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COMPOSITIONS CONTAINING, METHODS INVOLVING, AND USES OF NON-NATURAL AMINO ACID LINKED DOLASTATIN DERIVATIVES

20180009845 · 2018-01-11

Assignee

Inventors

Cpc classification

International classification

Abstract

Disclosed herein are non-natural amino acids and dolastatin analogs 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. Typically, the modified dolastatin analogs include at least one oxime, carbonyl, dicarbonyl, and/or hydroxylamine group. Further disclosed are methods for using such non-natural amino acid dolastatin analogs and modified non-natural amino acid dolastatin analogs, including therapeutic, diagnostic, and other biotechnology use.

Claims

1-24. (canceled)

25. A compound comprising Formula (VIII) or (IX), wherein the compound is a trastuzumab antibody conjugated to a dolastatin, wherein the conjugation occurs via a non-naturally encoded amino acid in the antibody, wherein Formula (VIII) or (IX) correspond to: ##STR00244## wherein: A is optional, and when present is lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, 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, —O—, —O-(alkylene or substituted alkylene)-, —S—, —S-(alkylene or substituted alkylene)-, —S(O).sub.k— where k is 1, 2, or 3, —S(O).sub.k(alkylene or substituted alkylene)-, —C(O)—, —C(O)-(alkylene or substituted alkylene)-, —C(S)—, —C(S)-(alkylene or substituted alkylene)-, —N(R′)—, —NR′-(alkylene or substituted alkylene)-, —C(O)N(R′)—, —CON(R′)-(alkylene or substituted alkylene)-, —CSN(R′)—, —CSN(R′)-(alkylene or substituted alkylene)-, —N(R′)CO-(alkylene or substituted alkylene)-, —N(R′)C(O)O—, —S(O).sub.kN(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—, —N(R′)S(O).sub.kN(R′)—, —N(R′)—N═, —C(R′)═N—, —C(R′)═N—N(R′)—, —C(R′)═N—N═, —C(R′).sub.2—N═N—, and —C(R′).sub.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; R.sub.1 is H, an amino protecting group, resin, at least one amino acid, polypeptide or polynucleotide; R.sub.2 is OH, an ester protecting group, resin, at least one amino acid, polypeptide or polynucleotide; R.sub.3 and R.sub.4 are each independently H, halogen, lower alkyl, or substituted lower alkyl, or R.sub.3 and R.sub.4 or two R.sub.3 groups optionally form a cycloalkyl or a heterocycloalkyl. Z has the structure of: ##STR00245## R.sub.5 is H, COR.sub.8, C.sub.1-C.sub.6alkyl, or thiazole; R.sub.8 is OH; R.sub.6 is OH or H; Ar is phenyl or pyridine; R.sub.7 is C.sub.1-C.sub.6 alkyl or hydrogen; L is a linker selected from the group consisting of a bond, -alkylene-, -alkylene-C(O)—, -(alkylene-O).sub.n-alkylene-, -(alkylene-O).sub.n-alkylene-C(O)—, -(alkylene-O).sub.n—(CH.sub.2).sub.n′—NHC(O)—(CH.sub.2).sub.n″—C(Me).sub.2-S—S—(CH.sub.2).sub.n′″—NHC(O)-(alkylene-O).sub.n″″-alkylene-, -(alkylene-O).sub.n-alkylene-W—, -alkylene-C(O)—W—, -(alkylene-O).sub.n-alkylene-J-, -alkylene′-J-(alkylene-O).sub.n-alkylene-, -(alkylene-O).sub.n-alkylene-J-alkylene′, -J-(alkylene-O).sub.n-alkylene-, -(alkylene-O).sub.n-alkylene-J-(alkylene-O).sub.n′-alkylene-J′-, —W—, -alkylene-W—, alkylene′-J-(alkylene-NMe).sub.n-alkylene-W—, and J-(alkylene-NMe).sub.n-alkylene-W—, -(alkylene-O).sub.n-alkylene-U-alkylene-C(O)—, -(alkylene-O).sub.n-alkylene-U-alkylene-; -J-alkylene-NMe-alkylene′—NMe-alkylene″-W—, and -alkylene-J-alkylene′-NMe-alkylene″-NMe-alkylene′″—W—; W has the structure of: ##STR00246## U has the structure of: ##STR00247## each n, n′, n″, n′″ and n″″ are independently integers from 0 to 20; wherein substituted means substituted with one or more substituents independently selected from: halo, C.sub.1-C.sub.10 alkyl, C.sub.2-C.sub.10 alkenyl, C.sub.2-C.sub.10 alkynyl, C.sub.1-C.sub.10 alkoxy, C.sub.5-C.sub.12 aralkyl, C.sub.3-C.sub.12 cycloalkyl, C.sub.4-C.sub.12 cycloalkenyl, phenyl, toluoyl, xylenyl, biphenyl, C.sub.2-C.sub.12 alkoxyalkyl, C.sub.5-C.sub.12 alkoxyaryl, C.sub.5-C.sub.12 aryloxyalkyl, C.sub.7-C.sub.12 oxyaryl, C.sub.1-C.sub.6 alkylsulfinyl, C.sub.1-C.sub.10 alkylsulfonyl, —(CH.sub.2).sub.m—O—(C.sub.1-C.sub.10 alkyl) wherein m is from 1 to 8, aryl, fluoroalkyl, heterocyclic radical, nitroalkyl, —NO.sub.2, —CN, —NR″C(O)—(C.sub.1-C.sub.10 alkyl), —C(O)—(C.sub.1-C.sub.10 alkyl), C.sub.2-C.sub.10 alkthioalkyl, —C(O)O—(C.sub.1-C.sub.10 alkyl), —OH, —SO.sub.2, ═S, —COOH, —NR″.sub.2, carbonyl, —C(O)—(C.sub.1-C.sub.10 alkyl)-CF.sub.3, —C(O)—CF.sub.3, —C(O)NR″.sub.2, —(C.sub.1-C.sub.10 aryl)-S—(C.sub.6-C.sub.10 aryl), —C(O)—(C.sub.6-C.sub.10 aryl), —(CH2).sub.m—O—(CH.sub.2).sub.m-O—(C.sub.1-C.sub.10 alkyl) wherein each m is from 1 to 8, —C(O)NR″.sub.2, —C(S)NR″.sub.2, —SO.sub.2NR″.sub.2, —NR″C(O)NR″.sub.2, —NR″C(S)NR″.sub.2; wherein each R″ group is independently selected from H, alkyl, aryl, or alkaryl, or an active metabolite, or a pharmaceutically acceptable prodrug or solvate thereof.

26-57. (canceled)

58. A method for derivatizing a dolastatin analog comprising Formula (I), (III), (IV), (V), or (VI), wherein the derivatized dolastatin analog is a trastuzumab antibody conjugated to a dolastatin, wherein the conjugation occurs via a non-naturally encoded amino acid in the antibody, wherein the method comprising contacting the dolastatin analog with a reagent of Formula (XXXVII), wherein Formula (I), (III), (IV), (V), or (VI) correspond to: ##STR00248## ##STR00249## wherein: Z has the structure of: ##STR00250## R.sub.5 is H, COR.sub.8, C.sub.1-C.sub.6alkyl, or thiazole; R.sub.8 is OH or —NH-(alkylene-O).sub.n—NH.sub.2; R.sub.6 is OH or H; Ar is phenyl or pyridine; R.sub.7 is C.sub.1-C.sub.6 alkyl or hydrogen; Y is NH.sub.2—O— or methyl and V is NH.sub.2—O—; L, L.sub.1, L.sub.2, L.sub.3, and L.sub.4 are each linkers selected from the group consisting of a bond, -alkylene-, -alkylene-C(O)—, -(alkylene-O).sub.n-alkylene-, -(alkylene-O).sub.n-alkylene-C(O)—, -(alkylene-O).sub.n—(CH.sub.2).sub.n′, —NHC(O)—(CH.sub.2).sub.n″—C(Me).sub.2-S—S—(CH.sub.2).sub.n′″—NHC(O)-(alkylene-O).sub.n″″-alkylene-, -(alkylene-O).sub.n-alkylene-W—, -alkylene-C(O)—W—, -(alkylene-O).sub.n-alkylene-J-, -alkylene′-J-(alkylene-O).sub.n-alkylene-, -(alkylene-O).sub.n-alkylene-J-alkylene′, -J-(alkylene-O).sub.n-alkylene-, -(alkylene-O).sub.n-alkylene-J-(alkylene-O).sub.n′-alkylene-J′-, —W—, -alkylene-W—, alkylene′-J-(alkylene-NMe).sub.n-alkylene-W—, and J-(alkylene-NMe).sub.n-alkylene-W—, -(alkylene-O).sub.n-alkylene-U-alkylene-C(O)—, -(alkylene-O).sub.n-alkylene-U-alkylene-; -J-alkylene-NMe-alkylene′-NMe-alkylene″-W—, and -alkylene-J-alkylene′-NMe-alkylene″-NMe-alkylene′″—W—; W has the structure of: ##STR00251## U has the structure of: ##STR00252## each J and J′ independently have the structure of: ##STR00253## or L is absent, Y is methyl, R.sub.5 is COR.sub.8, and R.sub.5 is —NH-(alkylene-O).sub.n—NH.sub.2; and each n, n′, n″, n′″ and n″″ are independently integers from 0 to 20; wherein Formula (XXXVII) corresponds to: ##STR00254## wherein: A is optional, and when present is lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, 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, —O—, —O-(alkylene or substituted alkylene)-, —S—, —S-(alkylene or substituted alkylene)-, —S(O).sub.k— where k is 1, 2, or 3, —S(O).sub.k(alkylene or substituted alkylene)-, —C(O)—, —C(O)-(alkylene or substituted alkylene)-, —C(S)—, —C(S)-(alkylene or substituted alkylene)-, —N(R′)—, —NR′-(alkylene or substituted alkylene)-, —C(O)N(R′)—, —CON(R′)-(alkylene or substituted alkylene)-, —CSN(R′)—, —CSN(R′)-(alkylene or substituted alkylene)-, —N(R′)CO-(alkylene or substituted alkylene)-, —N(R′)C(O)O—, —S(O).sub.kN(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—, —N(R′)S(O).sub.kN(R′)—, —N(R′)—N═, —C(R′)═N—, —C(R′)═N—N(R′)—, —C(R′)═N—N═, —C(R′).sub.2—N═N—, and —C(R′).sub.2—N(R′)—N(R′)—, where each R′ is independently H, alkyl, or substituted alkyl; each R′ is independently H, alkyl, or substituted alkyl; K is ##STR00255## R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; R.sub.1 is H, an amino protecting group, resin, at least one amino acid, polypeptide or polynucleotide; R.sub.2 is OH, an ester protecting group, resin, at least one amino acid, polypeptide or polynucleotide; R.sub.3 and R.sub.4 are each independently H, halogen, lower alkyl, or substituted lower alkyl, or R.sub.3 and R.sub.4 or two R.sub.3 groups optionally form a cycloalkyl or a heterocycloalkyl.

59. The method of claim 58, wherein the derivatized dolastatin analog comprises at least one oxime containing amino acid having the structure of Formula (VIII), (IX), (X), (XI), (XII), or (XIII): ##STR00256## ##STR00257##

60. The method of claim 58, wherein the dolastatin analog is contacted with the reagent of Formula (XXXVII) in aqueous solution under mildly acidic conditions.

61. A compound of claim 25 of Formula (VIII) or (IX), wherein the conjugation occurs via a non-naturally encoded amino acid at amino acid position 122 of the antibody; A is arylene; B is absent; R is alkyl; R.sub.1 and R.sub.2 is a trastuzumab antibody; R.sub.3 and R.sub.4 are H; Z has the structure of: ##STR00258## R.sub.5 is thiazole; R.sub.6 is H; Ar is phenyl; R.sub.7 is C.sub.1-C.sub.6alkyl or hydrogen; L is -(alkylene-O).sub.n-alkylene-.

62. A pharmaceutical composition comprising a compound of claim 61 and a pharmaceutically acceptable carrier, excipient, or binder.

63. A compound, or salt thereof, of claim 58 comprising Formula (I) wherein: Z has the structure of ##STR00259## R.sub.5 is thiazole; R.sub.6 is H; Ar is phenyl; R.sub.7 is methyl; Y is hydroxylamine; L is —CH.sub.2CH.sub.2— or —(CH.sub.2CH.sub.2O)CH.sub.2CH.sub.2—: and n is 1, 2, or 3.

64. A compound, or salt thereof, of claim 58 comprising Formula (I) wherein: Z has the structure of: ##STR00260## R.sub.5 is COR.sub.8 R.sub.6 is H; R.sub.8 is OH; Ar is phenyl; R.sub.7 is methyl; Y is hydroxylamine; L is —CH.sub.2CH.sub.2— or —(CH.sub.2CH.sub.2O).sub.nCH.sub.2CH.sub.2—; and n is 1, 2, or 3.

65. A compound, or salt thereof, of claim 58 comprising Formula (I) wherein: Z has the structure of: ##STR00261## R.sub.5 is COR.sub.8 R.sub.6 is H; R.sub.8 is OH; AR is phenyl; R.sub.7 is H; Y is hydroxylamine; L is —CH.sub.2CH.sub.2—.

66. A compound, or salt thereof, of claim 58 comprising Formula (I) wherein: Z has the structure of: ##STR00262## R.sub.5 is H R.sub.6 is H; Ar is pyridine; R.sub.7 is H or methyl; Y is hydroxylamine; and L is —CH.sub.2CH.sub.2—.

67. The compound of claim 58, wherein the antibody is an antibody fragment.

68. A pharmaceutical composition according to claim 25 and a pharmaceutically acceptable carrier, excipient, or binder.

69. A compound, or salt thereof, comprising Formula (X), (XI), (XII) or (XIII), wherein the compound is a trastuzumab antibody conjugated to a dolastatin, wherein the conjugation occurs via a non-naturally encoded amino acid in the antibody, wherein Formula (X), (XI), (XII) or (XIII), correspond to: ##STR00263## ##STR00264## wherein: A is optional, and when present is lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, 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, —O—, —O-(alkylene or substituted alkylene)-, —S—, —S-(alkylene or substituted alkylene)-, —S(O).sub.k— where k is 1, 2, or 3, —S(O).sub.k(alkylene or substituted alkylene)-, —C(O)—, —C(O)-(alkylene or substituted alkylene)-, —C(S)—, —C(S)-(alkylene or substituted alkylene)-, —N(R′)—, —NR′-(alkylene or substituted alkylene)-, —C(O)N(R′)—, —CON(R′)-(alkylene or substituted alkylene)-, —CSN(R′)—, —CSN(R′)-(alkylene or substituted alkylene)-, —N(R′)CO-(alkylene or substituted alkylene)-, —N(R′)C(O)O—, —S(O).sub.kN(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—, —N(R′)S(O).sub.kN(R′)—, —N(R′)—N═, —C(R′)═N—, —C(R′)═N—N(R′)—, —C(R′)═N—N═, —C(R′).sub.2—N═N—, and —C(R′).sub.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; R.sub.1 is H, an amino protecting group, resin, at least one amino acid, polypeptide or polynucleotide; R.sub.2 is OH, an ester protecting group, resin, at least one amino acid, polypeptide or polynucleotide; R.sub.3 and R.sub.4 are each independently H, halogen, lower alkyl, or substituted lower alkyl, or R.sub.3 and R.sub.4 or two R.sub.3 groups optionally form a cycloalkyl or a heterocycloalkyl. Z has the structure of: ##STR00265## R.sub.5 is H, COR.sub.8, C.sub.1-C.sub.6alkyl, or thiazide; R.sub.8 is OH; R.sub.6 is OH or H; Ar is phenyl or pyridine; R.sub.7 is C.sub.1-C.sub.6 alkyl or hydrogen; L.sub.1, L.sub.2, L.sub.3 and L.sub.4 are each linkers selected from the group consisting of a bond, -alkylene-, -alkylene-C(O)—, -(alkylene-O).sub.n-alkylene-, -(alkylene-O).sub.n-alkylene-C(O)—, -(alkylene-O).sub.n—(CH.sub.2).sub.n′—NHC(O)—(CH.sub.2).sub.n″—C(Me).sub.2-S—S—(CH.sub.2).sub.n′″—NHC(O)-(alkylene-O).sub.n″″-alkylene-, -(alkylene-O).sub.n-alkylene-W—, -alkylene-C(O)—W—, -(alkylene-O).sub.n-alkylene-J-, -alkylene′-J-(alkylene-O).sub.n-alkylene-, -(alkylene-O).sub.n′-alkylene-J-alkylene′, -J-(alkylene-O).sub.n-alkylene-, -(alkylene-O).sub.n-alkylene-J-(alkylene-O).sub.n′-alkylene-J′-, —W—, -alkylene-W—, alkylene′-J-(alkylene-NMe).sub.n-alkylene-W—, and J-(alkylene-NMe).sub.n-alkylene-W—, -(alkylene-O).sub.n-alkylene-U-alkylene-C(O)—, -(alkylene-O).sub.n-alkylene-U-alkylene-; -J-alkylene-NMe-alkylene′-NMe-alkylene″-W—, and -alkylene-J-alkylene′-NMe-alkylene″-NMe-alkylene′″—W—; W has the structure of: ##STR00266## U has the structure of: ##STR00267## each J and J′ independently have the structure of: ##STR00268## each n, n′, n″, n′″ and n″″ are independently integers from 0 to 20; wherein substituted means substituted with one or more substituents independently selected from: halo, C.sub.1-C.sub.10 alkyl, C.sub.2-C.sub.10 alkenyl, C.sub.2-C.sub.10 alkynyl, C.sub.1-C.sub.10 alkoxy, C.sub.5-C.sub.12 aralkyl, C.sub.3-C.sub.12 cycloalkyl, C.sub.4-C.sub.12 cycloalkenyl, phenyl, toluolyl, xylenyl, biphenyl, C.sub.2-C.sub.12 alkoxyalkyl, C.sub.5-C.sub.12 alkoxyaryl, C.sub.5-C.sub.12 aryloxyalkyl, C.sub.7-C.sub.12 ox aryl, C.sub.1-C.sub.6 alkylsulfonyl, C.sub.1-C.sub.10 alkylsulfonyl, —(CH.sub.2).sub.m—O—(C.sub.1-C.sub.10 alkyl) wherein m is from 1 to 8, aryl, fluoroalkyl, heterocyclic radical, nitroalkyl, —NO.sub.2, —CN, —NR″C(O)—(C.sub.1-C.sub.10 alkyl), —C(O)—(C.sub.1-C.sub.10 alkyl), C.sub.2-C.sub.10 alkthioalkyl, —C(O)O—(C.sub.1-C.sub.10 alkyl), —OH, —SO.sub.2, ═S, —COOH, —NR″.sub.2, carbonyl, —C(O)—(C.sub.1-C.sub.10 alkyl)-CF.sub.3, —C(O)—CF.sub.3, —C(O)NR′.sub.2, —(C.sub.1-C.sub.10 aryl)-S—(C.sub.6-C.sub.10 aryl), —C(O)—(C.sub.6-C.sub.10 aryl), —(CH.sub.2).sub.m—O—(CH.sub.2).sub.m—O—(C.sub.1-C.sub.10 alkyl) wherein each m is from 1 to 8, —C(O)NR″.sub.2, —C(S)NR″.sub.2, —SO.sub.2NR″.sub.2, —NR″C(O)NR″.sub.2, —NR″C(S)NR″.sub.2; wherein each R″ group is independently selected from H, alkyl, aryl, or alkaryl.

