natural amino acids). See, e. g., the toxicity assays in, e. g., the US. Patent Publication No. 2004/198637 entitled “Protein Arrays,” which is herein incorporated by reference in its entirety, and Liu, D.R. & Schultz, P.G. (1999) Progress toward the evolution of an organism with an expanded genetic code. PNAS United States 96:4780-4785. Although uptake is easily analyzed with various assays, an alternative to ing non-natural amino acids that are amenable to cellular uptake pathways is to provide thetic pathways to create amino acids in vivo.

Typically, the non-natural amino acid produced Via cellular uptake as described herein is produced in a concentration sufficient for ent protein biosynthesis, including but not d to, a natural ar amount, but not to such a degree as to affect the concentration of the other amino acids or exhaust cellular resources. Typical concentrations ed in this manner are about 10 mM to about 0.05 IX. Biosynthesis 0fN0n-Nataral Amino Acids Many biosynthetic pathways already exist in cells for the tion of amino acids and other compounds. While a biosynthetic method for a particular non-natural amino acid may not exist in nature, ing 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 can be generated in host cell by adding new enzymes or modifying existing host cell pathways. Additional new enzymes include naturally occurring enzymes or artificially evolved enzymes. For example, the biosynthesis of p- aminophenylalanine (as presented in an example in entitled “In vivo incorporation of unnatural amino acids”) relies 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 cell with a plasmid sing the genes. The genes, when sed in the cell, provide an enzymatic pathway to size the desired compound. Examples of the types of enzymes that are optionally added are provided . Additional enzymes sequences are found, for example, in k. Artificially evolved enzymes can be added into a cell in the same manner. In this manner, the cellular machinery and resources of a cell are manipulated to produce non-natural amino acids.

A variety of methods are available for producing novel enzymes for use in thetic pathways or for evolution of existing pathways. For example, recursive recombination, including but not limited to, as ped by Maxygen, Inc. (available on the world wide web at www.maxygen.com), can be used to p novel enzymes and ys. See, e. g., Stemmer (1994), Rapid evolution of a protein in vitro by DNA Shuflling, m370(4):389-391; and, Stemmer, (1994), DNA Shuflling by random ntation and reassembly: In vitro recombination for molecular evolution, Proc. Natl. Acad. Sci.

M, 91:10747-10751. Similarly DesignPathTM, developed by Genencor (available on the world wide web at genencor.com) is optionally used for metabolic pathway engineering, including but not limited to, to engineer a pathway to create a non-natural amino acid in a cell. This technology reconstructs existing ys in host organisms using a combination of new genes, including but not limited to those identified through functional genomics, molecular evolution and design. Diversa Corporation (available on the world wide web at diversa.com) also provides technology for rapidly screening libraries of genes and gene pathways, including but not limited to, to create new pathways for biosynthetically producing non-natural amino acids.

Typically, the non-natural amino acid produced with an engineered biosynthetic pathway as described herein is produced in a concentration sufficient for efficient protein biosynthesis, including but not limited to, a l cellular amount, but not to such a degree as to affect the tration of the other amino acids or exhaust cellular resources. Typical concentrations produced in vivo in this manner are about 10 mM to about 0.05 mM. Once a cell is transformed with a d comprising the genes used to produce enzymes desired for a c pathway and a non-natural amino acid is generated, in vivo selections are optionally used to r optimize the production of the non-natural amino acid for both ribosomal protein sis and cell growth.

X. onal Synthetic Methodology The non-natural amino acids described herein may be synthesized using methodologies described in the art or using the techniques bed herein or by a combination thereof. As an aid, the following table provides various ng electrophiles and nucleophiles Which may be combined to create a desired functional group. The information provided is meant to be illustrative and not limiting to the synthetic techniques described .

Table 1: Examples of Covalent Linkages and Precursors Thereof Carboxamides Activated esters am1nes/an111nes Carboxamides acyl azides amines/anilines Carboxamides acyl halides /anilines Esters acyl halides alcohols/phenols Esters acyl nitriles alcohols/phenols Carboxamides acyl nitriles amines/anilines lmines Aldehydes amines/anilines ones aldehydes or ketones Hydrazines OXimes des or ketones Hydroxylamines Alkyl amines alkyl halides amines/anilines Esters alkyl halides carboxylic acids Thioethers alkyl halides Thiols Ethers alkyl halides alcohols/phenols Thioethers alkyl sulfonates Thiols Esters alkyl sulfonates carboxylic acids Ethers alkyl sulfonates alcohols/phenols Esters Anhydrides alcohols/phenols Carboxamides Anhydrides amines/anilines Thiophenols aryl halides Thiols Aryl amines aryl halides Amines Thioethers Azindines Thiols Boronate esters Boronates Glycols Carboxamides carboxylic acids amines/anilines Esters carboxylic acids Alcohols hydrazines Hydrazides carboxylic acids N—acylureas or Anhydrides carbodiimides carboxylic acids Esters diazoalkanes carboxylic acids hers Epoxides Thiols Thioethers haloacetamides Thiols iazines iazines amines/anilines Triazinyl ethers halotriazines alcohols/phenols Amidines imido esters amines/anilines Ureas lsocyanates amines/anilines Urethanes lsocyanates alcohols/phenols Thioureas isothiocyanates amines/anilines hers Maleimides Thiols Phosphite esters phosphoramidites ls Silyl ethers silyl halides ls Alkyl amines sulfonate esters amines/anilines 2012/039468 Thioethers sulfonate esters ate esters sulfonate esters Alcohols Sulfonamides sulfonyl halides sulfonyl halides phenols/alcohols In l, carbon electrophiles are susceptible to attack by complementary nucleophiles, including carbon nucleophiles, wherein an ing nucleophile brings an on pair to the carbon electrophile in order to form a new bond between the nucleophile and the carbon electrophile.