70. The compound of claim 25, wherein R.sub.1 is an antibody.

71. The compound of claim 70, wherein the antibody is trastuzumab.

72. The compound of claim 25, wherein R.sub.2 is an antibody.

73. The compound of claim 72, wherein antibody is trastuzumab.

74. The compound of claim 25, wherein the trastuzumab antibody comprises at least one non-naturally encoded amino acid.

75. The method of claim 58, wherein the trastuzumab antibody comprises at least one non-naturally encoded amino acid.

76. The method of claim 58, wherein R.sub.1 and R.sub.2 are each independently a trastuzumab antibody.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0302] 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 principles of the invention are utilized, and the accompanying drawings of which:

[0303] FIG. 1 presents a graphical illustration of Her-Tox binding to the Her2 receptor.

[0304] FIG. 2 presents a graphical illustration of the expression of Anti-Her2 variants determined by ELISA analysis.

[0305] FIG. 3 presents a graphical illustration of the expression of Anti-Her2 variants determined by ELISA analysis.

[0306] FIG. 4 presents a graphical illustration of the cell proliferation assay with HCC 1954 breast cancer cell line and dolastatin linker derivatives.

[0307] FIG. 5 presents a graphical illustration of the analysis of the cell proliferation assay with HCC 1954 breast cancer cell line and trastuzumab-tox conjugates.

[0308] FIG. 6 presents a graphical illustration of the analysis of the cell proliferation assay with SKOV-3 ovarian cancer cell line and dolastatin linker derivatives.

[0309] FIG. 7 presents a graphical illustration of the analysis of the cell proliferation assay with SKOV-3 ovarian cancer cell line and trastuzumab-tox conjugates.

[0310] FIG. 8 presents a graphical illustration of the analysis of the cell proliferation assay with MDA-MB-468 breast cancer cell line and dolastatin linker derivatives.

[0311] FIG. 9 presents a graphical illustration of the analysis of the cell proliferation assay with MDA-MB-468 breast cancer line and trastuzumab-tox conjugates.

[0312] FIG. 10 presents a graphical illustration of tumor volume measurement (mm.sup.3) after a single IC dose (3.3 mg/kg, 10 mg/kg, 20 mg/kg) of trastuzumab-linked dolastatin derivatives.

[0313] FIG. 11 presents assay formats used to measure trastuzumab-linked dolastatin derivatives concentration in SD rat serum.

[0314] FIG. 12 presents graphical illustrations of serum concentrations (ng/mL) of trastuzumab-linked dolastatin derivatives after single IV injections.

[0315] FIG. 13 presents a graphical illustration of serum concentrations (ng/mL) of trastuzumab-linked dolastatin derivatives after single IV injections. This assay detects antibody binding to the ErbB2 receptor.

[0316] FIG. 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.

[0317] FIG. 15 presents graphical illustrations of the change in rat body weight and tumor volume after treatment with trastuzumab-linked dolastatin derivatives.

[0318] FIG. 16 presents graphical illustrations of anti-tumor efficacy of trastuzumab, Her2-HS122-NCD1 and Her2-HS122/LK145-HCD1 against established tumors of HCC1954 in SCID-bg mice. Mice were administered a single IV injection on day 1 (arrow). Data points represent group average tumor volume and error bars represent standard error of the mean (SEM).

[0319] FIG. 17 presents graphical illustrations of anti-tumor efficacy of dolastatin linker derivatives in the MDA361DYT2 Breast (2+) Xenograft model.

[0320] FIG. 18 presents graphical illustrations of anti-tumor efficacy of dolastatin linker derivatives in the MDA361DYT2 Breast (2+) Xenograft model.

DETAILED DESCRIPTION OF THE INVENTION

[0321] While preferred embodiments of the present invention 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 departing 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 equivalents be covered thereby.

[0322] I. Introduction

[0323] Recently, an entirely 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 biosynthetic machinery of the prokaryote Escherichia coli (E. coli) (e.g., L. Wang, et al., (2001), Science 292:498-500) and the eukaryote Saccharomyces cerevisiae (S. cerevisiae) (e.g., J. Chin et al., Science 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 photoisomerizable 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. See, e.g., J. W. Chin et al., (2002), Journal of the American Chemical Society 124:9026-9027 (incorporated by reference in its entirety); J. W. Chin, & P. G. Schultz, (2002), ChemBioChem 3(11):1135-1137 (incorporated by reference in its entirety); J. W. Chin, et al., (2002), PNAS United States of America 99(17):11020-11024 (incorporated by reference in its entirety); and, L. Wang, & P. G. Schultz, (2002), Chem. Comm., 1-11 (incorporated by reference in its entirety). These studies have demonstrated that it is possible to selectively and routinely introduce chemical functional groups that are not found in proteins, that are chemically inert to all of the functional groups found in the 20 common, genetically-encoded amino acids and that may be used to react efficiently and selectively to form stable covalent linkages.

[0324] II. Overview

[0325] At one level, described herein are the tools (methods, compositions, techniques) for creating and using dolastatin linker derivatives or analogs comprising at least one carbonyl, dicarbonyl, 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. At another level, described herein are the tools (methods, compositions, techniques) for creating and using dolastatin linker derivatives 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, phenazine, pyrazole, triazole, etc.).

[0326] Such dolastatin linker derivatives comprising non-natural 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, however the overlap is not complete and thus both functionalities are cited above.

[0327] Provided herein in some embodiments, is a toxic group linker derivative comprising a carbonyl, dicarbonyl, 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, described herein are the tools (methods, compositions, techniques) for creating and using toxic group derivatives 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, phenazine, pyrazole, triazole, etc.).

[0328] In some embodiments, such toxic derivatives comprising non-natural 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. 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 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, however the overlap is not complete and thus both functionalities are cited above.

[0329] Certain embodiments of the present invention describe 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 compared to the free toxic group or toxic group derivatives comprising labile linkages, while retaining pharmacological 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-10).

[0330] III. Dolastatin Linker Derivatives

[0331] At one level, described herein are the tools (methods, compositions, techniques) for creating and using a dolastatin linker derivatives or analogs comprising 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 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, however the overlap is not complete and thus both functionalities are cited above.

[0332] 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 example only, as part of high-throughput screening process (in which case numerous polypeptides may be designed, synthesized, characterized and/or tested) or based on the interests of the researcher. The new dolastatin linker derivative may also be designed based on the structure of a known or partially characterized polypeptide. By way of example only, dolastatin has been the subject of intense 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 polypeptide, 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, modulating immunogenicity, modulating biological activity, or extending the circulation time. In addition, such modifications include, by way of example only, providing additional functionality to the polypeptide, incorporating an antibody, and any combination of the aforementioned modifications.

[0333] Also described herein are dolastatin linker derivatives that have or can be modified to contain an oxime, carbonyl, dicarbonyl, or hydroxylamine group. Included with this aspect are methods for producing, purifying, characterizing and using such dolastatin linker derivatives

[0334] 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 dolastatin linker derivative can be the same or different, for example, there can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more different sites in the 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.

[0335] A. Structure and Synthesis of Dolastatin Linker Derivatives: Electrophilic and Nucleophilic Groups

[0336] Dolastatin derivatives with linkers containing a hydroxylamine (also called an aminooxy) 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 semicarbazides, 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, including but not limited to, ketones, aldehydes or other functional groups with similar chemical reactivity. See, e.g., Shao, J. and Tam, J., J. Am. Chem. Soc. 117:3893-3899 (1995); H. Hang and C. Bertozzi, Acc. Chem. Res. 34(9): 727-736 (2001). 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.). (Described in U.S. Pat. No. 7,807,619 which is incorporated by reference herein to the extent relative to the reaction).

[0337] Thus, in certain embodiments described herein are dolastatin derivatives with linkers comprising a hydroxylamine, aldehyde, protected aldehyde, ketone, protected ketone, 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 converted into a hydroxylamine group), or a protected hydroxylamine group (which has reactivity similar to a hydroxylamine group upon deprotection). In some embodiments, the dolastatin derivatives with linkers comprise azides, alkynes or cycloalkynes. Such dolastatin linker derivatives include compounds having the structure of Formula (I), (III), (IV), (V), and (VI):

##STR00054## ##STR00055##

[0338] wherein [0339] Z has the structure of:

##STR00056## [0340] R.sub.5 is H, COR.sub.8, C.sub.1-C.sub.6alkyl, or thiazole; [0341] R.sub.8 is OH or —NH-(alkylene-O).sub.n—NH.sub.2; [0342] R.sub.6 is OH or H; [0343] Ar is phenyl or pyridine; [0344] R.sub.7 is C.sub.1-C.sub.6alkyl or hydrogen; [0345] Y and V are each selected from the group consisting of an hydroxylamine, methyl, aldehyde, protected aldehyde, ketone, protected ketone, thioester, ester, dicarbonyl, hydrazine, azide, amidine, imine, diamine, keto-amine, keto-alkyne, alkyne, cycloalkyne, and ene-dione; [0346] L, L.sub.1, L.sub.2, L.sub.3, and L.sub.4 are each linkers selected from the group consisting of a bond, -alkylene-, -alkylene-C(O)—, -alkylene-J-, -(alkylene-O).sub.n-alkylene-, -(alkylene-O).sub.n-alkylene-C(O)—, -(alkylene-O).sub.n-J-, -(alkylene-O).sub.n-J-alkylene-, -(alkylene-O).sub.n—(CH.sub.2).sub.n′—NHC(O)—(CH.sub.2).sub.n″—C(Me).sub.2-S—S—(CH.sub.2).sub.n′″—NHC(O)-(alkylene-O).sub.n″″-alkylene-, -(alkylene-O).sub.n-alkylene-W—, -alkylene-C(O)—W—, -(alkylene-O).sub.n-alkylene-J-, -alkylene′-J-(alkylene-O).sub.n-alkylene-, -(alkylene-O).sub.n-alkylene-J-alkylene′, -J-(alkylene-O).sub.n-alkylene-, -(alkylene-O).sub.n-alkylene-J-(alkylene-O).sub.n′-alkylene-J′-, —W—, -alkylene-W—, alkylene′-J-(alkylene-NMe).sub.n-alkylene-W—, -J-(alkylene-NMe).sub.n-alkylene-W—, -(alkylene-O).sub.n-alkylene-U-alkylene-C(O)—, -(alkylene-O).sub.n-alkylene-U-alkylene-; -J-alkylene-NMe-alkylene′-NMe-alkylene″-W—, and -alkylene-J-alkylene′-NMe-alkylene″-NMe-alkylene′″—W—; [0347] W has the structure of:

##STR00057## [0348] U has the structure of:

##STR00058## [0349] each J and J′ independently have the structure of:

##STR00059## [0350] each n, n′ n″, n′″ and n″″ are independently integers greater than or equal to one; and [0351] or L is absent, Y is methyl, R.sub.5 is COR.sub.8, and R.sub.5 is —NH-(alkylene-O).sub.n—NH.sub.2. [0352] Such dolastatin 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.

[0353] In certain embodiments of compounds of Formula (I), (III), and (V), R.sub.5 is thiazole or carboxylic acid. In certain embodiments of compounds of Formula (I), (III), and (V), R.sub.5 is hydrogen. In certain embodiments of compounds of Formula (I), (III), and (V), R.sub.5 is methyl, ethyl, propyl, iso-propyl, butyl, iso-butyl, sec-butyl, tert-butyl, pentyl, or hexyl. In certain embodiments of compounds of Formula (I), (III), and (V), R.sub.5 is —NH-(alkylene-O).sub.n—NH.sub.2, wherein alkylene is —CH.sub.2—, —CH.sub.2CH.sub.2—, —CH.sub.2CH.sub.2CH.sub.2—, —CH.sub.2CH.sub.2CH.sub.2CH.sub.2—, —CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2—, —CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2—, —CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2—CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2—, —CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2—, —CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2—, —CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2—, or —CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2—. In certain embodiments of compounds of Formula (IV) and (VI), R.sub.5 is —NH-(alkylene-O).sub.n—NH.sub.2, wherein n is 0, 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.

[0354] In some embodiments, Y is azide. In other embodiments, Y is cycloalkyne. In specific embodiments, the cyclooctyne has a structure of:

##STR00060## [0355] each R.sub.19 is independently selected from the group consisting of C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 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; and [0356] q is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11.

[0357] In certain embodiments of compounds of Formula (I), (III), and (V), R.sub.6 is H. In some embodiments of compounds of Formula (I), (III), and (V), R.sub.6 is hydroxy.

[0358] In certain embodiments of compounds of Formula (I), (III), and (V), Ar is phenyl.

[0359] In certain embodiments of compounds of Formula (I), (III), (IV), (V), and (VI), R.sub.7 is methyl, ethyl, propyl, iso-propyl, butyl, sec-butyl iso-butyl, tert-butyl, pentyl, or hexyl. In certain embodiments of compounds of Formula (I), (III), (IV), (V), and (VI), R.sub.7 is hydrogen.

[0360] In certain embodiments of compounds of Formula (I), (III), and (V), Y is hydroxylamine, aldehyde, protected aldehyde, ketone, protected ketone, thioester, ester, dicarbonyl, hydrazine, amidine, imine, diamine, keto-amine, keto-alkyne, or ene-dione.

[0361] In certain embodiments of compounds of Formula (IV) and (VI), V is a hydroxylamine, methyl, aldehyde, protected aldehyde, ketone, protected ketone, thioester, ester, dicarbonyl, hydrazine, amidine, imine, diamine, keto-amine, keto-alkyne, and ene-dione.

[0362] In certain embodiments of compounds of Formula (I), (III), (IV), (V), and (VI), each L, L.sub.1, L.sub.2, L.sub.3, and L.sub.4 is independently a cleavable linker or non-cleavable linker. In certain embodiments of compounds of Formula (I), (III), (IV), (V), and (VI), each L, L.sub.1, L.sub.2, L.sub.3, and L.sub.4 is independently a oligo(ethylene glycol) derivatized linker.

[0363] In certain embodiments of compounds of Formula (I), (III), (IV), (V), and (VI), each alkylene, alkylene′, alkylene″, and alkylene′″ independently is —CH.sub.2—, —CH.sub.2CH.sub.2—, —CH.sub.2CH.sub.2CH.sub.2—, —CH.sub.2CH.sub.2CH.sub.2CH.sub.2—, —CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2—, —CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH—, —CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2—CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2—, —CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2—, —CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2—, —CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2—, or —CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH—. In certain embodiments of compounds of Formula (XIV), (XV), (XVI), (XVII), and (XVIII), each n, n′, n″, n′″, and n″″ is 0, 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.

[0364] B. Structure and Synthesis of Dolastatin Linker Derivatives: Hydroxylamine Groups

[0365] Thus, in certain embodiments described herein are dolastatin derivatives 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 converted 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 (I):

##STR00061## [0366] wherein: [0367] Z has the structure of:

##STR00062## [0368] R.sub.5 is H, COR.sub.8, C.sub.1-C.sub.6alkyl, or thiazole; [0369] R.sub.8 is OH or —NH-(alkylene-O).sub.n—NH.sub.2; [0370] R.sub.6 is OH or H; [0371] Ar is phenyl or pyridine; [0372] R.sub.7 is C.sub.1-C.sub.6alkyl or hydrogen; [0373] Y is NH.sub.2—O— or methyl; [0374] L is a linker selected from the group consisting of -alkylene-, -alkylene-C(O)—, -(alkylene-O).sub.n-alkylene-, -(alkylene-O).sub.n-alkylene-C(O)—, -(alkylene-O).sub.n—(CH.sub.2).sub.n′—NHC(O)—(CH.sub.2).sub.n″—C(Me).sub.2-S—S—(CH.sub.2).sub.n′″—NHC(O)-(alkylene-O).sub.n″″-alkylene-, -(alkylene-O).sub.n-alkylene-W—, -alkylene-C(O)—W—, -(alkylene-O).sub.n-alkylene-U-alkylene-C(O)—, and -(alkylene-O).sub.n-alkylene-U-alkylene-; [0375] W has the structure of:

##STR00063## [0376] U has the structure of:

##STR00064## [0377] or L is absent, Y is methyl, R.sub.5 is COR.sub.8, and R.sub.5 is —NH-(alkylene-O).sub.n—NH.sub.2; and [0378] each n, n′, n″, n′″ and n″″ are independently integers greater than or equal to one. Such dolastatin 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.

[0379] In certain embodiments of compounds of Formula (I), R.sub.5 is thiazole. In certain embodiments of compounds of Formula (I), R.sub.5 is hydrogen. In certain embodiments of compounds of Formula (I), R.sub.5 is methyl, ethyl, propyl, iso-propyl, butyl, iso-butyl, sec-butyl, tert-butyl, pentyl, or hexyl. In certain embodiments of compounds of Formula (I), R.sub.5 is —NH-(alkylene-O).sub.n—NH.sub.2, wherein alkylene is —CH.sub.2—, —CH.sub.2CH.sub.2—, —CH.sub.2CH.sub.2CH.sub.2—, —CH.sub.2CH.sub.2CH.sub.2CH.sub.2—, —CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2—, —CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2—, —CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2—, —CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2—, CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2—, —CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2—, —CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2—, or —CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2—. In certain embodiments of compounds of Formula (I), R.sub.5 is —NH-(alkylene-O).sub.n—NH.sub.2, wherein n is 0, 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.

[0380] In certain embodiments of compounds of Formula (I), R.sub.6 is H. In some embodiments of compounds of Formula (I), R.sub.6 is hydroxy.

[0381] In certain embodiments of compounds of Formula (I), Ar is phenyl.

[0382] In certain embodiments of compounds of Formula (I), R.sub.7 is methyl, ethyl, propyl, iso-propyl, butyl, sec-butyl iso-butyl, tert-butyl, pentyl, or hexyl. In certain embodiments of compounds of Formula (I), R.sub.7 is hydrogen.

[0383] 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), V is a hydroxylamine, methyl, aldehyde, protected aldehyde, ketone, protected ketone, thioester, ester, dicarbonyl, hydrazine, amidine, imine, diamine, keto-amine, keto-alkyne, and ene-dione.

[0384] In certain embodiments of compounds of Formula (I), each L is independently a cleavable linker or non-cleavable linker. In certain embodiments of compounds of Formula (I), each L is independently a oligo(ethylene glycol) derivatized linker.