Non-limiting examples of carbon nucleophiles include, but are not limited to alkyl, alkenyl, aryl and alkynyl Grignard, organolithium, organozinc, alkyl-, alkenyl , aryl- and alkynyl-tin reagents (organostannanes), alkyl-, alkenyl-, aryl- and alkynyl-borane reagents (organoboranes and organoboronates); these carbon nucleophiles have the age of being kinetically stable in water or polar organic solvents. Other non-limiting examples of carbon nucleophiles include phosphorus ylids, enol and enolate reagents; these carbon nucleophiles have the advantage of being relatively easy to te from precursors well known to those skilled in the art of synthetic organic chemistry. Carbon nucleophiles, when used in conjunction with carbon electrophiles, engender new carbon-carbon bonds between the carbon nucleophile and carbon electrophile.

Non-limiting examples of non-carbon nucleophiles suitable for coupling to carbon electrophiles include but are not limited to primary and secondary amines, thiols, tes, and thioethers, alcohols, des, azides, semicarbazides, and the like. These non-carbon nucleophiles, when used in conjunction with carbon electrophiles, typically generate heteroatom linkages ), wherein X is a oatom, including, but not limited to, oxygen, sulfur, or nitrogen.

EXAMPLES Example 1: Synthesis ound 1 Scheme 1 H0\/\o/\/0\/\0/\/0H 1_2 DIAD PPh3 0::NON°\/\o/\/0\/\o/\/0H HCI .nH H Elf-'01: HflNthpfirMN No\/\0/\/ \/\0/\CH0 O /\ OMe O OMe O 1-5 N\—/_S l NaBH3CN wwwgg:1ka0 OMeO OMeO N\_/, s 16] NH2NH2 WWW?»R%EOMeO OMe O Compound 1-3: Tetra (ethylene glycol ) 1-1 (10g, 51.5mmol), N—hydroxyphthalimide 1-2 ( 8.4g, 51.15mmol) and triphenylphosphine ( 17.6g, 67mmol) were dissolved in 300 mL of tetrahydrofiJran followed by addition of DIAD (12.8 mL, 61.78 mol) at 0°C. The resulting solution was stirred at room ature overnight, and then concentrated to dryness. The residue was purified by flash column chromatography to give 5.47g (31%) of compound 1-3.

Compound 1-4: To a solution of compound 1-3 (200mg, 0.59mmol) in 15mL dichloromethane was added Dess-Martin Periodinane ( 300mg, 0.71 mmol). The reaction e was stirred at ambient temperature overnight. The reaction was quenched with the solution of sodium bisulfite in 15 mL of saturated sodium bicarbonate. The mixture was ted. The organic layer was washed with ted sodium bicarbonate, brine, dried over sodium sulfate, d and concentrated in vacuo. The e was purified by flash column chromatography to give 150mg(75%) of compound 1-4.

Compound 1-6: To a solution of monomethyldolastatin hydrochloride salt 1-5 (50mg, 0.062 mmol) in lmL of DMF was added compound 1-4 (63mg, 0.186mmol) and 70uL of acetic acid, followed by addition of 8 mg of sodium cyanoborohydride. The resulting mixture was stirred at ambient temperature for 2 hours. The reaction mixture was d with water and d by HPLC to give 60mg (80%) of compound 1-6. MS (ESI) m/z 547 [M+2H], 1092 [M+H].

Compound 1: Compound 1-6 (60mg, 0.05mmol) was dissolved in 1 mL of DMF. 32 uL of hydrazine was added. The resulting solution was stirred at ambient temperature for 1 hour. The reaction was quenched with lN hloride solution. The reaction mixture was purified by HPLC to give 33 mg (55%) of compound 1. MS (ESI) m/z 482 [M+2H], 962 [M+H]. e 2: Synthesis ofCompound 2 Scheme 2 H°\/\o/\/o\/\OH 2-1 DIAD PPh3 l “6.199%099gs:lcgimwwOMeO OMe O NaBH3CN 658.25%99%Q999:OMeO OMe O l NHZNHZ Wow;9099999: OMe O OMe O Compound 2 was synthesized via a similar synthetic route as described in Example 1. MS (ESI) m/z 460 [M+2H], 918 [M+H].

Example 3: Synthesis ofCompound 3 Scheme 3 NOH o 0 6?:10 N’O\/\0/\/0H H0/\/1\/\OH \ DIAD, PPh3 0 u;21ka5 OMe O OMe 0 @1133 NaBH3CN inowwmfim?OMeO OMe O NH2NH2 Wtwt]? OMe O OMe O Compound 3 was synthesized Via similar tic route to Example 1. MS (ESI) m/z 438 [M+2H], 974 [M+H].

Example 4: Synthesis ofCompound 4 nd 4-2: To a on of Val (OtBu)-OH.HCl 4-1 (1 g, 4.77 mmol) and bromoethanol (304.7 uL, 4.3 mmol) in 10 mL of DMF was added 1.68 ml of DIEA. The reaction mixture was stirred at room temperature for 2 days. 4.8 mmol of BocZO was added to the on mixture, followed by 0.84 mL of DIEA. The reaction mixture was stirred at room temperature for 2 days. The reaction mixture was concentrated in vacuo and extracted with ethyl acetate, and washed with water, brine, dried over sodium e and concentrated in vacuo. The residue was purified by flash column chromatography to give 0.66 g of compound 4-2.

Scheme 4 0 Boc O Boc O ’VBr "'2N 1. N ‘ OJ<L> HON\Jkok_> HCI i 2. Boc20 A A NOTNE—Jk HCI Boc “kg/LN“:I 0Me o 0Me 00 +:2/ONOgo/\/I a A OtBu /\ HATU o prthrngJ’ka—I‘JW:30H \ Boc o /\ 0Me0 0Me0 OtBu o H “H H \N o /\ 0Me o 0Me0 HCI 0 OH 0 H “H H N/°\/\~ “Q‘fifirQfi/‘WN \ I O /=\ I 0Me O 0Me O ; O OH HNQLN H ‘\\H N N 2N’O\/\N a 0 /\ I OMeO OMe o 0 OH ] Compound 4-3: To a solution of compound 4-2 (500 mg, 1.58 mmol), N—hydroxyphthalimide (261 mg, 1.6 mmol) and triphenylphosphine (538 mg, 2.05 mmol) in 15 mL THF was added DIAD (394 uL, 1.9 mol) at at 0°C. The resulting solution was stirred at room temperature overnight, and then concentrated in vacuo. The residue was d by flash column chromatography to give 0.68 g of compound 4-3.

Compound 4-4: Compound 4-3 was dissolved in 15 mL 4N HCl/Dioxane. The reaction mixture was stirred at room temperature for 2 days and concentrated in vacuo. The residue was dissolved in DMF and treated with BocZO (230 uL, 1 mmol) and DIEA (352 uL, 2 mmol). The reaction mixture was stirred at room temperature for 2 days. The reaction mixture was purified by HPLC to give 100 mg of nd 4-4. nd 4-5: To a solution of compound Boc-Val-Dil-methleap-OH in DMF is added phe(OtBu)-OH.HCl, HATU and N—methylmorpholine. The reaction mixture is stirred at room temperature for 4 hours. The reaction e is trated in vacuo and extracted with ethyl acetate (100 mLXl, 50 mL X2). The organic layer is combined and washed with brine, dried over sodium sulfate and concentrated in vacuo. The residue is purified by flash tography. The resulting compound is treated with HCl/EtOAC to give nd 4-5.

Compound 4-6: To a solution of compound 4-5 in DMF is added compound 4-4, HATU and DIEA. The on mixture is stirred at room temperature for 4 hours. The reaction mixture is concentrated in vacuo and extracted with ethyl acetate (100 mLXl, 50 mL X2). The organic layer is combined and washed with brine, dried over sodium sulfate and concentrated in vacuo. The residue is purified by flash chromatography to give compound 4-6. nd 4-7: Compound 4-6 is dissolved in 15 mL 4N HCl/Dioxane. The reaction mixture is stirred at room temperature for 2 hours and concentrated in vacuo to give compound 4-7. nd 4-8: To a solution of compound 4-7 in 1 mL of DMF is added formylaldehyde and acetic acid, followed by on of sodium cyanoborohydride. The resulting mixture is stirred at ambient temperature for 2 hours. The on mixture is diluted with water and purified by HPLC to give compound 4-8.

Compound 4: Compound 4-8 is dissolved in 1 mL of DMF. Hydrazine is added. The resulting solution is d at ambient temperature for 1 hour. The reaction is quenched with lN hydrochloride solution. The reaction mixture is purified by HPLC to give nd 4.

Example 5: Synthesis ofCompound 5 ] Compound 4-7 is dissolved in 1 mL of DMF. Hydrazine is added. The resulting solution is stirred at ambient temperature for 1 hour. The reaction is quenched with lN hydrochloride solution. The reaction mixture is purified by HPLC to give Compound 5.

Example 6: Synthesis ofCompound 6 Scheme 6 ”‘3' Phe-OMe.HC| X0YN/{Lt oH —. ON\)LN —.

OMe O OMe O OMe o OMe 00 HzN/iokN OMe O OMe O COOMe Boc-N--methyl--Va| BocO OMeO OMeo COOMe OMeO OMeO COOH CW\/\0 Ir“: @0950Ali?6° o A OMeO OMeO COOH 6-6 I NaBH3CN Wo/Vowo/Vow OHEJDLN 670‘NHZNHZ OMeO OMeOo H2“oN/\/o\/\o/\/O\/\:ngEmm‘mMg; OMe O OMe O Compound 6-2: To a solution of compound 6-1 (500 mg, 0.875 mmol) in 3 mL of DMF was added 283 mg of phenylalanine hydrochloride, 433mg of HATU and 581 uL of N—methylmorpholine. The reaction mixture was stirred at room temperature for 4 hours. The reaction mixture was concentrated in vacuo and extracted with ethyl acetate (100 mLXl, 50 mL X2). The c layer was ed and washed with brine, dried over sodium sulfate and concentrated in vacuo. The residue was purified by flash chromatography to give 560 mg (76%) of compound 6-2.

Compound 6-3: Compound 6-2 was dissolved in 15 mL 4N HCl/Dioxane. The reaction mixture was stirred at room temperature for 2 hours and concentrated in vacuo to give 511 mg of compound 6-3.

Compound 6-4: To a solution of compound 6-3 (368 mg, 0.55 mmol) in 3 mL of DMF was added 255 mg of Boc-N—methyl valine, 314mg of HATU and 303 uL of N—methylmorpholine. The reaction mixture was stirred at room temperature for 4 hours. The reaction mixture was concentrated in vacuo and extracted with ethyl acetate (100 mLXl, 50 mL X2). The organic layer was ed and washed with brine, dried over sodium e and concentrated in vacuo. The e was purified by flash chromatography to give 370 mg (79%) of compound 6-4.

] Compound 6-5: To a solution of compound 6-4 (170mg) in 10 mL MeOH was added 5eq of 1N LiOH. The reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was acidified by lNHCl and extracted with ethyl acetate washed with brine, dried over sodium e and concentrated in vacuo to give 150 mg (90%) of compound 6-5.

Compound 6-6: Compound 6-5 was dissolved in 4N HCl/Dioxane. The reaction mixture was stirred at room temperature for 2 hours and concentrated in vacuo and purified by HPLC to give 150mg of compound 6-6.