[0385] In certain embodiments of compounds of Formula (I), alkylene is —CH.sub.2—, —CH.sub.2CH.sub.2—, —CH.sub.2CH.sub.2CH.sub.2—, —CH.sub.2CH.sub.2CH.sub.2CH.sub.2—, —CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2—, —CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2—, —CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2—, —CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2—, CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2—, —CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2—, —CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2—, or —CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2—. In certain embodiments of compounds of Formula (I), each n, n′, n″, n′″, and n″″ is 0, 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.

[0386] In certain embodiments, dolastatin linker derivatives include compounds having the structure of Formula (II):

##STR00065##

In some embodiments of compounds of Formula (II), L is -(alkylene-O).sub.n-alkylene-. In some embodiments, each alkylene is —CH.sub.2CH.sub.2—, n is equal to 3, and R.sub.7 is methyl. In some embodiments, L is -alkylene-. In some embodiments of compounds of Formula (II), each alkylene is —CH.sub.2CH.sub.2— and R.sub.7 is methyl or hydrogen. In some embodiments of compounds of Formula (II), L is -(alkylene-O).sub.n-alkylene-C(O)—. In some embodiments of compounds of Formula (II), each alkylene is —CH.sub.2CH.sub.2—, n is equal to 4, and R.sub.7 is methyl. In some embodiments of compounds of Formula (II), L is -(alkylene-O).sub.n—(CH.sub.2).sub.n′—NHC(O)—(CH.sub.2).sub.n″—C(Me).sub.2-S—S—(CH.sub.2).sub.n′″—NHC(O)-(alkylene-O).sub.n″″-alkylene-. In some embodiments of compounds of Formula (II), each alkylene is —CH.sub.2CH.sub.2—, n is equal to 1, n′ is equal to 2, n″ is equal to 1, n′″ is equal to 2, n″″ is equal to 4, and R.sub.7 is methyl. Such dolastatin 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.

[0387] In certain embodiments of compounds of Formula (II), each L is independently a cleavable linker or non-cleavable linker. In certain embodiments of compounds of Formula (II), each L is independently a oligo(ethylene glycol) derivatized linker.

[0388] In certain embodiments of compounds of Formula (II), R.sub.7 is methyl, ethyl, propyl, iso-propyl, butyl, sec-butyl iso-butyl, tert-butyl, pentyl, or hexyl. In certain embodiments of compounds of Formula (II), R.sub.7 is hydrogen.

[0389] In certain embodiments of compounds of Formula (II), alkylene is —CH.sub.2—, —CH.sub.2CH.sub.2—, —CH.sub.2CH.sub.2CH.sub.2—, —CH.sub.2CH.sub.2CH.sub.2CH.sub.2—, —CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2—, —CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2—, —CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2—, —CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2—, CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2—, —CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2—, —CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2—, or —CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2—. In certain embodiments of compounds of Formula (II), each n, n′, n″, n′″, and n″″ is 0, 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.

[0390] Such dolastatin linker derivatives include compounds having the structure of Formula (III), (IV), (V) or (VI):

##STR00066## ##STR00067##

[0391] wherein: [0392] Z has the structure of:

##STR00068## [0393] R.sub.5 is H, COR.sub.8, C.sub.1-C.sub.6alkyl, or thiazole; [0394] R.sub.8 is OH; [0395] R.sub.6 is OH or H; [0396] Ar is phenyl or pyridine; [0397] R.sub.7 is C.sub.1-C.sub.6alkyl or hydrogen; [0398] Y is NH.sub.2—O—; [0399] V is —O—NH.sub.2 [0400] L.sub.1, L.sub.2, L.sub.3, and L.sub.4 are each linkers independently selected from the group consisting of a bond, -alkylene-, -(alkylene-O).sub.n-alkylene-J-, -alkylene′-J-(alkylene-O).sub.n-alkylene-, -J-(alkylene-O).sub.n-alkylene-, -(alkylene-O).sub.n-alkylene-J-(alkylene-O).sub.n′-alkylene-J′-, -(alkylene-O).sub.n-alkylene-J-alkylene′-, —W—, -alkylene-W—, alkylene′-J-(alkylene-NMe)-alkylene-W—, -J-(alkylene-NMe).sub.n-alkylene-W—, -J-alkylene-NMe-alkylene′-NMe-alkylene″-W—, and -alkylene-J-alkylene′-NMe-alkylene″-NMe-alkylene′″—W—; [0401] W has the structure of:

##STR00069## [0402] each J and J′ independently have the structure of:

##STR00070##  and [0403] each n and n′ are independently integers greater than or equal to one. [0404] Such dolastatin 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.

[0405] In certain embodiments of compounds of Formula (III), (IV), (V) or (VI), R.sub.5 is thiazole. In certain embodiments of compounds of Formula (III), (IV), (V) or (VI), R.sub.6 is H. In certain embodiments of compounds of Formula (III), (IV), (V) or (VI), Ar is phenyl. In certain embodiments of compounds of Formula (III), (IV), (V) or (VI), R.sub.7 is methyl. In certain embodiments of compounds of Formula (III), (IV), (V) or (VI), n and n′ are integers from 0 to 20. In certain embodiments of compounds of Formula (III), (IV), (V) or (VI), n and n′ are integers from 0 to 10. In certain embodiments of compounds of Formula (III), (IV), (V) or (VI), n and n′ are integers from 0 to 5.

[0406] In certain embodiments of compounds of Formula (III) and (V), R.sub.5 is thiazole or carboxylic acid. In certain embodiments of compounds of Formula (III) and (V), R.sub.5 is hydrogen. In certain embodiments of compounds of Formula (III) and (V), R.sub.5 is methyl, ethyl, propyl, iso-propyl, butyl, iso-butyl, sec-butyl, tert-butyl, pentyl, or hexyl. In certain embodiments of compounds of Formula (III) and (V), R.sub.5 is —NH-(alkylene-O).sub.n—NH.sub.2, wherein alkylene is —CH.sub.2—, —CH.sub.2CH.sub.2—, —CH.sub.2CH.sub.2CH.sub.2—, —CH.sub.2CH.sub.2CH.sub.2CH.sub.2—, —CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2—, —CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2—, —CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2—, —CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2—, —CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2—, CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2—, —CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2—, or —CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2—. In certain embodiments of compounds of Formula (III) and (V), R.sub.5 is —NH-(alkylene-O).sub.n—NH.sub.2, wherein n is 0, 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.

[0407] In certain embodiments of compounds of Formula (III), (IV), (V) and (VI), R.sub.6 is H. In some embodiments of compounds of Formula (III), (IV), (V) and (VI), R.sub.6 is hydroxy.

[0408] In certain embodiments of compounds of Formula (III), (IV), (V) and (VI), Ar is phenyl.

[0409] In certain embodiments of compounds of Formula (III), (IV), (V) and (VI), R.sub.7 is methyl, ethyl, propyl, iso-propyl, butyl, sec-butyl iso-butyl, tert-butyl, pentyl, or hexyl. In certain embodiments of compounds of Formula (III), (IV), (V) and (VI), R.sub.7 is hydrogen.

[0410] In certain embodiments of compounds of Formula (III) and (V), 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 (IV) and (VI), V is a hydroxylamine, methyl, aldehyde, protected aldehyde, ketone, protected ketone, thioester, ester, dicarbonyl, hydrazine, amidine, imine, diamine, keto-amine, keto-alkyne, and ene-dione.

[0411] In certain embodiments of compounds of Formula (XIV), (XV), (XVI), (XVII), and (XVIII), each L, L.sub.1, L.sub.2, L.sub.3, and L.sub.4 is independently a cleavable linker or non-cleavable linker. In certain embodiments of compounds of Formula (XIV), (XV), (XVI), (XVII), and (XVIII), each L, L.sub.1, L.sub.2, L.sub.3, and L.sub.4 is independently a oligo(ethylene glycol) derivatized linker.

[0412] In certain embodiments of compounds of Formula (III), (IV), (V) and (VI), each alkylene, alkylene′, alkylene″, and alkylene′″ independently is —CH.sub.2—, —CH.sub.2CH.sub.2—, —CH.sub.2CH.sub.2CH.sub.2—, —CH.sub.2CH.sub.2CH.sub.2CH.sub.2—, —CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2—, —CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2—, —CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2—CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2—, —CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2—, —CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2—, —CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2—, or —CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2—. In certain embodiments of compounds of Formula (III), (IV), (V) and (VI), alkylene is methylene, ethylene, propylene, butylenes, pentylene, hexylene, or heptylene.

[0413] In certain embodiments of compounds of Formula (III), (IV), (V) and (VI), each n and n′ independently is 0, 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.

[0414] In certain embodiments, dolastatin linker derivatives include compounds having the structure of Formula (VII):

##STR00071##

[0415] In certain embodiments of compounds of Formula (VII), L.sub.1 is -(alkylene-O).sub.n-alkylene-J-, L.sub.2 is -alkylene′-J′-(alkylene-O).sub.n′-alkylene-, L.sub.3 is -J″-(alkylene-O).sub.n″-alkylene-, alkylene is —CH.sub.2CH.sub.2—, alkylene′ is —(CH.sub.2).sub.4—, n is 1, n′ and n″ are 3, J has the structure of

##STR00072##

J′ and J″ have the structure of

##STR00073##

and R.sub.7 is methyl. In certain embodiments of compounds of Formula (VII), L.sub.1 is -J-(alkylene-O).sub.n-alkylene-, L.sub.2 is -(alkylene-O).sub.n′-alkylene-J′-alkylene′-, L.sub.3 is -(alkylene-O).sub.n″-alkylene-J″-, alkylene is —CH.sub.2CH.sub.2—, alkylene′ is —(CH.sub.2).sub.4—, n is 1, n′ and n″ are 4, and J, J′ and J″ have the structure of

##STR00074##

Such dolastatin 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.

[0416] In certain embodiments, compounds of Formula (I)-(VII) are stable in aqueous solution for at least 1 month under mildly acidic conditions. In certain embodiments, compounds of Formula (I)-(VII) are stable for at least 2 weeks under mildly acidic conditions. In certain embodiments, compound of Formula (I)-(VII) are stable for at least 5 days under mildly acidic conditions. In certain embodiments, such acidic conditions are pH 2 to 8.

[0417] The methods and compositions provided and described herein include polypeptides comprising dolastatin linker derivative containing at least one carbonyl or dicarbonyl group, oxime group, hydroxylamine group, or protected or masked forms thereof. 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 modified by utilizing chemistry methodologies described herein or suitable for the particular functional groups or substituents present in the dolastatin linker derivative.

[0418] The dolastatin 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 derivative of polyethylene glycol; a second protein or polypeptide or polypeptide analog; an antibody or antibody fragment; and any combination thereof.

[0419] In certain embodiments, the dolastatin linker derivatives, linkers and reagents described herein, including compounds of Formulas (I)-(VII) 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 conditions. 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.

[0420] 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 dolastatin linker derivatives. Included within this aspect, by way of example only, are therapeutic, diagnostic, assay-based, industrial, cosmetic, plant biology, environmental, energy-production, consumer-products, and/or military uses which would benefit from a dolastatin linker derivative comprising a “modified or unmodified” non-natural amino acid polypeptide or protein.

[0421] Non-limiting examples of dolastatin linker derivatives include:

##STR00075## ##STR00076## ##STR00077## ##STR00078## ##STR00079## ##STR00080##

[0422] IV. Non-Natural Amino Acid Derivatives

[0423] 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 functional group on the sidechain of the non-natural 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, serine, threonine, tryptophan, tyrosine, and valine), or at least orthogonal to the chemical reactivity of the naturally occurring 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 common, 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 physiological 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 properties is the purpose of the modification/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 non-natural 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 include protected or masked carbonyl or dicarbonyl 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.

[0424] 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 functional 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, aldehyde-containing amino acids, amino acids comprising polyethylene glycol or other polyethers, heavy atom substituted amino acids, chemically cleavable and/or photocleavable amino acids, amino acids with an elongated 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.

[0425] In some embodiments, non-natural amino acids comprise a saccharide moiety. Examples 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 replaced 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, 2-deoxygalactose and the like.

[0426] The chemical moieties incorporated into polypeptides via incorporation 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 reagents in vivo and in vitro. A heavy atom non-natural amino acid, for example, can be useful for phasing x-ray structure data. The site-specific introduction of heavy atoms using non-natural amino acids also provides selectivity and flexibility in choosing positions for heavy atoms. Photoreactive non-natural amino acids (including but not limited 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 isotopically 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 resonance and vibrational spectroscopy.

[0427] A. Structure and Synthesis of Non-Natural Amino Acid Derivatives: Carbonyl, Carbonyl Like, Masked Carbonyl, and Protected Carbonyl Groups

[0428] 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 carbonyl- 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 reactivity similar to a carbonyl- or dicarbonyl-group upon deprotection). Such amino acids include amino acids having the structure of Formula (XXXVII):

##STR00081## [0429] wherein: [0430] 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; [0431] 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).sub.k— where k is 1, 2, or 3, —S(O).sub.k(alkylene or substituted alkylene)-, —C(O)—, —NS(O).sub.2—, —OS(O).sub.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′)—, —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).sub.kN(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—, —N(R′)S(O).sub.kN(R′)—, —N(R′)—N═, —C(R′)═N—, —C(R′)═N—N(R′)—, —C(R′)═N—N═, —C(R′).sub.2—N═N—, and —C(R′).sub.2—N(R′)—N(R′)—, where each R′ is independently H, alkyl, or substituted alkyl; [0432] K is

##STR00082## [0433] R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; [0434] each R″ is independently H, alkyl, substituted alkyl, or a protecting group, or when more than one R″ group is present, two R″ optionally form a heterocycloalkyl; [0435] R.sub.1 is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and [0436] R.sub.2 is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide; [0437] each of R.sub.3 and R.sub.4 is independently H, halogen, lower alkyl, or substituted lower alkyl, or R.sub.3 and R.sub.4 or two R.sub.3 groups optionally form a cycloalkyl or a heterocycloalkyl; [0438] or the -A-B—K—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; [0439] or the —K—R group together forms a monocyclic or bicyclic cycloalkyl or heterocycloalkyl comprising at least one carbonyl group, including a dicarbonyl group, protected carbonyl group, including a protected dicarbonyl group, or masked carbonyl group, including a masked dicarbonyl group; [0440] with a proviso that when A is phenylene and each R.sub.3 is H, B is present; and that when A is —(CH.sub.2).sub.4- and each R.sub.3 is H, B is not —NHC(O)(CH.sub.2CH.sub.2)—; and that when A and B are absent and each R.sub.3 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 polynucleotide and optionally post translationally modified.

[0441] In certain embodiments, 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 conditions. In certain embodiments, such acidic conditions are pH 2 to 8.

[0442] In certain embodiments of compounds of Formula (XXXVII), B is lower alkylene, substituted 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).sub.2(alkylene or substituted alkylene)-. In certain embodiments of compounds of Formula (XXXVII), B is —O(CH.sub.2)—, —CH═N—, —CH═N—NH—, —NHCH.sub.2—, —NHCO—, —C(O)—, —C(O)—(CH.sub.2)—, —CONH—(CH.sub.2)—, —SCH.sub.2—, —S(═O)CH.sub.2—, or —S(O).sub.2CH.sub.2—. In certain embodiments of compounds of Formula (XXXVII), R is C.sub.1-6 alkyl or cycloalkyl. In certain embodiments of compounds of Formula (XXXVII) R is —CH.sub.3, —CH(CH.sub.3).sub.2, or cyclopropyl. In certain embodiments of compounds of Formula (XXXVII), R.sub.1 is H, tert-butyloxycarbonyl (Boc), 9-Fluorenylmethoxycarbonyl (Fmoc), N-acetyl, tetrafluoroacetyl (TFA), or benzyloxycarbonyl (Cbz). In certain embodiments of compounds of Formula (XXXVII), R.sub.1 is a resin, amino acid, polypeptide, antibody, or polynucleotide. In certain embodiments of compounds of Formula (XXXVII), R.sub.2 is OH, O-methyl, O-ethyl, or O-t-butyl. In certain embodiments of compounds of Formula (XXXVII), R.sub.2 is a resin, amino acid, polypeptide, antibody, or polynucleotide. In certain embodiments of compounds of Formula (XXXVII), R.sub.2 is a polynucleotide. In certain embodiments of compounds of Formula (XXXVII), R.sub.2 is ribonucleic acid (RNA).

[0443] In certain embodiments of compounds of Formula (XXXVII),

##STR00083##

is selected from the group consisting of: [0444] (i) A is substituted lower alkylene, C.sub.4-arylene, substituted arylene, 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 alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, —O—, —O-(alkylene or substituted alkylene)-, —S—, —S(O)—, —S(O).sub.2—, —NS(O).sub.2—, —OS(O).sub.2—, —C(O)—, —C(O)-(alkylene or substituted alkylene)-, —C(S)—, —N(R′)—, —C(O)N(R′)—, —CON(R′)-(alkylene or substituted alkylene)-, —CSN(R′)—, —N(R′)CO-(alkylene or substituted alkylene)-, —N(R′)C(O)O—, —N(R′)C(S)—, —S(O)N(R′), —S(O).sub.2N(R′), —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—, —N(R′)S(O)N(R′)—, —N(R′)S(O).sub.2N(R′)—, —N(R′)—N═, —C(R′)═N—N(R′)—, —C(R′)═N—N═, —C(R′).sub.2—N═N—, and —C(R′).sub.2—N(R′)—N(R′)—; [0445] (ii) A is optional, and when present is substituted lower alkylene, C.sub.4-arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene; [0446] 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).sub.2—, —NS(O).sub.2—, —OS(O).sub.2—, —C(O)—, —C(O)-(alkylene or substituted alkylene)-, —C(S)—, —N(R′)—, —C(O)N(R′)—, —CON(R′)-(alkylene or substituted alkylene)-, —CSN(R′)—, —N(R′)CO-(alkylene or substituted alkylene)-, —N(R′)C(O)O—, —N(R′)C(S)—, —S(O)N(R′), —S(O).sub.2N(R′), —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—, —N(R′)S(O)N(R′)—, —N(R′)S(O).sub.2N(R′)—, —N(R′)—N═, —C(R′)═N—N(R′)—, —C(R′)═N—N═, —C(R′).sub.2—N═N—, and —C(R′).sub.2—N(R′)—N(R′)—; [0447] (iii) A is lower alkylene; [0448] B is optional, and when present 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).sub.2—, —NS(O).sub.2—, —OS(O).sub.2—, —C(O)—, —C(O)-(alkylene or substituted alkylene)-, —C(S)—, —N(R′)—, —C(O)N(R′)—, —CSN(R′)—, —CON(R′)-(alkylene or substituted alkylene)-, —N(R′)C(O)O—, —N(R′)C(S)—, —S(O)N(R′), —S(O).sub.2N(R′), —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—, —N(R′)S(O)N(R′)—, —N(R′)S(O).sub.2N(R′)—, —N(R′)—N═, —C(R′)═N—N(R′)—, —C(R′)═N—N═, —C(R′).sub.2—N═N—, and —C(R′).sub.2—N(R′)—N(R′)—; and [0449] (iv) A is phenylene; [0450] 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).sub.2—, —NS(O).sub.2—, —OS(O).sub.2—, —C(O)—, —C(O)-(alkylene or substituted alkylene)-, —C(S)—, —N(R′)—, —C(O)N(R′)—, —CON(R′)-(alkylene or substituted alkylene)-, —CSN(R′)—, —N(R′)CO-(alkylene or substituted alkylene)-, —N(R′)C(O)O—, —N(R′)C(S)—, —S(O)N(R′), —S(O).sub.2N(R′), —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—, —N(R′)S(O)N(R′)—, —N(R′)S(O).sub.2N(R′)—, —N(R′)—N═, —C(R′)═N—N(R′)—, —C(R′)═N—N═, —C(R′).sub.2—N═N—, and —C(R′).sub.2—N(R′)—N(R′)—; [0451] K is

##STR00084## [0452] each R′ is independently H, alkyl, or substituted alkyl; [0453] R.sub.1 is optional, and when present, is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and [0454] R.sub.2 is optional, and when present, is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide; and [0455] each R.sub.3 and R.sub.4 is independently H, halogen, lower alkyl, or substituted lower alkyl; [0456] R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;

[0457] In addition, amino acids having the structure of Formula (XXXVIII) are included:

##STR00085## [0458] wherein: [0459] 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; [0460] 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).sub.k— where k is 1, 2, or 3, —S(O).sub.k(alkylene or substituted alkylene)-, —C(O)—, —NS(O).sub.2—, —OS(O).sub.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′)—, —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).sub.kN(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—, —N(R′)S(O).sub.kN(R′)—, —N(R′)—N═, —C(R′)═N—, —C(R′)═N—N(R′)—, —C(R′)═N—N═, —C(R′).sub.2—N═N—, and —C(R′).sub.2—N(R′)—N(R′)—, where each R′ is independently H, alkyl, or substituted alkyl; [0461] R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; [0462] R.sub.1 is H, an amino protecting group, resin, at least one amino acid, polypeptide, or polynucleotide; and [0463] R.sub.2 is OH, an ester protecting 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 —(CH.sub.2).sub.4—, B is not —NHC(O)(CH.sub.2CH.sub.2)—; 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 incorporated into a non-natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post translationally modified.