Compound 6-7: To a on of compound 6-6 (50mg, 0.062 mmol) in lmL of DMF was added compound 1-4 (63mg, 0.186mmol) and 70uL of acetic acid, ed by addition of 8 mg of sodium orohydride. The resulting mixture was stirred at ambient temperature for 2 hours. The reaction mixture was diluted with water and purified by HPLC to give 60mg (80%) of compound 6-7.

Compound 6: Compound 6-7 (60mg, 0.05mmol) was dissolved in 1 mL of DMF. 32 [LL of hydrazine was added. The resulting solution was stirred at ambient temperature for 1 hour. The on was quenched with lN hydrochloride solution. The reaction mixture was purified by HPLC to give 33 mg (55%) of Compound 6.

Example 7: sis ofCompound 7 Scheme 7 Hadrypfi? {WATTOMeO OMe O l NaBH3CN Cngwfiwd%@%OMe0 OMeO O OH I NHZNHZ 0mmEmma OMe O OMe 00 nd 7 was synthesized via similar synthetic route to Compound 1. MS (ESI) m/z 440 [M+2H], 879 [M+H].

Example 8: Synthesis ofCompound 8 Scheme8 HCI Hdk ,nH N/°\/\o/\¢° HN N \ l o/‘\'a OMeO + OMeoo 0 3'3 / OHMHINaBH3CN No/\/O\/\N ONZLN {3?} OMe o OMeO O OH NH2NH2 H2No/\/O\/\“If“?mmrN OMe o OMeOo OH Compound 8 was sized Via similar synthetic route to Compound 1. MS (ESI) m/z 418 [M+2H], 835 [M+H].

Example 9: Synthesis ofCompound 9 Scheme 9 :13:ij o H / ?0c 0 /E\ | + N‘O’VN OH OMe o OMe o , N g 9.1 /\ O ZI "’I Z ZI 2\$0:H / / —Z O ZI I Z ZI 2\$0—2022* / Illin- / —Z I I I Z Z Z \$02022* Z / llluu Compound 9-1: To a solution of nd Boc-Val-Dil-methleap-OH in DMF is added 4- (2—Aminoethyl) pyridine, HATU and N—methylmorpholine. The reaction mixture is stirred at room temperature for 4 hours. The reaction mixture is concentrated in vacuo and extracted With ethyl acetate (100 mLXl, 50 mL X2). The organic layer is combined and washed With brine, dried over sodium sulfate and concentrated in vacuo. The residue is purified by flash chromatography. The resulting compound is d with HCl/EtOAC to give compound 9-1.

Compound 9-2:To a solution of compound 9-1 in DMF is added compound 4-4, HATU and DIEA. The reaction mixture is stirred at room temperature for 4 hours. The reaction mixture is trated in vacuo and extracted With ethyl acetate (100 mLXl, 50 mL X2). The organic layer is combined and washed With brine, dried over sodium sulfate and concentrated in vacuo. The residue is purified by flash chromatography to give compound 9-2.

Compound 9-3:Compound 9-2 is dissolved in 15 mL 4N HCl/Dioxane. The reaction e is stirred at room temperature for 2 hours and concentrated in vacuo to give compound 9-3.

Compound 9-4: To a solution of compound 9-3 in 1mL of DMF is added formylaldehyde and acetic acid, followed by addition of sodium cyanoborohydride. The resulting mixture is stirred at ambient temperature for 2 hours. The reaction mixture is diluted With water and purified by HPLC to give compound 9-4.

Compound 9: Compound 9-4 is dissolved in 1 mL of DMF. Hydrazine is added. The resulting on is stirred at ambient temperature for 1 hour. The reaction is quenched With 1N hydrochloride solution. The reaction mixture is purified by HPLC to give compound 9.

Example 10: Synthesis ofCompound 10 Scheme 10 o H H N/o\/\H NEJLNH N N I | \ o A OMeO OMeO HCI IN NH2NHzi HNHJLN H H N N H N/O\/\H2 o /-\| OMeO OMeO l/N Compound 10: Compound 9-3 is dissolved in 1 mL of DMF. Hydrazine is added. The resulting solution is stirred at t temperature for 1 hour. The reaction is quenched With 1N hloride solution. The reaction mixture is purified by HPLC to give Example 10.

Table 1. ures of Compounds 1-10 1 o/\/0\/\o/\/o\/\“If“A91R09? OMe o OMe o N\_/s \\\H 2 HZN’O\/\o/\/O\/\NI‘/N\i)ol\RMOMe o OMe oN 2012/039468 H H H ’0 \\\ H2N \/\'il NgL'ifiW/N N o A OMeO OMeO 0 0H Example 11: Analysis t0x binding to HER2 receptor Her2-Fc was immobilized on CM5 chip to a density of N 280 RU. Flow rate was adjusted to 50 ul/min with HBS-EP as buffer. HerTox variants were injected for 3 min with 15 dissociation phase. 30sec pulse of 20mM HCl was used for regeneration. The Bivalent Analyte model was utilize to fit the data (Figure 1). Analysis of the data indicates that HerTox HA121-NC2D: Kd N 60 pM (chi2=4) and HerTox HA121-NC1D: Kd N 300 pM (chi2=l4).

Example 12: Transient Transfection CHO-S culture is seeded at 0.75x10A6/mL approximately 16 hours pre-transfection in FreeStyle Cho medium. Cells are ready to transfect the next day when the cell count has reached 1.4 — 1.6x10A6/mL. When cells reach target count, 400 mM pAF stock is added to a 1.4mM final culture tration. PEI/DNA complex is prepared as bed: DNA (1.42 ug/1x10A6 cells) is dissolved in RPMI (5% (V/V) of total culture volume), DNA/RPMI mixture is incubated at room temperature for 2 minutes, PEI stock (1 mg/mL) is added to DNA solution at a 3:1 ratio (mL PEI/ug DNA), and the mixture is incubated at room temperature for 5 min. The culture is gently added to the e and swirled. The flasks are transferred to a 32°C tor. At day 6 post-transfection a western blot , analysis is performed. At day 7 post-transfection, the supernatant is harvested. e 13: Anti-Her2 variant sion test 30 ml shaker cultures, CHO-S in FreeStyle medium; 56 ug DNA in PEI reagent were used. 1.5 mM pAF was also used. At day 6, the supernatant was harvested. Titer was determined by PC ELISA. (Figures 2 and 3) Example 14: In vitro tion ofProliferation Assay At day 1, the cells were seeded. The media was aspirated and the T-225 culture flasks of cells was rinsed with 30mL PBS -/. PBS was aspirated and 6 mL of 0.25% Trypsin—EDTA was added to each flask. The flasks were incubated at 37C, 5%C02 for 2-5 minutes. Adherent cells were dislodged by hitting flask and trypsin was neutralized by adding 14mL culture medium. The cell suspension was mixed and transered to a 50 mL l tube. The cells were spun down at 1200 rpm, 5 min, room temperature.

The resultant cell pellet was resuspended in 12 mL of culture medium. The cells were counted in a tometer. Cells were seeded at appropriate cell densities into 96-well flat-bottom, clear plates and incubated overnight at 37°C, 5% C02 to allow cells to attach. Plating volume was 80uL/well. 80uL/well of e medium was utilized as the “no cell” control.

Cell plating density examples include: BT474 (high Her2) — 20,000 cells/well in MEM (50/50), 10% FBS, P/S MDA—MB-468 (Her2 negative) — 6,000 cells/well in MEM (50/50), 10% FBS, P/S HCC1954 — 5,000 cells/well in RPMI 1640, 10% FBS, P/S SKOV-3 — 6,000 cells/well in RPMI 1640, 10% FBS, P/S HT29 — 20,000 cells/well in McCoy’ s 5A, 10% FBS, P/S At day 2, the test samples were added to the cells. The test samples were diluted in culture medium to a 9X stock concentration in Column 2 of a round-bottom 96-well plate. 3X serial dilutions were made from Column 2 to Column 11 with a multi-channel pipettor. 10uL/well of the above samples was added in duplicate to the appropriate wells of the seeded plate. The total volume in the wells was about 90uL/well. 10uL/well of culture medium was added to avoid edge effects for sample wells. Media l wells were included in the inner wells where no sample was added (just 10uL/well of culture medium) to be used in proliferation calculations. The plates were incubated at 37°C, 5%C02 for 72 hrs.

At day 5, eration readout occurred. To detect cell proliferation, 10uL/well of WST-8 reagent was added (Cell Counting Kit-8, cat# CK04-20, Dojindo Labs) to all wells. The plate was ted for 4 hrs at 37°C, 5%C02. The plates were measured for absorbance at OD 450nm. Calculation of proliferation inhibition of test samples: subtract the “no cell control” OD450 value from all wells’ OD450 values; calculate average of the Media Control (untreated) wells; calculate each sample’s % Media Control value using the formula: (OD450 sample/OD450 avg % Media Control) *100; calculate the average, rd deviation, and %CV of each sample’s duplicate % Media Control ; plot each samples average % Media Control value t sample concentration; calculate IC50 values using 4- parameter logistic fit regression analysis to determine potency of test samples.

Figure 4 and Figure 5 illustrate results from the in vitro proliferation assay with dolastatin linker derivatives and breast cancer line HCC1954, HER2 +++. Figures 6 and 7 illustrate results from the in vitro proliferation assay with dolastatin linker derivatives and n cancer line SKOV-3, HER2 +++. s 8 and 9 rate results from the in vitro proliferation assay with dolastatin linker tives and breast cancer line MDS-MB-468, HER2 ve.

TABLE 2. HER-Tox Proliferation Assay Summary: IC50 Values [nM] after 72hr drug treatment Data Set I Experiment Date 9/4/10 9/4/10 9/4/10 9/4/10 9/4/10 9/4/10 9/13/10 9/13/10 9/13/10 HER2 exp. (literature/in house) +++/+++ +++/+++ ?/+++ +++/++++ +++/+++ +++/++++ +++/+++ +++/+++ ? In Vivo sensitivity to + + - ? _ ? + _ ? HCC195 4 NCI—N87 30 Dolastatin 0.1 0.06 0.2 >30 0.2 >30 0.1 0.1 no fit NC-Dl 2.7 0.9 11 >100 3.7 >100 2.9 2.1 no fit NC-D2 2.4 1.5 5.2 >100 3.4 >100 2.8 2.3 no fit PHC-D2 Herceptin >300 >300 >300 >300 >300 >300 >300 >300 >300 Mab HA121- NC-Dl 0.2 0.1 >10 >10 (0.04)* >10 0.2 no fit 0.3 Mab HA121- NC-D2 1.0 0.3 >10 >10 0.3 >10 0.4 no fit 0.7 Mab HA121- PHC-D2 Fab K136pAF >10 >10 >10 >10 >10 >10 Fab K136-NC- D1 1.8 0.5 >10 >10 2.5 >10 Data Set 11 Experiment Date 0 9/24/10 10/8/10 10/8/10 10/8/10 HER2 exp. (literature/in house) ++/? H/H: H/Hl :H/H: -/? In vivo sensitivity to Herceptin unlikely -- -- - ? Sam le HT29 BT474 HCC1954 SKOV-3 MDA-MB-468 Dolastatin 4 2.5 0.04 0.2 <0.01 NC-Dl 4 (45)* <0.01 <0.1 <0.1 NC-D2 4 3 <0.01 (0.1)* 0.3 PHC-D2 12 7 2 8 2 Herceptin >300 >300 >300 >300 >300 Mab HA121- NC-Dl >10 1 0.2 1.3 >30 Mab HA121- NC-D2 >10 0.8 0.03 (1.3)* >30 TABLE 3. In vitro Cellular Data Small Violecule, IC50, nVI ADC, IC50, nM EGFR C225‘HC‘ C225‘NC‘ Cancer Cell Line KRAS BRAF MMD NC-D-l C-D-l NC-D-2 C225—NC-D-1 expression D-1 D-2 Skin A431 wt wt : 0.1 8.22 16.54 0.09 0.12 0.19 Co1o205 wt mut -- 0.25 6.81 40.03 >100 >100 HCT-116 mut wt 0.13 2.14 24.86 5.7 51.73 >100 62.8 Colon HT-29 wt mut 0.1 4.3 1.7 36.9 16.7 SW620 mut wt — 0.14 5 3.2 121 56.8 HCT-15 mut wt ++ 2.65 31.03 >100 >100 >300 >300 A549* mut wt --+ 0.19 6.44 39.82 39.82 >100 >100 Lung H2122 mut wt -- 0.11 12.71 31.76 31.76 >100 >100 H460 mut wt + 0.48 10.4 95.1 95.1 >300 >300 Prostate DU145 wt wt --+ 0.24 4.8 20.51 20.51 >100 >100 Example 15: In vivo anti-tumor efficacy 0fHer2-ADCs in HCCI954 (human breast carcinoma) xenograft animal model HCCI954 (human breast carcinoma) cells were obtained from American Type Culture Collection (Manassas, VA) and cultured in RPMI + 10% FBS, 37°C, 5% C02 until 80% confluent. Cells were harvested by trypsinization and suspended in PBS at l X 108 cells/mL.