[0464] In addition, amino acids having the structure of Formula (XXXIX) are included:

##STR00086## [0465] wherein: [0466] B 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).sub.k— where k is 1, 2, or 3, —S(O).sub.k(alkylene or substituted alkylene)-, —C(O)—, —NS(O).sub.2—, —OS(O).sub.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′)—, —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).sub.kN(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—, —N(R′)S(O).sub.kN(R′)—, —N(R′)—N═, —C(R′)═N—, —C(R′)═N—N(R′)—, —C(R′)═N—N═, —C(R′).sub.2—N═N—, and —C(R′).sub.2—N(R′)—N(R′)—, where each R′ is independently H, alkyl, or substituted alkyl; [0467] R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; [0468] R.sub.1 is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and [0469] R.sub.2 is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide; [0470] each R.sub.a is independently selected from the group consisting of H, halogen, alkyl, substituted alkyl, —N(R′).sub.2, —C(O).sub.kR′ where k is 1, 2, or 3, —C(O)N(R′).sub.2, —OR′, and —S(O).sub.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.

[0471] In addition, the following amino acids are included:

##STR00087## ##STR00088##

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 non-natural amino acid polypeptide, polymer, polysaccharide, or a polynucleotide and optionally post translationally modified.

[0472] In addition, the following amino acids having the structure of Formula (XXXX) are included:

##STR00089## [0473] wherein [0474] —NS(O).sub.2—, —OS(O).sub.2—, 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).sub.k— where k is 1, 2, or 3, —S(O).sub.k(alkylene or substituted alkylene)-, —C(O)—, —C(O)-(alkylene or substituted alkylene)-, —C(S)—, —C(S)-(alkylene or substituted alkylene)-, —N(R′)—, —NR′-(alkylene or substituted alkylene)-, —C(O)N(R′)—, —CON(R′)-(alkylene or substituted alkylene)-, —CSN(R′)—, —CSN(R′)-(alkylene or substituted alkylene)-, —N(R′)CO-(alkylene or substituted alkylene)-, —N(R′)C(O)O—, —S(O).sub.kN(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—, —N(R′)S(O).sub.kN(R′)—, —N(R′)—N═, —C(R′)═N—, —C(R′)═N—N(R′)—, —C(R′)═N—N═, —C(R′).sub.2—N═N—, and —C(R′).sub.2—N(R′)—N(R′)—, where each R′ is independently H, alkyl, or substituted alkyl; [0475] R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; [0476] R.sub.1 is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and [0477] R.sub.2 is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide; [0478] each R.sub.a is independently selected from the group consisting of H, halogen, alkyl, substituted alkyl, —N(R′).sub.2, —C(O).sub.kR′ where k is 1, 2, or 3, —C(O)N(R′).sub.2, —OR′, and —S(O).sub.kR′, where each R′ is independently H, alkyl, or substituted alkyl; and n is 0 to 8; [0479] with a proviso that when A is —(CH.sub.2).sub.4-, B is not —NHC(O)(CH.sub.2CH.sub.2)—. 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.

[0480] In addition, the following amino acids are included:

##STR00090## ##STR00091##

wherein such compounds are optionally amino protected, optionally carboxyl protected, 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 polynucleotide and optionally post translationally modified.

[0481] In addition, the following amino acids having the structure of Formula (XXXXI) are included:

##STR00092## [0482] wherein, [0483] 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; [0484] 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).sub.k— where k is 1, 2, or 3, —S(O).sub.k(alkylene or substituted alkylene)-, —C(O)—, —NS(O).sub.2—, —OS(O).sub.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′)—, —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).sub.kN(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—, —N(R′)S(O).sub.kN(R′)—, —N(R′)—N═, —C(R′)═N—, —C(R′)═N—N(R′)—, —C(R′)═N—N═, —C(R′).sub.2—N═N—, and —C(R′).sub.2—N(R′)—N(R′)—, where each R′ is independently H, alkyl, or substituted alkyl; [0485] R.sub.1 is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and [0486] R.sub.2 is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide. [0487] 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.

[0488] In addition, the following amino acids having the structure of Formula (XXXXII) are included:

##STR00093## [0489] wherein, [0490] 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).sub.k— where k is 1, 2, or 3, —S(O).sub.k(alkylene or substituted alkylene)-, —C(O)—, —NS(O).sub.2—, —OS(O).sub.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′)—, —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).sub.kN(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—, —N(R′)S(O).sub.kN(R′)—, —N(R′)—N═, —C(R′)═N—, —C(R′)═N—N(R′)—, —C(R′)═N—N═, —C(R′).sub.2—N═N—, and —C(R′).sub.2—N(R′)—N(R′)—, where each R′ is independently H, alkyl, or substituted alkyl; [0491] R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; [0492] R.sub.1 is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and [0493] R.sub.2 is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide; wherein each R.sub.a is independently selected from the group consisting of H, halogen, alkyl, substituted alkyl, —N(R′).sub.2, —C(O).sub.kR′ where k is 1, 2, or 3, —C(O)N(R′).sub.2, —OR′, and —S(O).sub.kR′, where each R′ is independently H, alkyl, or substituted alkyl. [0494] 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.

[0495] In addition, the following amino acids are included:

##STR00094## ##STR00095##

wherein such compounds are optionally amino protected, optionally carboxyl protected, 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 polynucleotide and optionally post translationally modified.

[0496] In addition, the following amino acids having the structure of Formula (XXXXIV) are included:

##STR00096## [0497] wherein, [0498] 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).sub.k— where k is 1, 2, or 3, —S(O).sub.k(alkylene or substituted alkylene)-, —C(O)—, —NS(O).sub.2—, —OS(O).sub.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′)—, —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).sub.kN(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—, —N(R′)S(O).sub.kN(R′)—, —N(R′)—N═, —C(R′)═N—, —C(R′)═N—N(R′)—, —C(R′)═N—N═, —C(R′).sub.2—N═N—, and —C(R′).sub.2—N(R′)—N(R′)—, where each R′ is independently H, alkyl, or substituted alkyl; [0499] R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; [0500] R.sub.1 is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and [0501] R.sub.2 is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide; [0502] each R.sub.a is independently selected from the group consisting of H, halogen, alkyl, substituted alkyl, —N(R′).sub.2, —C(O).sub.kR′ where k is 1, 2, or 3, —C(O)N(R′).sub.2, —OR′, and —S(O).sub.kR′, where each R′ is independently H, alkyl, or substituted alkyl; and n is 0 to 8. [0503] 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.

[0504] In addition, the following amino acids are included:

##STR00097##

wherein such compounds are optionally amino protected, optionally carboxyl protected, 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 polynucleotide and optionally post translationally modified.

[0505] 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 structure of Formula (XXXXV) are included:

##STR00098## [0506] wherein, [0507] 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; [0508] 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).sub.k— where k is 1, 2, or 3, —S(O).sub.k(alkylene or substituted alkylene)-, —C(O)—, —NS(O).sub.2—, —OS(O).sub.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′)—, —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).sub.kN(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—, —N(R′)S(O).sub.kN(R′)—, —N(R′)—N═, —C(R′)═N—, —C(R′)═N—N(R′)—, —C(R′)═N—N═, —C(R′).sub.2—N═N—, and —C(R′).sub.2—N(R′)—N(R′)—, where each R′ is independently H, alkyl, or substituted alkyl; [0509] R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; [0510] R.sub.1 is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and [0511] R.sub.2 is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide. [0512] 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.

[0513] In addition, the following amino acids having the structure of Formula (XXXXVI) are included:

##STR00099## [0514] wherein, [0515] 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).sub.k— where k is 1, 2, or 3, —S(O).sub.k(alkylene or substituted alkylene)-, —C(O)—, —NS(O).sub.2—, —OS(O).sub.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′)—, —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).sub.kN(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—, —N(R′)S(O).sub.kN(R′)—, —N(R′)—N═, —C(R′)═N—, —C(R′)═N—N(R′)—, —C(R′)═N—N═, —C(R′).sub.2—N═N—, and —C(R′).sub.2—N(R′)—N(R′)—, where each R′ is independently H, alkyl, or substituted alkyl; [0516] R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; [0517] R.sub.1 is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and [0518] R.sub.2 is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide; [0519] wherein each R.sub.a is independently selected from the group consisting of H, halogen, alkyl, substituted alkyl, —N(R′).sub.2, —C(O).sub.kR′ where k is 1, 2, or 3, —C(O)N(R′).sub.2, —OR′, and —S(O).sub.kR′, where each R′ is independently H, alkyl, or substituted alkyl. [0520] 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.

[0521] In addition, the following amino acids are included:

##STR00100##

wherein such compounds are optionally amino protected and carboxyl protected, or a salt thereof. 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.

[0522] In addition, the following amino acids having the structure of Formula (XXXXVII) are included:

##STR00101## [0523] wherein, [0524] 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).sub.k— where k is 1, 2, or 3, —S(O).sub.k(alkylene or substituted alkylene)-, —C(O)—, —NS(O).sub.2—, —OS(O).sub.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′)—, —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).sub.kN(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—, —N(R′)S(O).sub.kN(R′)—, —N(R′)—N═, —C(R′)═N—, —C(R′)═N—N(R′)—, —C(R′)═N—N═, —C(R′).sub.2—N═N—, and —C(R′).sub.2—N(R′)—N(R′)—, where each R′ is independently H, alkyl, or substituted alkyl; [0525] R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; [0526] R.sub.1 is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and [0527] R.sub.2 is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide; [0528] each R.sub.a is independently selected from the group consisting of H, halogen, alkyl, substituted alkyl, —N(R′).sub.2, —C(O).sub.kR′ where k is 1, 2, or 3, —C(O)N(R′).sub.2, —OR′, and —S(O).sub.kR′, where each R′ is independently H, alkyl, or substituted alkyl; and n is 0 to 8. [0529] 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.

[0530] In addition, the following amino acids are included:

##STR00102## ##STR00103##

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 polynucleotide and optionally post translationally modified.

[0531] In addition, the following amino acids having the structure of Formula (XXXXVIII) are included:

##STR00104## [0532] wherein: [0533] 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; [0534] R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; [0535] R.sub.1 is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and [0536] R.sub.2 is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide; [0537] X.sub.1 is C, S, or S(O); and L is alkylene, substituted alkylene, N(R′)(alkylene) or N(R′)(substituted alkylene), where R′ is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl. [0538] 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.

[0539] In addition, the following amino acids having the structure of Formula (XXXXIX) are included:

##STR00105## [0540] wherein: [0541] 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; [0542] R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; [0543] R.sub.1 is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and [0544] R.sub.2 is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide; [0545] L is alkylene, substituted alkylene, N(R′)(alkylene) or N(R′)(substituted alkylene), where R′ is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl. [0546] 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.

[0547] In addition, the following amino acids having the structure of Formula (XXXXX) are included:

##STR00106## [0548] wherein: [0549] 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; [0550] R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; [0551] R.sub.1 is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and [0552] R.sub.2 is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide; [0553] L is alkylene, substituted alkylene, N(R′)(alkylene) or N(R′)(substituted alkylene), where R′ is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl. [0554] 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.

[0555] In addition, the following amino acids having the structure of Formula (XXXXXI) are included:

##STR00107## [0556] wherein: [0557] 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; [0558] R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; [0559] R.sub.1 is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and [0560] R.sub.2 is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide; [0561] X.sub.1 is C, S, or S(O); and n is 0, 1, 2, 3, 4, or 5; and each R.sup.8 and R.sup.9 on each CR.sup.8R.sup.9 group is independently selected from the group consisting of H, alkoxy, alkylamine, halogen, alkyl, aryl, or any R.sup.8 and R.sup.9 can together form═O or a cycloalkyl, or any to adjacent R.sup.8 groups can together form a cycloalkyl. [0562] 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.

[0563] In addition, the following amino acids having the structure of Formula (XXXXXII) are included:

##STR00108## [0564] wherein: [0565] 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; [0566] R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; [0567] R.sub.1 is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and [0568] R.sub.2 is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide; [0569] n is 0, 1, 2, 3, 4, or 5; and each R.sup.8 and R.sup.9 on each CR.sup.8R.sup.9 group is independently selected from the group consisting of H, alkoxy, alkylamine, halogen, alkyl, aryl, or any R.sup.8 and R.sup.9 can together form ═O or a cycloalkyl, or any to adjacent R.sup.8 groups can together form a cycloalkyl. [0570] 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.

[0571] In addition, the following amino acids having the structure of Formula (XXXXXIII) are included:

##STR00109## [0572] wherein: [0573] 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; [0574] R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; [0575] R.sub.1 is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and [0576] R.sub.2 is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide; [0577] n is 0, 1, 2, 3, 4, or 5; and each R.sup.8 and R.sup.9 on each CR.sup.8R.sup.9 group is independently selected from the group consisting of H, alkoxy, alkylamine, halogen, alkyl, aryl, or any R.sup.8 and R.sup.9 can together form ═O or a cycloalkyl, or any to adjacent R.sup.8 groups can together form a cycloalkyl. [0578] 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.

[0579] In addition, the following amino acids having the structure of Formula (XXXXXIV) are included:

##STR00110## [0580] wherein: [0581] 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; [0582] R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; [0583] R.sub.1 is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and [0584] R.sub.2 is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide; [0585] X.sub.1 is C, S, or S(O); and L is alkylene, substituted alkylene, N(R′)(alkylene) or N(R′)(substituted alkylene), where R′ is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl. [0586] 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.

[0587] In addition, the following amino acids having the structure of Formula (XXXXXV) are included:

##STR00111## [0588] wherein: [0589] 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; [0590] R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; [0591] R.sub.1 is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and [0592] R.sub.2 is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide; [0593] L is alkylene, substituted alkylene, N(R′)(alkylene) or N(R′)(substituted alkylene), where R′ is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl. [0594] 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.

[0595] In addition, the following amino acids having the structure of Formula (XXXXXVI) are included:

##STR00112## [0596] wherein: [0597] 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; [0598] R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; [0599] R.sub.1 is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and [0600] R.sub.2 is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide; [0601] L is alkylene, substituted alkylene, N(R′)(alkylene) or N(R′)(substituted alkylene), where R′ is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl. [0602] 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.

[0603] In addition, amino acids having the structure of Formula (XXXXXVII) are included:

##STR00113## [0604] wherein: [0605] 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; [0606] M is —C(R.sub.3)—,

##STR00114##  where (a) indicates bonding to the A group and (b) indicates bonding to respective carbonyl groups, R.sub.3 and R.sub.4 are independently chosen from H, halogen, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl, or R.sub.3 and R.sub.4 or two R.sub.3 groups or two R.sub.4 groups optionally form a cycloalkyl or a heterocycloalkyl; [0607] R is H, halogen, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; [0608] T.sub.3 is a bond, C(R)(R), O, or S, and R is H, halogen, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; [0609] R.sub.1 is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and [0610] R.sub.2 is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide. [0611] 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.

[0612] In addition, amino acids having the structure of Formula (XXXXXVIII) are included:

##STR00115## [0613] wherein: [0614] M is —C(R.sub.3)—,

##STR00116##  where (a) indicates bonding to the A group and (b) indicates bonding to respective carbonyl groups, R.sub.3 and R.sub.4 are independently chosen from H, halogen, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl, or R.sub.3 and R.sub.4 or two R.sub.3 groups or two R.sub.4 groups optionally form a cycloalkyl or a heterocycloalkyl; [0615] R is H, halogen, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; [0616] T.sub.3 is a bond, C(R)(R), O, or S, and R is H, halogen, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; [0617] R.sub.1 is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and [0618] R.sub.2 is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide; each R.sub.a is independently selected from the group consisting of H, halogen, alkyl, substituted alkyl, —N(R′).sub.2, —C(O).sub.kR′ where k is 1, 2, or 3, —C(O)N(R′).sub.2, —OR′, and —S(O).sub.kR′, where each R′ is independently H, alkyl, or substituted alkyl. [0619] 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.

[0620] In addition, amino acids having the structure of Formula (XXXXXIX) are included:

##STR00117## [0621] wherein: [0622] R is H, halogen, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; and [0623] T.sub.3 is O, or S. [0624] 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.

[0625] In addition, amino acids having the structure of Formula (XXXXXX) are included:

##STR00118## [0626] wherein: [0627] R is H, halogen, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl.

[0628] In addition, the following amino acids having structures of Formula (XXXXXX) are included:

##STR00119## [0629] 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.

[0630] The carbonyl or dicarbonyl functionality can be reacted selectively with a hydroxylamine-containing reagent under mild conditions 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). Moreover, the unique reactivity of the carbonyl or dicarbonyl group allows for selective modification 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).

[0631] The synthesis of p-acetyl-(+/−)-phenylalanine and m-acetyl-(+/−)-phenylalanine is described in Zhang, Z., et al., Biochemistry 42: 6735-6746 (2003), incorporated by reference. Other carbonyl- or dicarbonyl-containing amino acids can be similarly prepared.

[0632] In some embodiments, a polypeptide comprising a non-natural amino acid is chemically modified 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 N-terminal serine or threonine (which may be normally present or may be exposed via chemical or enzymatic digestion) can be used to generate an aldehyde functionality under mild oxidative cleavage conditions using periodate. See, e.g., Gaertner, et. al., Bioconjug. 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.

[0633] 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, 5-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 involves 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. U.S. Pat. No. 6,423,685.

[0634] B. Structure and Synthesis of Non-Natural Amino Acids: Dicarbonyl, Dicarbonyl-like, Masked Dicarbonyl, and Protected Dicarbonyl Groups

[0635] Amino acids with an electrophilic reactive group allow for a variety of reactions to link molecules via nucleophilic 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 similar 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 dicarbonyl group upon deprotection). Such amino acids include amino acids having the structure of Formula (XXXVII):

##STR00120## [0636] wherein: [0637] 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; [0638] 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″—, —S(O).sub.k(alkylene or substituted alkylene)-, where k is 1, 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 substituted alkyl; [0639] K is

##STR00121## [0640] T.sub.1 is a bond, optionally substituted C.sub.1-C.sub.4 alkylene, optionally substituted C.sub.1-C.sub.4 alkenylene, or optionally substituted heteroalkyl; [0641] wherein each optional substituents is independently selected from 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; [0642] T.sub.2, is selected from the group consisting of lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, —O—, —O-(alkylene or substituted alkylene)-, —S—, —S-(alkylene or substituted alkylene)-, —S(O).sub.k— where k is 1, 2, or 3, —S(O).sub.k(alkylene or substituted alkylene)-, —C(O)—, —C(O)-(alkylene or substituted alkylene)-, —C(S)—, —C(S)-(alkylene or substituted alkylene)-, —N(R′)—, —NR′-(alkylene or substituted alkylene)-, —C(O)N(R′)—, —CON(R′)-(alkylene or substituted alkylene)-, —CSN(R′)—, —CSN(R′)-(alkylene or substituted alkylene)-, —N(R′)CO-(alkylene or substituted alkylene)-, —N(R′)C(O)O—, —S(O).sub.kN(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—, —N(R′)S(O).sub.kN(R′)—, —N(R′)—N═, —C(R′)═N—, —C(R′)═N—N(R′)—, —C(R′)═N—N═, —C(R′).sub.2—N═N—, and —C(R′).sub.2—N(R′)—N(R′)—, where each R′ is independently H, alkyl, or substituted alkyl; [0643] T.sub.3 is

##STR00122##  where each X.sub.1 is independently selected from the group consisting of —O—, —S—, —N(H)—, —N(R)—, —N(Ac)—, and —N(OMe)-; X.sub.2 is —OR, —OAc, —SR, —N(R).sub.2, —N(R)(Ac), —N(R)(OMe), or N.sub.3, and where each R′ is independently H, alkyl, or substituted alkyl; [0644] R is H, halogen, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; [0645] R.sub.1 is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and [0646] R.sub.2 is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide; [0647] or the -A-B—K—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; [0648] or the —K—R group together forms a monocyclic or bicyclic cycloalkyl or heterocycloalkyl comprising at least one carbonyl group, including a dicarbonyl group, protected carbonyl group, including a protected dicarbonyl group, or masked carbonyl group, including a masked dicarbonyl group.

[0649] Non-limiting example of dicarbonyl amino acids having the structure of Formula (XXXVII) include:

##STR00123## ##STR00124## ##STR00125## ##STR00126##

[0650] The following amino acids having structures of Formula (XXXVII) are also included:

##STR00127## ##STR00128## ##STR00129## [0651] 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.

[0652] C. Structure and Synthesis of Non-Natural Amino Acids: Ketoalkyne, Ketoalkyne—like, Masked Ketoalkyne, Protected Ketoalkyne Groupk, Alkyne, and Cycloalkyne Groups

[0653] Amino acids containing reactive groups with dicarbonyl-like reactivity allow for the linking of molecules via nucleophilic addition reactions. Such electrophilic reactive groups include a ketoalkyne group, a ketoalkyne-like group (which has reactivity similar to a ketoalkyne group and is structurally similar to a ketoalkyne group), a masked ketoalkyne group (which can be readily converted into a ketoalkyne group), or a protected ketoalkyne group (which has reactivity similar to a ketoalkyne group upon deprotection). In some embodiments, amino acids containing reactive 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 Huisgen cycloaddition, etc.) Such amino acids include amino acids having the structure of Formula (XXXXXXI-A) or (XXXXXXI-B):

##STR00130## [0654] wherein: [0655] 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; [0656] 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″—, —S(O).sub.k(alkylene or substituted alkylene)-, where k is 1, 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 substituted alkyl; [0657] G is optional, and when present is

##STR00131## [0658] T.sub.4 is a carbonyl protecting group including, but not limited to,

##STR00132##

[0659] where each X.sub.1 is independently selected from the group consisting of —O—, —S—, —N(H)—, —N(R)—, —N(Ac)—, and —N(OMe)-; X.sub.2 is —OR, —OAc, —SR, —N(R).sub.2, —N(R)(Ac), —N(R)(OMe), or N.sub.3, and where each R′ is independently H, alkyl, or substituted alkyl; [0660] R is H, halogen, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; [0661] R.sub.1 is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; [0662] R.sub.2 is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide; [0663] each of R.sub.3 and R.sub.4 is independently H, halogen, lower alkyl, or substituted lower alkyl, or R.sub.3 and R.sub.4 or two R.sub.3 groups optionally form a cycloalkyl or a heterocycloalkyl; [0664] each R.sub.19 is independently selected from the group consisting of C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 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; and [0665] q is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11.

[0666] D. Structure and Synthesis of Non-Natural Amino Acids: Ketoamine, Ketoamine-like, Masked Ketoamine, and Protected Ketoamine Groups

[0667] Amino acids containing reactive groups with dicarbonyl-like reactivity allow for the linking of molecules via nucleophilic addition reactions. Such reactive groups include 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 ketoamine group (which can be readily converted into a ketoamine group), or a protected ketoamine group (which has reactivity similar to a ketoamine group upon deprotection). Such amino acids include amino acids having the structure of Formula (XXXXXXII):

##STR00133## [0668] wherein: [0669] 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; [0670] 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″—, —S(O).sub.k(alkylene or substituted alkylene)-, where k is 1, 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 substituted alkyl; [0671] G is

##STR00134## [0672] T.sub.1 is an optionally substituted C.sub.1-C.sub.4 alkylene, an optionally substituted C.sub.1-C.sub.4 alkenylene, or an optionally substituted heteroalkyl; [0673] T.sub.4 is a carbonyl protecting group including, but not limited to,

##STR00135##  where each X.sub.1 is independently selected from the group consisting of —O—, —S—, —N(H)—, —N(R′)—, —N(Ac)—, and —N(OMe)-; X.sub.2 is —OR, —OAc, —SR′, —N(R′).sub.2, —N(R′)(Ac), —N(R′)(OMe), or N.sub.3, and where each R′ is independently H, alkyl, or substituted alkyl; [0674] R is H, halogen, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; [0675] R.sub.1 is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and [0676] R.sub.2 is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide; [0677] each of R.sub.3 and R.sub.4 is independently H, halogen, lower alkyl, or substituted lower alkyl, or R.sub.3 and R.sub.4 or two R.sub.3 groups optionally form a cycloalkyl or a heterocycloalkyl.

[0678] Amino acids having the structure of Formula (XXXXXXII) include amino acids having the structure of Formula (XXXXXXIII) and Formula (XXXXXXIV):

##STR00136## [0679] wherein each R.sub.a is independently selected from the group consisting of H, halogen, alkyl, substituted alkyl, —N(R′).sub.2, —C(O).sub.kR′ where k is 1, 2, or 3, —C(O)N(R′).sub.2, —OR′, and —S(O).sub.kR′, where each R′ is independently H, alkyl, or substituted alkyl.

[0680] E. Structure and Synthesis of Non-Natural Amino Acids: Diamine, Diamine-Like, Masked Diamine, Protected Amines and Azides

[0681] Amino acids with a nucleophilic reactive group allow for a variety of reactions to link molecules via electrophilic addition reactions among others. Such nucleophilic reactive groups include a diamine group (including a hydrazine group, an amidine group, an imine group, a 1,1-diamine group, a 1,2-diamine group, a 1,3-diamine group, and a 1,4-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.).

[0682] 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 carbonyl-containing non-natural amino acid polypeptides, including by way of example 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 additional 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 polypeptide, including a nitrogen-containing heterocycle-derivatized polypeptide in a site-specific fashion. In a further or additional embodiment, the method for the preparation of hydrazine-substituted dolastatin derivatives provides access to a wide variety of site-specifically derivatized polypeptides. In a further or additional embodiment are methods for synthesizing hydrazine-functionalized polyethyleneglycol (PEG) linked dolastatin derivatives.

[0683] Such amino acids include amino acids having the structure of Formula (XXXVII-A) or (XXXVII-B):

##STR00137## [0684] wherein: [0685] 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; [0686] 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″—, —S(O).sub.k(alkylene or substituted alkylene)-, where k is 1, 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 substituted alkyl; [0687] K is

##STR00138##  where: [0688] R.sub.8 and R.sub.9 are independently selected from H, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, or amine protecting group; [0689] T.sub.1 is a bond, optionally substituted C.sub.1-C.sub.4 alkylene, optionally substituted C.sub.1-C.sub.4 alkenylene, or optionally substituted heteroalkyl; [0690] T.sub.2 is optionally substituted C.sub.1-C.sub.4 alkylene, optionally substituted C.sub.1-C.sub.4 alkenylene, optionally substituted heteroalkyl, optionally substituted aryl, or optionally substituted heteroaryl; [0691] 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 heteroaryl, alkaryl, substituted alkaryl, aralkyl, or substituted aralkyl; [0692] R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; [0693] R.sub.1 is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and [0694] R.sub.2 is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide; [0695] each of R.sub.3 and R.sub.4 is independently H, halogen, lower alkyl, or substituted lower alkyl, or R.sub.3 and R.sub.4 or two R.sub.3 groups optionally form a cycloalkyl or a heterocycloalkyl; [0696] or the -A-B—K—R groups together form a bicyclic or tricyclic cycloalkyl or heterocycloalkyl comprising at least one diamine group, protected diamine group or masked diamine group; [0697] or the —B—K—R groups together form a bicyclic or tricyclic cycloalkyl or cycloaryl or heterocycloalkyl comprising at least one diamine group, protected diamine group or masked diamine group; [0698] 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; [0699] wherein at least one amine group on -A-B—K—R is optionally a protected amine.

[0700] In one aspect are compounds comprising the structures 1 or 2:

##STR00139## [0701] wherein: [0702] 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; [0703] 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″—, —S(O).sub.k(alkylene or substituted alkylene)-, where k is 1, 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 substituted alkyl; [0704] T.sub.1 is a bond or CH.sub.2; and T.sub.2 is CH; [0705] 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 heteroaryl, alkaryl, substituted alkaryl, aralkyl, or substituted aralkyl; [0706] R.sub.1 is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; and [0707] R.sub.2 is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide; [0708] each of R.sub.3 and R.sub.4 is independently H, halogen, lower alkyl, or substituted lower alkyl, or R.sub.3 and R.sub.4 or two R.sub.3 groups optionally form a cycloalkyl or a heterocycloalkyl; [0709] or the -A-B-diamine containing moiety together form a bicyclic cycloalkyl or heterocycloalkyl comprising at least one diamine group, protected diamine group or masked diamine group; [0710] or the —B-diamine containing moiety groups together form a bicyclic or tricyclic cycloalkyl or cycloaryl or heterocycloalkyl comprising at least one diamine group, protected diamine group or masked diamine group; [0711] wherein at least one amine group on -A-B-diamine containing moiety is optionally a protected amine; [0712] or an active metabolite, salt, or a pharmaceutically acceptable prodrug or solvate thereof.

[0713] The following non-limiting examples of amino acids having the structure of Formula (XXXVII) are included:

##STR00140## ##STR00141## [0714] 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/or optionally post translationally modified.

[0715] In certain embodiments, 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 conditions. In certain embodiments, such acidic conditions are pH about 2 to about 8.

[0716] 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).sub.2(alkylene or substituted alkylene)-. In certain embodiments of compounds of Formula (XXXVII), B is —O(CH.sub.2)—, —CH═N—, CH═NNH—, —NHCH.sub.2—, —NHCO—, C(O)—, C(O)(CH.sub.2)—, CONH(CH.sub.2)—, —SCH.sub.2—, —S(═O)CH.sub.2—, or —S(O).sub.2CH.sub.2—. In certain embodiments of compounds of Formula (XXXVII), R is C.sub.1-6 alkyl or cycloalkyl. In certain embodiments of compounds of Formula (XXXVII) R is —CH.sub.3, —CH(CH3).sub.2, or cyclopropyl. In certain embodiments of compounds of Formula (XXXVII), R.sub.1 is H, tert-butyloxycarbonyl (Boc), 9-Fluorenylmethoxycarbonyl (Fmoc), N-acetyl, tetrafluoroacetyl (TFA), or benzyloxycarbonyl (Cbz). In certain embodiments of compounds of Formula (XXXVII), R.sub.1 is a resin, amino acid, polypeptide, or polynucleotide. In certain embodiments of compounds of Formula (XXXVII), R.sub.1 is an antibody, antibody fragment or monoclonal antibody. In certain embodiments of compounds of Formula (XXXVII), R.sub.2 is OH, O-methyl, O-ethyl, or O-t-butyl. In certain embodiments of compounds of Formula (XXXVII), R.sub.2 is a resin, at least one amino acid, polypeptide, or polynucleotide. In certain embodiments of compounds of Formula (XXXVII), R.sub.2 is an antibody, antibody fragment or monoclonal antibody.

[0717] The following non-limiting examples of amino acids having the structure of Formula (XXXVII) are also included:

##STR00142## ##STR00143##

[0718] Non-Limiting Examples of Protected Amino Acids Having the Structure of Formula (XXXVII) Include:

##STR00144##

[0719] F. Structure and Synthesis of Non-Natural Amino Acids: Aromatic A mines

[0720] Non-natural amino acids with nucleophilic reactive groups, such as, by way of example only, an aromatic amine group (including secondary and tertiary amine groups), 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 an aromatic amine group upon deprotection) allow for a variety of reactions to link molecules via various 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 structure of Formula (XXXXXXV):

##STR00145## [0721] wherein:

##STR00146##  is selected from the group consisting of a monocyclic aryl ring, a bicyclic aryl ring, a multicyclic aryl ring, a monocyclic heteroaryl ring, a bicyclic heteroaryl ring, and a multicyclic heteroaryl ring; [0722] A is independently CR.sub.a, or N; [0723] B is independently CR.sub.a, N, O, or S; [0724] each R.sub.a is independently selected from the group consisting of H, halogen, alkyl, —NO.sub.2, —CN, substituted alkyl, —N(R′).sub.2, —C(O).sub.kR′, —C(O)N(R′).sub.2, —OR′, and —S(O).sub.kR′, where k is 1, 2, or 3; and n is 0, 1, 2, 3, 4, 5, or 6; [0725] R.sub.1 is H, an amino protecting group, resin, at least one amino acid, polypeptide, or polynucleotide; and [0726] R.sub.2 is OH, an ester protecting group, resin, at least one amino acid, polypeptide, or polynucleotide; [0727] each of R.sub.3 and R.sub.4 is independently H, halogen, lower alkyl, or substituted lower alkyl, or R.sub.3 and R.sub.4 or two R.sub.3 groups optionally form a cycloalkyl or a heterocycloalkyl; [0728] M is H or —CH.sub.2R.sub.5; or the M-N—C(R.sub.5) moiety may form a 4 to 7 membered ring structure; [0729] R.sub.5 is alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, alkylalkoxy, substituted alkylalkoxy, polyalkylene oxide, substituted polyalkylene oxide, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocycle, substituted heterocycle, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl, —C(O)R″, —C(O)OR″, —C(O)N(R″).sub.2, —C(O)NHCH(R″).sub.2, -(alkylene or substituted alkylene)-N(R″).sub.2, -(alkenylene or substituted alkenylene)-N(R″).sub.2, -(alkylene or substituted alkylene)-(aryl or substituted aryl), -(alkenylene or substituted alkenylene)-(aryl or substituted aryl), -(alkylene or substituted alkylene)-ON(R″).sub.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, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocycle, substituted heterocycle, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl, or —C(O)OR′; [0730] or two R.sub.5 groups optionally form a cycloalkyl or a heterocycloalkyl; [0731] or R.sub.5 and any R.sub.a optionally form a cycloalkyl or a heterocycloalkyl; and [0732] each R′ is independently H, alkyl, or substituted alkyl. [0733] 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 alkylated. [0734] The structure

##STR00147##

(as presented in all examples herein) does not present the relative orientations of “A,” “B,” “NH-M” and “R.sub.a”; rather these four features of this structure may be oriented in any chemically-sound manner (along with other features of this structure), as illustrated by example herein.

[0735] Non-natural amino acids containing an aromatic amine moiety having the structure of Formula (A) include non-natural amino acids having the structures:

##STR00148##

wherein, each A′ is independently selected from CR.sub.a, N, or

##STR00149##

and

[0736] up to two A′ may be

##STR00150##

with the remaining A′ selected from CR.sub.a, or N. [0737] 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 alkylated.

[0738] 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),

##STR00151##

wherein; G is an amine protecting group, including, but not limited to,

##STR00152## [0739] 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 reductively alkylated.

[0740] Non-natural amino acids containing an aromatic amine moiety have the following structures:

##STR00153## [0741] wherein each R.sub.a is independently selected from the group consisting of H, halogen, alkyl, —NO.sub.2, —CN, substituted alkyl, —N(R′).sub.2, —C(O).sub.kR′, —C(O)N(R′).sub.2, —OR′, and —S(O).sub.kR′, where k is 1, 2, or 3; [0742] M is H or —CH.sub.2R.sub.5; or the M-N—C(R.sub.5) moiety may form a 4 to 7 membered ring structure; [0743] R.sub.1 is H, an amino protecting group, resin, amino acid, polypeptide, or polynucleotide; [0744] R.sub.2 is OH, an ester protecting group, resin, amino acid, polypeptide, or polynucleotide; [0745] R.sub.5 is alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, alkylalkoxy, substituted alkylalkoxy, polyalkylene oxide, substituted polyalkylene oxide, cycloalkyl, substituted cycloalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocycle, substituted heterocycle, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl, —C(O)R″, —C(O)OR″, —C(O)N(R″).sub.2, —C(O)NHCH(R″).sub.2, -(alkylene or substituted alkylene)-N(R″).sub.2, -(alkenylene or substituted alkenylene)-N(R″).sub.2, -(alkylene or substituted alkylene)-(aryl or substituted aryl), -(alkenylene or substituted alkenylene)-(aryl or substituted aryl), -(alkylene or substituted alkylene)-ON(R″).sub.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, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocycle, substituted heterocycle, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl, or —C(O)OR′; [0746] or R.sub.5 and any R.sub.a optionally form a cycloalkyl or a heterocycloalkyl; and [0747] 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.