Female, SClD-beige mice, 5-8 weeks old, were obtained from Charles River Laboratories.

HCCl954 cells (human, breast carcinoma, ATCC, # CRL-2338) were mixed l:l with el (BD Biosciences, Bedford MA) and injected subcutaneously into the mice. When tumors reached an average size of 100—200 mm, mice were sorted into groups of 9-10 mice each. r ements were taken twice weekly until the end of the study. To estimate tumor volume, two orthogonal diameters were measured with rs and the values entered into the formula, (I X W X, W)/2= V, (Where W=the shortest diameter, L=the t diameter and VIVolume), to obtain an estimated . The tumor volume was converted to tumor weight in the Excel data file by assuming l mm3 = l mg. nt was based on a study design of tumor growth inhibition (TGl). When the mean tumor volume of the control group reached approx. LOGO mm3 all mice were euthanized or day 28, whichever came first.

WO 66559 Mice were given a single IV injection (tail vein) on day 1 of dosing. Test article was dissolved at 4 mg/mL, 2 mg/mL and 0.66 mg/mL and administered at a dose volume of 5 mL/kg to deliver 20, 10 and 3.3 mg/kg. Test articles were: Herceptin® clinical grade (Trastuzumab), Her2-HS122- NCDl (Ab:Drug ratio = 1:2, non-cleavable linker), and Her2-HS122/LK145-HCD1 (Ab:Drug ratio = 1:4, cleavable ) See Figure 16.

Example I 6: In vivo Studies 0fHer2-D0lastatin linked Derivative HCC1954 cells were utilized for this study with 107 cells/mouse in Matrigel, SC in the right flank. Mice were SClD-bg female 4-8 weeks. Grouping was performed at day 5 after cell tation (tumors ~100 mm3): sorted into 11 groups of 10 mice each. A single IV dose was given on day 1 of dosing with each compound at 3 dose levels, 20 mg/kg, 10 mg/kg and 3.3 mg/kg. Tumor volume was monitored until the endpoint was reached (1,000 mm3 or 60 days). (Figure 10) Paclitaxel 25 mg/kg, IV, qod X 5 was employed as the control chemotherapy. The vehicle = 50 mM histamine, 0.1 M NaCl, 5% trehalose, pH 6. Herceptin® clinical grade (Trastuzumab), Her2-HS122-NCD1 (non-cleavable linker) and Her2-HS122/LK145-HCD1 able ) were tested. (Figures Hand 12) Table 4. Calculation of T/C ed / Control) for the HCC1954 study at day 28 Median Tumor olume Group 11 Schedule (mm3) T/C 1# 10 e - iv qd x 1 486 -- 2 10 trastuzumab 3.3 iv qd x 1 405 0.833 3 10 zumab 10 iv qd x 1 446 0.918 4 10 trastuzumab 20 iv qd x 1 385 0.792 10 Her-HS]22-NC1D-002 3.3 iv qd x 1 40 0.082 6 10 Her-HS]22-NC1D-002 10 iv qd x 1 14 0.029 7 10 Her-HS]22-NC1D-002 20 iv qd x 1 18 0.037 8 10 122/LK145-HC1D-001 3.3 iv qd x 1 40 0.082 9 10 Her-H8122/LK145-HC1D-001 10 iv qd x 1 25 0.051 10 Her-H8122/LK145-HC1D-001 20 iv qd x 1 18 0.037 11 10 paclitaxel 25 iv qod x 5 18 0.037 Example I 7: Pharmacokinetic studies Assay was performed that detected antibody binding to ErbB2 receptor. (Figure 13) Assay was performed that detected at least two dolastatins linked to an antibody (Figure 14). 13 1 Figure 15.

Example 18: Treatmentfor Breast Cancer Human Clinical Trial of the Safety and/or Efficacy of Trastuzumab-Linked Dolastatin Derivative for Breast Cancer y Objective: To compare the safety and pharmacokinetics of administered composition comprising trastuzumab-linked dolastatin derivative.

Study Design: This study will be a Phase I, single-center, open-label, randomized dose escalation study followed by a Phase II study in breast cancer patients. Patients should not have had exposure to trastuzumab-linked atin derivative prior to the study entry. ts must not have ed treatment for their cancer within 2 weeks of beginning the trial. Treatments include the use of chemotherapy, hematopoietic grth s, and ic therapy such as monoclonal antibodies.