[0748] 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 protected or masked amine moieties include, but are not limited to, imines, hydrazines, nitro, or azide substituents.

[0749] The reducing agents used to reduce such protected or masked amine moieties include, but are not limited to, TCEP, Na.sub.2S, Na.sub.2S.sub.2O.sub.4, LiAlH.sub.4, NaBH.sub.4 or NaBCNH.sub.3.

[0750] V. Non-Natural Amino Acid Linked Dolastatin Derivatives

[0751] In another aspect described herein are methods, strategies and techniques for incorporating at least one such dolastatin linker derivatives into a non-natural amino acid. Also included with this aspect are methods for producing, purifying, characterizing and using such dolastatin 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.

[0752] 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 certain embodiments, dolastatin linker derivatives with at least one non-natural amino acid or modified 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 polypeptide. 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 post-translational 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 site(s) on the polypeptide.

[0753] In other embodiments, the post-translational modification does not utilize the cellular machinery, but the functionality 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 combination thereof) comprising a second reactive group to the at least one non-natural amino acid comprising a first reactive group (including 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 suitable for the particular reactive groups. In certain 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 post-translational modification is made in vitro not utilizing the cellular machinery. Also included 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 modified non-natural amino acids.

[0754] 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 post-translational modifications. In certain embodiments, 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).

[0755] In certain embodiments, the polypeptide or non-natural amino acid linked dolastatin derivative 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 normally made by another host cell type. In certain embodiments, the polypeptide includes 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 modification 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, acylation, lipid-modification, palmitoylation, palmitate 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 (including but not limited to, where the oligosaccharide comprises (GlcNAc-Man).sub.2-Man-GlcNAc-GlcNAc, and the like). In another embodiment, 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 localization sequence, an epitope tag, a FLAG tag, a polyhistidine tag, a GST fusion, and/or the like. Also included with this aspect are methods for producing, purifying, 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 oligosaccharide 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, prokaryotes 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 non-natural 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 polypeptide that would be glycosylated when produced under conditions in which naturally-occurring polypeptides are glycosylated). Of course, such non-glycosylated forms of normally-glycosylated non-natural amino acid polypeptides (or indeed any polypeptide described herein) may be in an unpurified form, a substantially purified form, or in an isolated form.

[0756] In certain embodiments, the non-natural amino acid polypeptide includes at least one post-translational modification that is made in the presence 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 accelerant is selected from the group consisting of:

##STR00154##

[0757] A. Chemical Synthesis of Non-Natural Amino Acid Linked Dolastatin Derivatives: Oxime-Containing Linked Dolastatin Derivatives

[0758] Non-natural amino acid dolastatin linked derivatives containing an oxime group allow for reaction with a variety of reagents that contain certain reactive carbonyl- or dicarbonyl- groups (including but not limited to, ketones, aldehydes, or other groups with similar 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 dolastatin 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).

[0759] Thus, in certain 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).

[0760] Such non-natural amino acid dolastatin linked derivatives include dolastatin linked derivatives having the structure of Formula (VIII) or (IX):

##STR00155## [0761] wherein: [0762] 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; [0763] 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).sub.k— where k is 1, 2, or 3, —S(O).sub.k(alkylene or substituted alkylene)-, —C(O)—, —C(O)-(alkylene or substituted alkylene)-, —C(S)—, —C(S)-(alkylene or substituted alkylene)-, —N(R′)—, —NR′-(alkylene or substituted alkylene)-, —C(O)N(R′)—, —CON(R′)-(alkylene or substituted alkylene)-, —CSN(R′)—, —CSN(R′)-(alkylene or substituted alkylene)-, —N(R′)CO-(alkylene or substituted alkylene)-, —N(R′)C(O)O—, —S(O).sub.kN(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—, —N(R′)S(O).sub.kN(R′)—, —N(R′)—N═, —C(R′)═N—, —C(R′)═N—N(R′)—, —C(R′)═N—N═, —C(R′).sub.2—N═N—, and —C(R′).sub.2—N(R′)—N(R′)—, where each R′ is independently H, alkyl, or substituted alkyl; [0764] R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; [0765] R.sub.1 is H, an amino protecting group, resin, at least one amino acid, polypeptide, or polynucleotide; [0766] R.sub.2 is OH, an ester protecting group, resin, at least one amino acid, polypeptide, or polynucleotide; [0767] R.sub.3 and R.sub.4 are each independently H, halogen, lower alkyl, or substituted lower alkyl, or R.sub.3 and R.sub.4 or two R.sub.3 groups optionally form a cycloalkyl or a heterocycloalkyl; [0768] Z has the structure of:

##STR00156## [0769] R.sub.5 is H, COR.sub.8, C.sub.1-C.sub.6alkyl, or thiazole; [0770] R.sub.8 is OH [0771] R.sub.6 is OH or H; [0772] Ar is phenyl or pyridine; [0773] R.sub.7 is C.sub.1-C.sub.6alkyl or hydrogen; [0774] L is a linker selected from the group consisting of -alkylene-, -alkylene-C(O)—, -(alkylene-O).sub.n-alkylene-, -(alkylene-O).sub.n-alkylene-C(O)—, -(alkylene-O).sub.n—(CH.sub.2).sub.n′—NHC(O)—(CH.sub.2).sub.n″—C(Me).sub.2-S—S—(CH.sub.2).sub.n′″—NHC(O)-(alkylene-O).sub.n″″-alkylene-, -(alkylene-O).sub.n-alkylene-W—, -alkylene-C(O)—W—, -(alkylene-O).sub.n-alkylene-U-alkylene-C(O)—, and -(alkylene-O).sub.n-alkylene-U-alkylene-; [0775] W has the structure of:

##STR00157## [0776] U has the structure of:

##STR00158##  and [0777] each n, n′, n″, n′″ and n″″ are independently integers greater than or equal to one;

[0778] or an active metabolite, or a pharmaceutically acceptable prodrug or solvate thereof.

[0779] In certain embodiments of compounds of Formula (VIII) and (IX), R.sub.5 is thiazole. In certain embodiments of compounds of Formula (VIII) and (IX), R.sub.6 is H. In certain embodiments of compounds of Formula (VIII) and (IX), Ar is phenyl. In certain embodiments of compounds of Formula (VIII) and (IX), R.sub.7 is methyl. In certain embodiments of compounds of Formula (VIII) and (IX), n is an integer from 0 to 20. In certain embodiments of compounds of Formula (VIII) and (IX), n is an integer from 0 to 10. In certain embodiments of compounds of Formula (VIII) and (IX), n is an integer from 0 to 5.

[0780] In certain embodiments of compounds of Formula (VIII) and (IX), R.sub.5 is thiazole. In certain embodiments of compounds of Formula (VIII) and (IX), R.sub.5 is hydrogen. In certain embodiments of compounds of Formula (VIII) and (IX), R.sub.5 is methyl, ethyl, propyl, iso-propyl, butyl, iso-butyl, sec-butyl, tert-butyl, pentyl, or hexyl. In certain embodiments of compounds of Formula (VIII) and (IX), R.sub.5 is —NH-(alkylene-O).sub.n—NH.sub.2, wherein alkylene is —CH.sub.2—, —CH.sub.2CH.sub.2—, —CH.sub.2CH.sub.2CH.sub.2—, —CH.sub.2CH.sub.2CH.sub.2CH.sub.2—, —CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2—, —CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2—, —CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2—, —CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2—, —CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2—, CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2—, —CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2—, or —CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2—. In certain embodiments of Formula (VIII) and (IX), alkylene is methylene, ethylene, propylene, butylenes, pentylene, hexylene, or heptylene.

[0781] In certain embodiments of compounds of Formula (VIII) and (IX), R.sub.5 is —NH-(alkylene-O).sub.n—NH.sub.2, wherein n is 0, 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.

[0782] In certain embodiments of compounds of Formula (VIII) and (IX), R.sub.6 is H.

[0783] In certain embodiments of compounds of Formula (VIII) and (IX), Ar is phenyl.

[0784] In certain embodiments of compounds of Formula (VIII) and (IX), R.sub.7 is methyl, ethyl, propyl, iso-propyl, butyl, sec-butyl iso-butyl, tert-butyl, pentyl, or hexyl. In certain embodiments of compounds of Formula (VIII) and (IX), R.sub.7 is hydrogen.

[0785] In certain embodiments of compounds of Formula (VIII) and (IX), each L is independently a cleavable linker or non-cleavable linker. In certain embodiments of compounds of Formula (VIII) and (IX), each L is independently a oligo(ethylene glycol) derivatized linker.

[0786] In certain embodiments of compounds of Formula (VIII) and (IX), each alkylene, alkylene′, alkylene″, and alkylene′″ independently is —CH.sub.2—, —CH.sub.2CH.sub.2—, —CH.sub.2CH.sub.2CH.sub.2—, —CH.sub.2CH.sub.2CH.sub.2CH.sub.2—, —CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2—, —CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2—, —CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2—, —CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2—, —CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2—, CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2—, —CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2—, or —CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2—. In certain embodiments of compounds of Formula (VIII) and (IX), alkylene is methylene, ethylene, propylene, butylenes, pentylene, hexylene, or heptylene.

[0787] In certain embodiments of compounds of Formula (VIII) and (IX), each n, n′, n″, n′″, and n″″ independently is 0, 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.

[0788] In certain embodiments of compounds of Formula (VIII) or (IX), R.sub.1 is a polypeptide. In certain embodiments of compounds of Formula (VIII) or (IX), R.sub.2 is a polypeptide. In certain embodiments of compounds of Formula (VIII) or (IX), the polypeptide is an antibody. In certain embodiments of compounds of Formula (VIII) or (IX), the antibody is herceptin.

[0789] Such non-natural amino acid dolastatin linked derivatives include dolastatin linked derivatives having the structure of Formula (X), (XI), (XII) or (XIII):

##STR00159## ##STR00160## [0790] wherein: [0791] 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; [0792] 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).sub.k— where k is 1, 2, or 3, —S(O).sub.k(alkylene or substituted alkylene)-, —C(O)—, —C(O)-(alkylene or substituted alkylene)-, —C(S)—, —C(S)-(alkylene or substituted alkylene)-, —N(R′)—, —NR′-(alkylene or substituted alkylene)-, —C(O)N(R′)—, —CON(R′)-(alkylene or substituted alkylene)-, —CSN(R′)—, —CSN(R′)-(alkylene or substituted alkylene)-, —N(R′)CO-(alkylene or substituted alkylene)-, —N(R′)C(O)O—, —S(O).sub.kN(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—, —N(R′)S(O).sub.kN(R′)—, —N(R′)—N═, —C(R′)═N—, —C(R′)═N—N(R′)—, —C(R′)═N—N═, —C(R′).sub.2—N═N—, and —C(R′).sub.2—N(R′)—N(R′)—, where each R′ is independently H, alkyl, or substituted alkyl; [0793] R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; [0794] R.sub.1 is H, an amino protecting group, resin, at least one amino acid, polypeptide, or polynucleotide; [0795] R.sub.2 is OH, an ester protecting group, resin, at least one amino acid, polypeptide, or polynucleotide; [0796] R.sub.3 and R.sub.4 are each independently H, halogen, lower alkyl, or substituted lower alkyl, or R.sub.3 and R.sub.4 or two R.sub.3 groups optionally form a cycloalkyl or a heterocycloalkyl; [0797] Z has the structure of:

##STR00161## [0798] R.sub.5 is H, CO.sub.2H, C.sub.1-C.sub.6alkyl, or thiazole; [0799] R.sub.6 is OH or H; [0800] Ar is phenyl or pyridine; [0801] R.sub.7 is C.sub.1-C.sub.6alkyl or hydrogen; [0802] L.sub.1, L.sub.2, L.sub.3, and L.sub.4 are each linkers independently selected from the group consisting of a bond, -alkylene-, -(alkylene-O).sub.n-alkylene-J-, -alkylene′-J-(alkylene-O).sub.n-alkylene-, -J-(alkylene-O).sub.n-alkylene-, -(alkylene-O).sub.n-alkylene-J-(alkylene-O).sub.n′-alkylene-J′-, -(alkylene-O).sub.n-alkylene-J-alkylene′-, —W—, -alkylene-W—, alkylene′-J-(alkylene-NMe).sub.n-alkylene-W—, -J-(alkylene-NMe).sub.n-alkylene-W—, -J-alkylene-NMe-alkylene′-NMe-alkylene″-W—, and -alkylene-J-alkylene′-NMe-alkylene″-NMe-alkylene′″—W—; [0803] W has the structure of:

##STR00162## [0804] each J and J′ independently have the structure of:

##STR00163##  and [0805] each n and n′ are independently integers greater than or equal to one.

[0806] In certain embodiments of compounds of Formula (X), (XI), (XII) or (XIII), R.sub.5 is thiazole or carboxylic acid. In certain embodiments of compounds of Formula (X), (XI), (XII) or (XIII), R.sub.6 is H. In certain embodiments of compounds of Formula (X), (XI), (XII) or (XIII), Ar is phenyl. In certain embodiments of compounds of Formula (X), (XI), (XII) or (XIII), R.sub.7 is methyl. In certain embodiments of compounds of Formula (X), (XI), (XII) or (XIII), n and n′ are integers from 0 to 20. In certain embodiments of compounds of Formula (X), (XI), (XII) or (XIII), n and n′ are integers from 0 to 10. In certain embodiments of compounds of Formula (X), (XI), (XII) or (XIII), n and n′ are integers from 0 to 5.

[0807] In certain embodiments of compounds of Formula (X), (XI), (XII) or (XIII), R.sub.5 is thiazole. In certain embodiments of compounds of Formula (X), (XI), (XII) or (XIII), R.sub.5 is hydrogen. In certain embodiments of compounds of Formula (X), (XI), (XII) or (XIII), R.sub.5 is methyl, ethyl, propyl, iso-propyl, butyl, iso-butyl, sec-butyl, tert-butyl, pentyl, or hexyl. In certain embodiments of compounds of Formula (X), (XI), (XII) or (XIII), R.sub.5 is —NH-(alkylene-O).sub.n—NH.sub.2, wherein alkylene is —CH.sub.2—, —CH.sub.2CH.sub.2—, —CH.sub.2CH.sub.2CH.sub.2—, —CH.sub.2CH.sub.2CH.sub.2CH.sub.2—, —CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2—, —CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2—, —CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2—, —CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2—, CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2—, —CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2—, —CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2—, or —CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2—. In certain embodiments of Formula (X), (XI), (XII) or (XIII), alkylene is methylene, ethylene, propylene, butylenes, pentylene, hexylene, or heptylene.

[0808] In certain embodiments of compounds of Formula (X), (XI), (XII) or (XIII), R.sub.5 is —NH-(alkylene-O).sub.n—NH.sub.2, wherein n is 0, 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.

[0809] In certain embodiments of compounds of Formula (X), (XI), (XII) or (XIII), R.sub.6 is H. In some embodiments of compounds of Formula (X), (XI), (XII) or (XIII), R.sub.6 is hydroxy.

[0810] In certain embodiments of compounds of Formula (X), (XI), (XII) or (XIII), Ar is phenyl.

[0811] In certain embodiments of compounds of Formula (X), (XI), (XII) or (XIII), R.sub.7 is methyl, ethyl, propyl, iso-propyl, butyl, sec-butyl iso-butyl, tert-butyl, pentyl, or hexyl. In certain embodiments of compounds of Formula (X), (XI), (XII) or (XIII), R.sub.7 is hydrogen.

[0812] In certain embodiments of compounds of Formula (X), (XI), (XII) or (XIII), each L.sub.1, L.sub.2, L.sub.3, and L.sub.4 is independently a cleavable linker or non-cleavable linker. In certain embodiments of compounds of Formula (X), (XI), (XII) or (XIII), each L.sub.1, L.sub.2, L.sub.3, and L.sub.4 is independently a oligo(ethylene glycol) derivatized linker.

[0813] In certain embodiments of compounds of Formula (X), (XI), (XII) or (XIII), each alkylene, alkylene′, alkylene″, and alkylene′″ independently is —CH.sub.2—, —CH.sub.2CH.sub.2—, —CH.sub.2CH.sub.2CH.sub.2—, —CH.sub.2CH.sub.2CH.sub.2CH.sub.2—, —CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2—, —CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2—, —CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2—CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2—, —CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2—, —CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2—, —CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2—, or —CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2—. In certain embodiments of compounds of Formula (X), (XI), (XII) or (XIII), alkylene is methylene, ethylene, propylene, butylenes, pentylene, hexylene, or heptylene.

[0814] In certain embodiments of compounds of Formula (X), (XI), (XII) or (XIII), each n and n′ independently is 0, 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.

[0815] In certain embodiments of compounds of Formula (X), (XI), (XII) or (XIII), R.sub.1 is a polypeptide. In certain embodiments of compounds of Formula (X), (XI), (XII) or (XIII), R.sub.2 is a polypeptide. In certain embodiments of compounds of Formula (X), (XI), (XII) or (XIII), the polypeptide is an antibody. In certain embodiments of compounds of Formula (X), (XI), (XII) or (XIII), the antibody is herceptin.

[0816] In certain embodiments, compounds of Formula (X), (XI), (XII) or (XIII) are stable in aqueous solution for at least 1 month under mildly acidic conditions. In certain embodiments, compounds of Formula (X), (XI), (XII) or (XIII) are stable for at least 2 weeks under mildly acidic conditions. In certain embodiments, compound of Formula (X), (XI), (XII) or (XIII) 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 modified.

[0817] Oxime-based non-natural amino acids may be synthesized 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 oxime-containing non-natural amino acid with certain carbonyl- or dicarbonyl-containing reagents.

[0818] B. Chemical Structure and Synthesis of Non-Natural Amino Acid Linked Dolastatin Derivatives: Alkylated Aromatic Amine Linked Dolastatin Derivatives

[0819] In one aspect are dolastatin linker derivatives for the chemical 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 embodiments, the dolastatin linker derivatives are functionalized on their sidechains 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 sidechain, including 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 further or additional embodiments, the non-natural amino acids comprise a masked sidechain, including a masked aromatic amine group.

[0820] 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 derivatives 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 polypeptide.