Patients must have recovered from all toxicities (to grade 0 or 1) associated with preVious treatment. All subjects are evaluated for safety and all blood collections for pharmacokinetic analysis are collected as scheduled. All studies are performed with institutional ethics committee approval and patient consent.

Phase 1: Patients e i.V. trastuzumab-linked atin derivative on days 1, 8, and 15 of each 28-day cycle. Doses of trastuzumab-linked dolastatin derivative may be held or modified for toxicity based on assessments as outlined below. Treatment repeats every 28 days in the absence of unacceptable toxicity. Cohorts of 3-6 patients receive escalating doses of trastuzumab-linked dolastatin tive until the maximum tolerated dose (MTD) for zumab-linked dolastatin derivative is determined. The MTD is defined as the dose preceding that at which 2 of 3 or 2 of 6 patients experience dose-limiting toxicity.

Dose limiting toxicities are determined according to the definitions and standards set by the National Cancer Institute (NCI) Common Terminology for Adverse Events (CTCAE) Version 3.0 t 9, 2006).

Phase 11: Patients receive trastuzumab-linked dolastatin derivative as in phase I at the MTD determined in phase 1. Treatment repeats every 4 weeks for 2-6 courses in the e of disease progression or ptable toxicity. After completion of 2 courses of study therapy, patients who achieve a complete or partial response may e an additional 4 courses. Patients who maintain stable e for more than 2 months after completion of 6 s of study therapy may receive an additional 6 courses at the time of disease progression, provided they meet original eligibility criteria.

Blood Sampling Serial blood is drawn by direct vein puncture before and after administration of trastuzumab-linked dolastatin derivative. Venous blood samples (5 mL) for determination of serum concentrations are obtained at about 10 minutes prior to dosing and at approximately the following times after dosing: days 1, 8, and 15. Each serum sample is diVided into two aliquots. All serum samples are stored at -20°C. Serum samples are d on dry ice.

Pharmacokinetics: Patients undergo plasma/serum sample tion for pharmacokinetic evaluation before beginning treatment and at days 1, 8, and 15. Pharmacokinetic parameters are 2012/039468 calculated by model independent methods on a Digital Equipment Corporation VAX 8600 computer system using the latest version of the BIOAVL software. The ing pharmacokinetics parameters are determined: peak serum concentration (Cmax); time to peak serum concentration (tmax); area under the tration-time curve (AUC) from time zero to the last blood ng time (AUC 042) calculated with the use of the linear trapezoidal rule; and al elimination half-life (t1/2), computed from the elimination rate constant. The elimination rate constant is estimated by linear sion of consecutive data points in the terminal linear region of the log-linear concentration-time plot. The mean, standard ion (SD), and coefficient of variation (CV) of the pharmacokinetic parameters are calculated for each treatment. The ratio of the parameter means (preserved formulation/non—preserved formulation) is calculated.

Patient Response to combination therapy: Patient response is ed via imaging with X- ray, CT scans, and MRI, and imaging is performed prior to beginning the study and at the end of the first cycle, with additional imaging performed every four weeks or at the end of subsequent cycles. Imaging modalities are chosen based upon the cancer type and feasibility/availability, and the same imaging modality is utilized for r cancer types as well as throughout each patient’s study course. Response rates are determined using the RECIST criteria. (Therasse et al, J. Natl. Cancer Inst. 2000 Feb 2; 92(3):205-16; http://ctep.cancer.gov/forms/TherasseRECISTJNCIpdf). ts also undergo /tumor biopsy to assess changes in progenitor cancer cell phenotype and clonogenic growth by flow cytometry, Western blotting, and IHC, and for changes in cytogenetics by FISH. After completion of study treatment, patients are followed periodically for 4 weeks.

Example 19: Treatmentfor Breast Cancer Human Clinical Trial of the Safety and Efficacy of zumab-Linked Dolastatin Derivative for Breast Cancer y Objective: Compare the efficacy and toxicity of trastuzumab-linked dolastatin derivative alone followed at disease progression by combination trastuzumab and paclitaxel vs first-line ation zumab and paclitaxel in women with HERZ-overexpressing metastatic breast cancer.

Study Design: This study is a randomized, multicenter study. Patients are stratified according to degree of HERZ/neu-overexpression (2+ vs 3+), prior anthracycline-containing nt treatment (no prior treatment vs prior treatment without radiotherapy to left chest wall vs prior treatment with radiotherapy to left chest wall), estrogen-receptor status (positive vs negative vs unknown), prior therapy (first-line vs second/third-line), and center. Patients are randomized to one of two treatment arms. Arm I: Patients receive trastuzumab-linked dolastatin derivative IV over 30-90 minutes weekly. At time of disease ssion, patients receive combination trastuzumab-linked dolastatin derivative IV and paclitaxel IV as in arm II. Arm II: Patients receive trastuzumab-linked dolastatin derivative IV over 30-90 minutes weekly. Paclitaxel is administered IV over 1 hour weekly for 3 weeks followed by 1 week of rest.

Treatment continues in both arms in the absence of disease progression or unacceptable toxicity.

Quality of life is assessed at ne and day 1 of courses 2, 3, 4, 5, 6, 8, 10, and 12. Patients are followed at 1, 3, and 6 months and then every 6 months thereafter.

Example 20: Treatmentfor Bladder Cancer Human Clinical Trial of the Safety and Efficacy of a Dolastatin Derivative described herein for Bladder Cancer Therapy Objective: Determine the acute ty of paclitaxel and radiotherapy with or without trastuzumab-linked dolastatin derivative in patients who have undergone prior transurethral r resection for muscle-invasive transitional cell carcinoma of the bladder.