[0821] In further or additional embodiments, the non-natural amino acids comprise aromatic amine sidechains where the aromatic amine is selected from an aryl amine or a heteroaryl amine. In a further or additional embodiment, the non-natural 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 phenylalanine or tyrosine (aromatic amino acids). In one embodiment, the non-natural amino acids have properties that are distinct from those of the natural amino acids. In one embodiment, such distinct properties are the chemical reactivity of the sidechain; in a further embodiment this distinct chemical reactivity permits the sidechain of the non-natural amino acid to undergo a reaction while being a unit of a polypeptide even though the sidechains of the naturally-occurring amino acid units in the same polypeptide do not undergo the aforementioned reaction. In a further embodiment, the sidechain of the non-natural amino acid has a chemistry orthogonal to those of the naturally-occurring amino acids. In a further embodiment, the sidechain of the non-natural amino acid comprises 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 embodiment, 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.

[0822] 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 generate substituted amine, including secondary and tertiary amine, linkages. Second, under these reaction conditions the chemistry 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 incorporated non-natural amino acids containing aromatic amine moieties or protected aldehyde moieties, including, by way of example, recombinant proteins. Such derivatized polypeptides and proteins can thereby be prepared as defined homogeneous products. Third, the mild conditions 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 reagent 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). Similarly, the mild conditions needed to effect the reaction of an aldehyde moiety on an amino acid, which has been incorporated into a polypeptide and deprotected, with an aromatic amine-containing reagent 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). Fourth, the reaction occurs rapidly at room temperature, which allows the use of many types of polypeptides or reagents that would otherwise be unstable at higher temperatures. Fifth, the reaction occurs readily 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, stoichiometric-like, or near-stoichiometric, so that it is unnecessary to add excess reagent or polypeptide to obtain a useful amount of reaction product. Seventh, the resulting amine can be produced regioselectively and/or regiospecifically, depending 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.

[0823] Non-natural amino acids with nucleophilic reactive groups, such as, by way of example only, an aromatic amine group (including secondary and tertiary amine groups), 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 reactions, including but not limited to, reductive alkylation reactions with aldehyde containing dolastatin linked derivatives. Such alkylated non-natural amino acid linked dolastatin derivatives include amino acids having the structure of Formula (XXV), (XXVI), (XXVII), (XXVIII), (XXIX), or (XXX):

##STR00164## ##STR00165## [0824] wherein: [0825] Z has the structure of:

##STR00166## [0826] R.sub.5 is H, CO.sub.2H, C.sub.1-C.sub.6alkyl, or thiazole; [0827] R.sub.6 is OH or H; [0828] Ar is phenyl or pyridine; [0829] R.sub.1 is H, an amino protecting group, resin, at least one amino acid, polypeptide, or polynucleotide; [0830] R.sub.2 is OH, an ester protecting group, resin, at least one amino acid, polypeptide, or polynucleotide; [0831] R.sub.4 is H, halogen, lower alkyl, or substituted lower alkyl; [0832] R.sub.7 is C.sub.1-C.sub.6alkyl or hydrogen; [0833] L, L.sub.1, L.sub.2, L.sub.3, and L.sub.4 are each linkers selected from the group consisting of a bond, -alkylene-, -alkylene-C(O)—, -(alkylene-O).sub.n-alkylene-, -(alkylene-O).sub.n-alkylene-C(O)—, -(alkylene-O).sub.n—(CH.sub.2).sub.n′—NHC(O)—(CH.sub.2).sub.n″—C(Me).sub.2-S—S—(CH.sub.2).sub.n′″—NHC(O)-(alkylene-O).sub.n″″-alkylene-, (alkylene-O).sub.n-alkylene-W—, -alkylene-C(O)—W—, -(alkylene-O).sub.n-alkylene-J-, -alkylene′-J-(alkylene-O).sub.n-alkylene-, -(alkylene-O).sub.n-alkylene-J-alkylene′, -J-(alkylene-O).sub.n-alkylene-, -(alkylene-O).sub.n-alkylene-J-(alkylene-O).sub.n′-alkylene-J′-, —W—, -alkylene-W—, alkylene′-J-(alkylene-NMe).sub.n-alkylene-W—, and J-(alkylene-NMe).sub.n-alkylene-W—, -(alkylene-O).sub.n-alkylene-U-alkylene-C(O)—, -(alkylene-O).sub.n-alkylene-U-alkylene-; -J-alkylene-NMe-alkylene′-NMe-alkylene″-W—, and -alkylene-J-alkylene′-NMe-alkylene″-NMe-alkylene′″—W—; [0834] W has the structure of:

##STR00167## [0835] U has the structure of:

##STR00168## [0836] each J and J′ independently have the structure of:

##STR00169## [0837] each n and n′ are independently integers greater than or equal to one; and [0838] each R.sub.16 is independently selected from the group consisting of hydrogen, halogen, alkyl, NO.sub.2, CN, and substituted alkyl. [0839] 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 polypeptide, polymer, polysaccharide, or a polynucleotide and optionally reductively alkylated.

[0840] In certain embodiments of compounds of Formula (XXV), (XXVI), (XXVII), (XXVIII), (XXIX), or (XXX), R.sub.5 is thiazole or carboxylic acid. In certain embodiments of compounds of Formula (XXV), (XXVI), (XXVII), (XXVIII), (XXIX), or (XXX), R.sub.6 is H. In certain embodiments of compounds of Formula (XXV), (XXVI), (XXVII), (XXVIII), (XXIX), or (XXX), Ar is phenyl. In certain embodiments of compounds of Formula (XXV), (XXVI), (XXVII), (XXVIII), (XXIX), or (XXX), R.sub.7 is methyl. In certain embodiments of compounds of Formula (XXV), (XXVI), (XXVII), (XXVIII), (XXIX), or (XXX), n is an integer from 0 to 20. In certain embodiments of compounds of Formula (XXV), (XXVI), (XXVII), (XXVIII), (XXIX), or (XXX), n is an integer from 0 to 10. In certain embodiments of compounds of Formula (XXV), (XXVI), (XXVII), (XXVIII), (XXIX), or (XXX) or (XXIV), n is an integer from 0 to 5.

[0841] In certain embodiments of compounds of Formula (XXV), (XXVI), (XXVII), (XXVIII), (XXIX), or (XXX), R.sub.5 is thiazole or carboxylic acid. In certain embodiments of compounds of Formula (XXV), (XXVI), (XXVII), (XXVIII), (XXIX), or (XXX), R.sub.5 is hydrogen. In certain embodiments of compounds of Formula (XXV), (XXVI), (XXVII), (XXVIII), (XXIX), or (XXX), R.sub.5 is methyl, ethyl, propyl, iso-propyl, butyl, iso-butyl, sec-butyl, tert-butyl, pentyl, or hexyl. In certain embodiments of compounds of Formula ((XXV), (XXVI), (XXVII), (XXVIII), (XXIX), or (XXX), R.sub.5 is —NH-(alkylene-O).sub.n—NH.sub.2, wherein alkylene is —CH.sub.2—, —CH.sub.2CH.sub.2—, —CH.sub.2CH.sub.2CH.sub.2—, —CH.sub.2CH.sub.2CH.sub.2CH.sub.2—, —CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2—, —CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2—, —CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2—, —CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2—, —CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2—, —CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2—, —CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2—, or —CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2—. In certain embodiments of Formula (XXV), (XXVI), (XXVII), (XXVIII), (XXIX), or (XXX), alkylene is methylene, ethylene, propylene, butylenes, pentylene, hexylene, or heptylene.

[0842] In certain embodiments of compounds of Formula (XXV), (XXVI), (XXVII), (XXVIII), (XXIX), or (XXX), R.sub.5 is —NH-(alkylene-O).sub.n—NH.sub.2, wherein n is 0, 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.

[0843] In certain embodiments of compounds of Formula (XXV), (XXVI), (XXVII), (XXVIII), (XXIX), or (XXX), R.sub.6 is H. In some embodiments of compounds of Formula (XXV), (XXVI), (XXVII), (XXVIII), (XXIX), or (XXX), R.sub.6 is hydroxy.

[0844] In certain embodiments of compounds of Formula (XXV), (XXVI), (XXVII), (XXVIII), (XXIX), or (XXX), Ar is phenyl.

[0845] In certain embodiments of compounds of Formula (XXV), (XXVI), (XXVII), (XXVIII), (XXIX), or (XXX), R.sub.7 is methyl, ethyl, propyl, iso-propyl, butyl, sec-butyl iso-butyl, tert-butyl, pentyl, or hexyl. In certain embodiments of compounds of Formula (XXV), (XXVI), (XXVII), (XXVIII), (XXIX), or (XXX), R.sub.7 is hydrogen.

[0846] In certain embodiments of compounds of Formula (XXV), (XXVI), (XXVII), (XXVIII), (XXIX), or (XXX), each L, L.sub.1, L.sub.2, L.sub.3, and L.sub.4 is independently a cleavable linker or non-cleavable linker. In certain embodiments of compounds of Formula (XXV), (XXVI), (XXVII), (XXVIII), (XXIX), or (XXX), each L, L.sub.1, L.sub.2, L.sub.3, and L.sub.4 is independently a oligo(ethylene glycol) derivatized linker.

[0847] In certain embodiments of compounds of Formula (XXV), (XXVI), (XXVII), (XXVIII), (XXIX), or (XXX), each alkylene, alkylene′, alkylene″, and alkylene′″ independently is —CH.sub.2—, —CH.sub.2CH.sub.2—, —CH.sub.2CH.sub.2CH.sub.2—, —CH.sub.2CH.sub.2CH.sub.2CH.sub.2—, —CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2—, —CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2—, —CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2—, —CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2—, CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2—, —CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2—, —CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2—, or —CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2—. In certain embodiments of compounds of Formula (XIX), (XX), (XXI), (XXII), (XXIII) or (XXIV), alkylene is methylene, ethylene, propylene, butylenes, pentylene, hexylene, or heptylene.

[0848] In certain embodiments of compounds of Formula (XXV), (XXVI), (XXVII), (XXVIII), (XXIX), or (XXX), each n, n′, n″, n′″, and n″″ independently is 0, 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.

[0849] In certain embodiments of compounds of Formula (XXV), (XXVI), (XXVII), (XXVIII), (XXIX), or (XXX), R.sub.1 is a polypeptide. In certain embodiments of compounds of Formula (XXV), (XXVI), (XXVII), (XXVIII), (XXIX), or (XXX), R.sub.2 is a polypeptide. In certain embodiments of compounds of Formula (XXV), (XXVI), (XXVII), (XXVIII), (XXIX), or (XXX), the polypeptide is an antibody. In certain embodiments of compounds of Formula (XXV), (XXVI), (XXVII), (XXVIII), (XXIX), or (XXX), the antibody is herceptin.

[0850] Compounds of Formula (XXV), (XXVI), (XXVII), (XXVIII), (XXIX), or (XXX) may be formed by the reductive alkylation of aromatic amine compounds with carbonyl containing reagents such as, by way of example, ketones, esters, thioesters, and aldehydes.

[0851] 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), (XXVI), (XXVII), (XXVIII), (XXIX), or (XXX). Such reactions may also be applied to non-natural amino acids incorporated into synthetic polymers, polysaccharides, or polynucleotides. Additionally, such reactions may be applied to non-incorporated non-natural amino acids. By way of example the reducing agent used to reduce masked amine moieties includes, but is not limited to, TCEP, Na.sub.2S, Na.sub.2S.sub.2O.sub.4, LiAlH.sub.4, B.sub.2H.sub.6, and NaBH.sub.4. By way of example only, reductive 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 (NaBCNH.sub.3). In addition, other reducing agents may be used for reductive alkylation including, but not limited to, TCEP, Na.sub.2S, Na.sub.2S.sub.2O.sub.4, LiAlH.sub.4, B.sub.2H.sub.6, and NaBH.sub.4.

[0852] A non-limiting exemplary syntheses of non-natural amino acid polypeptides containing amino acids of Formula (XXV), (XXVI), (XXVII), (XXVIII), (XXIX), or (XXX) by reductive alkylation of secondary 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 non-natural amino acids incorporated into synthetic polymers, polysaccharides, or polynucleotides. Additionally, such reactions may be applied to non-incorporated non-natural amino acids. By way of example only, reductive 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 (NaBCNH.sub.3). In addition, other reducing agents may be used for reductive alkylation including, but not limited to, TCEP, Na.sub.2S, Na.sub.2S.sub.2O.sub.4, LiAlH.sub.4, B.sub.2H.sub.6, and NaBH.sub.4.

[0853] C. Chemical Synthesis of Non-Natural Amino Acid Linked Dolastatin Derivatives: Heteroaryl-Containing Linked Dolastatin Derivatives

[0854] In one aspect are non-natural amino acids for the chemical derivatization of dolastatin linked derivatives based upon the reactivity of a dicarbonyl group, including a group containing at 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, and/or at least one thioester group, and wherein the dicarbonyl group can be a 1,2-dicarbonyl group, a 1,3-dicarbonyl group, or a 1,4-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 diamine group, including a hydrazine group, an amidine group, an imine group, a 1,1-diamine group, a 1,2-diamine group, a 1,3-diamine group, and a 1,4-diamine group. In further 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 embodiments, the non-natural amino acids are functionalized on their sidechains such that their reaction with a derivatizing molecule generates a linkage, including a heterocyclic-based linkage, including a nitrogen-containing heterocycle, and/or an aldol-based linkage. In further or additional embodiments 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 non-natural 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).

[0855] In further or additional embodiments, the non-natural amino acids comprise dicarbonyl sidechains where the carbonyl is selected 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 capable of forming a heterocycle, including a nitrogen-containing heterocycle, upon treatment with an appropriately functionalized reagent. In a further or additional embodiment, the non-natural amino acids resemble a natural amino acid in structure but contain one of the aforementioned functional groups. In another or further embodiment the non-natural amino acids resemble phenylalanine or tyrosine (aromatic amino acids); while in a separate embodiment, the non-natural amino acids resemble alanine and leucine (hydrophobic amino acids). In one embodiment, the non-natural amino acids have properties that are distinct from those of the natural amino acids. In one embodiment, such distinct properties are the chemical reactivity of the sidechain, in a further embodiment this distinct chemical reactivity permits the sidechain of the non-natural amino acid to undergo a reaction while being a unit of a polypeptide even though the sidechains of the naturally-occurring amino acid units in the same polypeptide do not undergo the aforementioned reaction. In a further embodiment, the sidechain of the non-natural amino acid has a chemistry orthogonal to those of the naturally-occurring amino acids. In a further embodiment, the sidechain of the non-natural amino acid comprises an electrophile-containing moiety; in a further embodiment, the electrophile-containing moiety on the sidechain of the non-natural amino acid can undergo nucleophilic attack to generate a heterocycle-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 length; if the latter, then the polypeptide may further incorporate naturally-occurring or non-natural amino acids.

[0856] In another aspect are diamine-substituted molecules, wherein the diamine group is selected from a hydrazine, an amidine, an imine, a 1,1-diamine, a 1,2-diamine, a 1,3-diamine and a 1,4-diamine group, for the production of derivatized non-natural amino acid linked dolastatin derivatives based upon a heterocycle, including a nitrogen-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 heterocycle, 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 non-natural 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 carbonyl 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 selected 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 further 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 non-natural amino acid can undergo nucleophilic attack to generate a heterocycle-derivatized protein, including a nitrogen-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 derivatizing molecule with the non-natural amino acid polypeptides. Further embodiments include any further modifications of the already modified non-natural amino acid polypeptides.

[0857] 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 nitrogen-containing heterocycle group. In a further embodiment are dicarbonyl-substituted molecules that can form such heterocycle, including a nitrogen-containing heterocycle groups with a diamine-containing non-natural amino acid polypeptide in a pH range between 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 further embodiment the dicarbonyl-substituted molecules are diketone-substitued molecules, in other aspects ketoaldehyde-substituted molecules, in other aspects ketoacid-substituted molecules, in other aspects ketoester-substituted molecules, including ketothioester-substituted molecules. In further embodiments, the dicarbonyl-substituted molecules 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 selectively 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 nitrogen-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 derivatizing molecule with the non-natural amino acid polypeptides. Further embodiments include any further modifications of the already modified non-natural amino acid polypeptides.

[0858] In one aspect are methods to derivatize proteins via the reaction of carbonyl and hydrazine reactants to generate a heterocycle-derivatized protein, 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 heterocycle-derivatized dolastatin, including a nitrogen-containing heterocycle-derivatized dolastatin. In additional or further embodiments are methods to derivatize ketone-containing dolastatin derivatives or aldehyde-containing dolastatin derivatives with hydrazine-functionalized non-natural amino acids. In yet additional or further aspects, the hydrazine-substituted molecule can include proteins, other polymers, and small molecules.

[0859] 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 derivative suitable for the derivatization of carbonyl-containing non-natural amino acid polypeptides, including by way of example only, ketone-, or aldehyde-containing non-natural amino acid polypeptides.

[0860] 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 additional embodiments, the non-natural amino acids are functionalized on their sidechains 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 1,2-dicarbonyl or 1,2-aryldiamine sidechains. In further or additional embodiments, the non-natural amino acids are selected from amino acids having protected or masked 1,2-dicarbonyl or 1,2-aryldiamine sidechains. Further included are equivalents to 1,2-dicarbonyl sidechains, or protected or masked equivalents to 1,2-dicarbonyl sidechains.

[0861] 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-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 derivatize 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 reaction 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 1,2-aryldiamine substituted dolastatin linker derivatives to form quinoxaline or phenazine linkages.

[0862] Provided herein in certain embodiments are derivatizing molecules for the production of toxic compounds comprising non-natural 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 certain 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 reactive 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 non-naturally encoded amino acids bearing azide and alkyne-containing side chains permits the resultant polypeptides to be modified 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 efficiently via Huisgen [32] cycloaddition 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 involving azide or alkyne-containing polypeptides can be carried out at room temperature under aqueous conditions by the addition of Cu(II) (e.g., in the form of a catalytic amount of CuSO.sub.4) in the presence of a reducing 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); Tornoe et al., J. Org. Chem. 67:3057-3064 (2002); Rostovtsev, Angew. Chem. Int. Ed. 41:2596-2599 (2002). Preferred reducing agents include ascorbate, metallic copper, quinine, hydroquinone, vitamin K, glutathione, cysteine, Fe.sup.2, Co.sup.2, and an applied electric potential.