Disease teristics: Histologically or cytologically is confirmed primary transitional cell carcinoma (TCC) of the bladder; histologic evidence of muscularis propria invasion; meets 1 of the following stage criteria: stage T2-4a; NX, NO, or N1; and MO disease or clinical stage T1, grade 3/3 disease AND requires definitive local therapy; tumor involvement of the prostatic urethra allowed ed the following criteria are met: tumor is visibly completely resected; no evidence of stromal invasion of the prostate, no evidence of distant metastases by chest x-ray or CT scan AND abdominal/pelvic CT scan; has undergone rethral bladder resection (as thorough as is judged safely possible) within the past 3-8 weeks, including bimanual examination with tumor mapping; sufficient tumor tissue available for HER2/neu analysis; not a candidate for radical cystectomy.

] Study Design: This study is a non-randomized, multicenter study. Patients are assigned to 1 of 2 ent groups according to HER2/neu status neu 2+ or 3+ ng [group 1] vs HER2/neu O or 1+ staining [group 2]).

Group 1: Patients receive paclitaxel IV over 1 hour on days 1, 8, 15, 22, 29, 36, and 43 and trastuzumab-linked dolastatin derivative 1V over 90 minutes on day 1 and then over 30 minutes on days 8, , 22, 29, 36, and 43. Patients also undergo radiotherapy once daily on days 1-5, 8-12, 15-19, 22-26, 29- 33, 36-40, 43-47, and 50. Treatment continues in the absence of disease progression or unacceptable Group 2: Patients receive paclitaxel and undergo herapy as in group 1. After completion of study treatment, patients are followed at 4-5 weeks, every 3 months for 1 year, every 4 months for 1 year, every 6 months for 3 years, and then annually thereafter.

Example 21: Treatmentfor Ovarian Cancer Human Clinical Trial of the Safety and y of Dolastatin Derivative described herein for Ovarian Cancer y Objective: Evaluate the safety and efficacy of a four week once weekly 1V dosage of composition sing a dolastatin derivative described herein in women with HER2-overexpressing ovarian cancer.

] Study Design: This study is a non-randomized, open-label, 11 week, multicenter study. This study will evaluate the safety profile of four once weekly IV dosage, the MTD, PK and immunogenicity of trastuzumab-linked dolastatin derivative. Patients are assigned to a single group. Patients receive one dose of trastuzumab-linked dolastatin derivative once a week for 4 weeks. Trastuzumab-linked dolastatin tive will be administered by IV infusion on Study Days 1, 8, 15, and 22. Urine samples will be taken on days 1 and 22.

] Blood Sampling Serial blood is drawn by direct vein re before and after administration of trastuzumab-linked dolastatin derivative. Venous blood samples (5 mL) for determination of serum concentrations are obtained at about 10 minutes prior to dosing and at approximately the following times after dosing: days 1, 2, 4, 5, 8, 15, 22, 36, 43 and 50. Each serum sample is divided into two ts. All serum samples are stored at -20°C. Serum samples are d on dry ice.

Treatment continues in the absence of disease progression or unacceptable toxicity. Quality of life is assessed at baseline and day l of courses 2, 3, 4, 5, 6, 8, 10, and 12. ts are followed on days 29. 36, 43, and 50. Patients will be asked about adverse events. Patients will have an imaging scan and ECG to evaluate tumor siz and heart on (day 43). At the termination of the study patients will have a physical exam day 50). Patients with evidence of disease regression may receive continued therapy until evidence of progression of disease is documented.

Claims (17)

THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS:

1. A compound of Formula (I), or a salt thereof: Me Me Me O Me H H L N N Y N Me (I) R7 O Me OMe O Me Me MeO NH O Z ; 5 Z has the structure of: R5 ; R5 is H, COR8, C1-C6alkyl, or thiazole; R8 is OH; R6 is OH or H; 10 Ar is phenyl or pyridine; R7 is C1-C6alkyl or hydrogen; Y is selected from the group ting of an hydroxylamine, methyl, aldehyde, protected aldehyde, , protected ketone, thioester, ester, dicarbonyl, hydrazine, amidine, imine, diamine, azide, keto-amine, keto-alkyne, alkyne, 15 cycloalkyne, and ene-dione; L is–alkylene– or –(alkylene–O)n–alkylene–; and n is an integer greater than or equal to one.

2. The compound of claim 1, wherein R5 is le.

3. The compound of claim 1, wherein R6 is H. 20

4. The compound of claim 1, wherein Ar is phenyl.

5. The nd of claim 1, wherein R7 is methyl.

6. The compound of claim 1, where n is an integer from 1 to 20.

7. The compound of claim 1, where n is an integer from 1 to 10.

8. The nd of claim 1, where n is an integer from 1 to 5.

9. The compound of claim 1, wherein Y is cyclooctyne.

10. The compound of claim 9, wherein the cyclooctyne has a structure of: (R19)q ; 5 each R19 is independently selected from the group consisting of C1-C6 alkyl, C1-C6 alkoxy, ester, ether, thioether, aminoalkyl, halogen, alkyl ester, aryl ester, amide, aryl amide, alkyl halide, alkyl amine, alkyl sulfonic acid, alkyl nitro, thioester, sulfonyl ester, lfonyl, nitrile, alkyl nitrile, and nitro; and q is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11. 10

11. The compound of claim 1, wherein Y is azide.

12. The compound of claim 1, wherein the compound is of Formula (II): Me Me Me O Me H H H2N L N N O N Me (II) R7 O Me OMe O Me Me MeO NH N Ph

13. The compound of claim 12, wherein L is –(alkylene–O) n–alkylene–.

14. The compound of claim 13, wherein each alkylene is 2–, n is equal to 3, and 15 R7 is methyl.

15. The compound of claim 12, wherein L is –alkylene–.

16. The nd of claim 15, wherein each alkylene is 2– and R7 is methyl or hydrogen.

17. A pharmaceutical composition comprising a compound of any of claims 1-16.