[0863] Such non-natural amino acid heteroaryl-linked dolastatin derivatives include amino acids having the structure of Formula (XXXI), (XXXII), (XXXIII), (XXXIV), (XXXV), or (XXXVI):

##STR00170## ##STR00171## [0864] wherein: [0865] Z has the structure of:

##STR00172## [0866] R.sub.5 is H, CO.sub.2H, C.sub.1-C.sub.6alkyl, or thiazole; [0867] R.sub.6 is OH or H; [0868] Ar is phenyl or pyridine; [0869] R.sub.1 is H, an amino protecting group, resin, at least one amino acid, polypeptide, or polynucleotide; [0870] R.sub.2 is OH, an ester protecting group, resin, at least one amino acid, polypeptide, or polynucleotide; [0871] R.sub.4 is H, halogen, lower alkyl, or substituted lower alkyl; [0872] R.sub.7 is C.sub.1-C.sub.6alkyl or hydrogen; [0873] L, L.sub.1, L.sub.2, L.sub.3, and L.sub.4 are each linkers selected from the group consisting of a bond, -alkylene-, -alkylene-C(O)—, -alkylene-J-, -(alkylene-O).sub.n-alkylene-, -(alkylene-O).sub.n-alkylene-C(O)—, -(alkylene-O).sub.n-J-, -(alkylene-O).sub.n-J-alkylene-, -(alkylene-O).sub.n—(CH.sub.2).sub.n′—NHC(O)—(CH.sub.2).sub.n″—C(Me).sub.2-S—S—(CH.sub.2).sub.n′″—NHC(O)-(alkylene-O).sub.n″″-alkylene-, -(alkylene-O).sub.n-alkylene-W—, -alkylene-C(O)—W—, -(alkylene-O).sub.n-alkylene-J-, -alkylene′-J-(alkylene-O).sub.n-alkylene-, -(alkylene-O).sub.n-alkylene-J-alkylene′, -J-(alkylene-O).sub.n-alkylene-, -(alkylene-O).sub.n-alkylene-J-(alkylene-O).sub.n′-alkylene-J′-, —W—, -alkylene-W—, alkylene′-J-(alkylene-NMe).sub.n-alkylene-W—, -J-(alkylene-NMe).sub.n-alkylene-W—, -(alkylene-O).sub.n-alkylene-U-alkylene-C(O)—, -(alkylene-O).sub.n-alkylene-U-alkylene-; -J-alkylene-NMe-alkylene′-NMe-alkylene″-W—, and -alkylene-J-alkylene′-NMe-alkylene″-NMe-alkylene′″—W—; [0874] W has the structure of:

##STR00173## [0875] U has the structure of:

##STR00174## [0876] each J and J′ independently have the structure of:

##STR00175## [0877] each n and n′ are independently integers greater than or equal to one; [0878] D has the structure of:

##STR00176## [0879] each R.sub.17 is independently selected from the group consisting of H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, alkylalkoxy, substituted alkylalkoxy, polyalkylene oxide, substituted polyalkylene oxide, aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl, -(alkylene or substituted alkylene)-ON(R″).sub.2, -(alkylene or substituted alkylene)-C(O)SR″, -(alkylene or substituted alkylene)-S—S-(aryl or substituted aryl), —C(O)R″, —C(O).sub.2R″, or —C(O)N(R″).sub.2, wherein each R″ is independently hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, alkaryl, substituted alkaryl, aralkyl, or substituted aralkyl; [0880] each Z.sub.1 is a bond, CR.sub.17R.sub.17, O, S, NR′, CR.sub.17R.sub.17—CR.sub.17R.sub.17, CR.sub.17R.sub.17—O, O—CR.sub.17R.sub.17, CR.sub.17R.sub.17—S, S—CR.sub.17R.sub.17, CR.sub.17R.sub.17—NR′, or NR′—CR.sub.17R.sub.17; [0881] each R′ is H, alkyl, or substituted alkyl; [0882] each Z.sub.2 is selected from the group consisting of a bond, —C(O)—, —C(S)—, optionally substituted C.sub.1-C.sub.3 alkylene, optionally substituted C.sub.1-C.sub.3 alkenylene, and optionally substituted heteroalkyl; [0883] each Z.sub.3 are independently selected from the group consisting of a bond, optionally substituted C.sub.1-C.sub.4 alkylene, optionally substituted C.sub.1-C.sub.4 alkenylene, optionally substituted heteroalkyl, —O—, —S—, —C(O)—, —C(S)—, and —N(R′)—; [0884] each T.sub.3 is a bond, C(R″)(R″), O, or S; with the proviso that when T.sub.3 is O or S, R″ cannot be halogen; [0885] each R″ is H, halogen, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; [0886] m and p are 0, 1, 2, or 3, provided that at least one of m or p is not 0; [0887] M.sub.2 is

##STR00177##  where (a) indicates bonding to the B group and (b) indicates bonding to respective positions within the heterocycle group; [0888] M.sub.3 is

##STR00178##  where (a) indicates bonding to the B group and (b) indicates bonding to respective positions within the heterocycle group; [0889] M.sub.4 is

##STR00179##  where (a) indicates bonding to the B group and (b) indicates bonding to respective positions within the heterocycle group; [0890] each R.sub.19 is independently selected from the group consisting of C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 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; [0891] q is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or 11; and [0892] each R.sub.16 is independently selected from the group consisting of hydrogen, halogen, alkyl, NO.sub.2, CN, and substituted alkyl.

[0893] In some embodiments, the compound of Formula (XXXI) include compounds having the structure of Formula (XXXI-A):

##STR00180##

[0894] In certain embodiments of compounds of Formula (XXXI), (XXXII), (XXXIII), (XXXIV), (XXXV), or (XXXVI), R.sub.5 is thiazole or carboxylic acid. In certain embodiments of compounds of Formula (XXXI), (XXXII), (XXXIII), (XXXIV), (XXXV), or (XXXVI), R.sub.6 is H. In certain embodiments of compounds of Formula (XXXI), (XXXII), (XXXIII), (XXXIV), (XXXV), or (XXXVI), Ar is phenyl. In certain embodiments of compounds of Formula (XXXI), (XXXII), (XXXIII), (XXXIV), (XXXV), or (XXXVI), R.sub.7 is methyl. In certain embodiments of compounds of Formula (XXXI), (XXXII), (XXXIII), (XXXIV), (XXXV), or (XXXVI), n is an integer from 0 to 20. In certain embodiments of compounds of Formula (XXXI), (XXXII), (XXXIII), (XXXIV), (XXXV), or (XXXVI), n is an integer from 0 to 10. In certain embodiments of compounds of Formula (XXXI), (XXXII), (XXXIII), (XXXIV), (XXXV), or (XXXVI), n is an integer from 0 to 5.

[0895] In certain embodiments of compounds of Formula (XXXI), (XXXII), (XXXIII), (XXXIV), (XXXV), or (XXXVI), R.sub.5 is thiazole or carboxylic acid. In certain embodiments of compounds of Formula (XXXI), (XXXII), (XXXIII), (XXXIV), (XXXV), or (XXXVI), R.sub.5 is hydrogen. In certain embodiments of compounds of Formula (XXXI), (XXXII), (XXXIII), (XXXIV), (XXXV), or (XXXVI), R.sub.5 is methyl, ethyl, propyl, iso-propyl, butyl, iso-butyl, sec-butyl, tert-butyl, pentyl, or hexyl. In certain embodiments of compounds of Formula (XXXI), (XXXII), (XXXIII), (XXXIV), (XXXV), or (XXXVI), R.sub.5 is —NH-(alkylene-O).sub.n—NH.sub.2, wherein alkylene is —CH.sub.2—, —CH.sub.2CH.sub.2—, —CH.sub.2CH.sub.2CH.sub.2—, —CH.sub.2CH.sub.2CH.sub.2CH.sub.2—, —CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2—, —CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2—, —CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2—, —CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2—, —CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2—, —CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2—, —CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2—, or —CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH—. In certain embodiments of Formula (XXXI), (XXXII), (XXXIII), (XXXIV), (XXXV), or (XXXVI), alkylene is methylene, ethylene, propylene, butylenes, pentylene, hexylene, or heptylene.

[0896] In certain embodiments of compounds of Formula (XXXI), (XXXII), (XXXIII), (XXXIV), (XXXV), or (XXXVI), R.sub.5 is —NH-(alkylene-O).sub.n—NH.sub.2, wherein n is 0, 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.

[0897] In certain embodiments of compounds of Formula (XXXI), (XXXII), (XXXIII), (XXXIV), (XXXV), or (XXXVI), R.sub.6 is H. In some embodiments of compounds of Formula (XXXI), (XXXII), (XXXIII), (XXXIV), (XXXV), or (XXXVI), R.sub.6 is hydroxy.

[0898] In certain embodiments of compounds of Formula (XXXI), (XXXII), (XXXIII), (XXXIV), (XXXV), or (XXXVI), Ar is phenyl.

[0899] In certain embodiments of compounds of Formula (XXXI), (XXXII), (XXXIII), (XXXIV), (XXXV), or (XXXVI), R.sub.7 is methyl, ethyl, propyl, iso-propyl, butyl, sec-butyl iso-butyl, tert-butyl, pentyl, or hexyl. In certain embodiments of compounds of Formula (XXXI), (XXXII), (XXXIII), (XXXIV), (XXXV), or (XXXVI), R.sub.7 is hydrogen.

[0900] In certain embodiments of compounds of Formula (XXXI), (XXXII), (XXXIII), (XXXIV), (XXXV), or (XXXVI), each L, L.sub.1, L.sub.2, L.sub.3, and L.sub.4 is independently a cleavable linker or non-cleavable linker. In certain embodiments of compounds of Formula (XXXI), (XXXII), (XXXIII), (XXXIV), (XXXV), or (XXXVI), each L, L.sub.1, L.sub.2, L.sub.3, and L.sub.4 is independently a oligo(ethylene glycol) derivatized linker.

[0901] In certain embodiments of compounds of Formula (XXXI), (XXXII), (XXXIII), (XXXIV), (XXXV), or (XXXVI), each alkylene, alkylene′, alkylene″, and alkylene′″ independently is —CH.sub.2—, —CH.sub.2CH.sub.2—, —CH.sub.2CH.sub.2CH.sub.2—, —CH.sub.2CH.sub.2CH.sub.2CH.sub.2—, —CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2—, —CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2—, —CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2—, —CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2—, CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2—, —CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2—, —CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2—, or —CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2CH.sub.2—. In certain embodiments of compounds of Formula (XXXI), (XXXII), (XXXIII), (XXXIV), (XXXV), or (XXXVI), alkylene is methylene, ethylene, propylene, butylenes, pentylene, hexylene, or heptylene.

[0902] In certain embodiments of compounds of Formula (XXXI), (XXXII), (XXXIII), (XXXIV), (XXXV), or (XXXVI), each n, n′, n″, n′″, and n″″ independently is 0, 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.

[0903] In certain embodiments of compounds of Formula (XXXI), (XXXII), (XXXIII), (XXXIV), (XXXV), or (XXXVI), R.sub.1 is a polypeptide. In certain embodiments of compounds of Formula (XXXI), (XXXII), (XXXIII), (XXXIV), (XXXV), or (XXXVI), R.sub.2 is a polypeptide. In certain embodiments of compounds of Formula (XXXI), (XXXII), (XXXIII), (XXXIV), (XXXV), or (XXXVI), the polypeptide is an antibody. In certain embodiments of compounds of Formula (XXXI), (XXXII), (XXXIII), (XXXIV), (XXXV), or (XXXVI), the antibody is herceptin.

[0904] Compounds of Formula (XXXI), (XXXII), (XXXIII), (XXXIV), (XXXV), or (XXXVI) may be formed by the reductive alkylation of aromatic amine compounds with carbonyl containing reagents such as, by way of example, ketones, esters, thioesters, and aldehydes.

[0905] The formation of such non-natural amino acid heterocycle-linked dolastatin derivatives having the structure of Formula (XXXI), (XXXII), (XXXIII), (XXXIV), (XXXV), or (XXXVI) includes, but is not limited to, (i) reactions of diamine-containing non-natural amino acids with dicarbonyl-containing dolastatin linked derivatives or reactions of diamine-containing non-natural amino acids with ketoalkyne-containing dolastatin 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.

[0906] Modification of dolastatin linked derivatives described herein with such reactions have any or all of the following advantages. First, diamines undergo condensation 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 embodiment a pH of about 8) to generate heterocycle, including a nitrogen-containing heterocycle, linkages. Under these conditions, the sidechains of the naturally occurring amino acids are unreactive. Second, such selective chemistry makes possible the site-specific derivatization of recombinant proteins: derivatized proteins can now be prepared as defined homogeneous products. Third, the mild conditions needed to effect the reaction of the diamines described herein with the dicarbonyl-containing polypeptides described herein generally do not irreversibly destroy the tertiary structure of the polypeptide (excepting, of course, where the purpose of the reaction is to destroy such tertiary structure). Fourth, the reaction occurs rapidly at room temperature, which allows the use of many types of polypeptides or reagents that would be unstable at higher temperatures. Fifth, the reaction occurs readily 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, near stoichiometric, or stoichiometric-like, so that it is unnecessary to add excess reagent or polypeptide to obtain a useful amount of reaction 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.

[0907] VI. Location of Non-Natural Amino Acids in Dolastatin Linker Derivatives

[0908] The methods and compositions described herein include incorporation of one or more non-natural amino acids into a dolastatin linker derivative. One or more non-natural 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, substituting 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 natural hydrophilic amino acids) and/or inserting the non-natural amino acid in a location that is not required for activity.

[0909] A variety of biochemical and structural approaches can be employed to select the desired sites for substitution 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 dolastatin 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, binding 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 identified 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. Another alternative would be to simply make serial substitutions in each position on the polypeptide 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 position for substitution with a non-natural amino acid into any polypeptide is suitable for use in the methods, techniques and compositions described herein.

[0910] The structure and activity of naturally-occurring mutants of a polypeptide that contain deletions can also be examined to determine 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 non-natural 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 relevant 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, www.rcsb.org), 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.

[0911] Exemplary sites of incorporation of a non-natural amino acid include, but are not limited to, those that are excluded from potential receptor binding regions, or regions for binding to binding proteins or ligands may be fully or partially solvent 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.

[0912] 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 dimensional crystal structure of a polypeptide with its associated ligand, receptor and/or binding proteins, a preference for conservative substitutions

[0913] In one embodiment, the methods described herein include incorporating into the dolastatin linker derivative, where the dolastatin linker derivative comprises a first reactive group; and contacting the dolastatin linker derivative with a molecule (including but not limited to a second protein or polypeptide or polypeptide analog; an antibody or antibody fragment; and any combination thereof) that comprises a second reactive group. In certain embodiments, the first reactive group is a hydroxylamine moiety and the second reactive group is a carbonyl 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 carbonyl 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.

[0914] In some cases, the dolastatin linker derivative incorporation(s) will be combined with other additions, substitutions, or deletions within the polypeptide to affect other chemical, physical, pharmacologic and/or biological traits. 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 affinity of the polypeptide 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 E. coli or other host cells) of the polypeptide. In some embodiments sites are selected for substitution with a naturally encoded or non-natural amino acid in addition to another site for incorporation of a non-natural amino acid for the purpose of increasing the polypeptide solubility following expression in E. coli, or other recombinant host cells. In some embodiments, the polypeptides comprise another addition, substitution, or deletion that modulates affinity for the associated ligand, binding proteins, and/or receptor, modulates (including but not limited to, increases or decreases) receptor dimerization, stabilizes receptor dimers, modulates circulating half-life, modulates release or bioavailability, facilitates purification, or improves or alters a particular route of administration. Similarly, the non-natural amino acid polypeptide can comprise chemical or enzyme cleavage 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 (including 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.

[0915] VII. HER2 Gene as Exemplar

[0916] The methods, compositions, strategies and techniques described herein are not limited to a particular type, class or family of polypeptides or proteins. Indeed, virtually any polypeptides may 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 polypeptide can be homologous to a therapeutic protein selected from the group consisting of: alpha-1 antitrypsin, angiostatin, antihemolytic factor, antibody, antibody fragment, monoclonal antibody (e.g., bevacizumab, cetuximab, panitumumab, infliximab, adalimumab, basiliximab, daclizumab, omalizumab, ustekinumab, etanercept, gemtuzumab, alemtuzumab, rituximab, trastuzumab, nimotuzumab, palivizumab, and abciximab), apolipoprotein, apoprotein, atrial natriuretic factor, atrial natriuretic polypeptide, atrial peptide, C—X—C chemokine, T39765, NAP-2, ENA-78, gro-a, gro-b, gro-c, IP-10, GCP-2, NAP-4, SDF-1, PF4, MIG, calcitonin, c-kit ligand, cytokine, CC chemokine, monocyte chemoattractant protein-1, monocyte chemoattractant protein-2, monocyte chemoattractant protein-3, monocyte inflammatory protein-1 alpha, monocyte inflammatory protein-1 beta, RANTES, 1309, R83915, R91733, HCC1, T58847, D31065, T64262, CD40, CD40 ligand, c-kit ligand, collagen, colony stimulating factor (CSF), complement factor 5a, complement inhibitor, complement receptor 1, cytokine, epithelial neutrophil activating peptide-78, MIP-16, MCP-1, epidermal growth factor (EGF), epithelial neutrophil activating peptide, erythropoietin (EPO), exfoliating toxin, Factor IX, Factor VII, Factor VIII, Factor X, fibroblast growth factor (FGF), fibrinogen, fibronectin, four-helical bundle protein, G-CSF, glp-1, GM-CSF, glucocerebrosidase, gonadotropin, growth factor, growth factor receptor, grf, hedgehog protein, hemoglobin, hepatocyte growth factor (hGF), hirudin, human growth hormone (hGH), human serum albumin, ICAM-1, ICAM-1 receptor, LFA-1, LFA-1 receptor, insulin, insulin-like growth factor (IGF), IGF-I, IGF-II, interferon (IFN), 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 growth factor (KGF), lactoferrin, leukemia inhibitory factor, luciferase, neurturin, neutrophil inhibitory factor (NIF), oncostatin M, osteogenic protein, oncogene product, paracitonin, parathyroid hormone, PD-ECSF, PDGF, peptide hormone, pleiotropin, protein A, protein G, pth, pyrogenic exotoxin A, pyrogenic exotoxin B, pyrogenic exotoxin C, pyy, relaxin, renin, SCF, small biosynthetic protein, soluble complement receptor I, soluble I-CAM 1, soluble interleukin receptor, soluble TNF receptor, somatomedin, somatostatin, somatotropin, streptokinase, superantigens, staphylococcal enterotoxin, SEA, SEB, SEC1, SEC2, SEC3, SED, SEE, steroid hormone receptor, superoxide dismutase, toxic shock syndrome toxin, thymosin alpha 1, tissue plasminogen activator, tumor growth factor (TGF), tumor necrosis factor, tumor necrosis factor alpha, tumor necrosis factor beta, tumor necrosis factor receptor (TNFR), VLA-4 protein, VCAM-1 protein, vascular endothelial growth factor (VEGF), urokinase, mos, ras, raf, met, p53, tat, fos, myc, jun, myb, rel, estrogen receptor, progesterone receptor, testosterone receptor, aldosterone receptor, LDL receptor, and corticosterone.

[0917] 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.

[0918] Thus, the following description of trastuzumab is provided for illustrative purposes and by way of example only, and not as a limit on the scope of the methods, compositions, strategies and techniques described herein. Further, reference to trastuzumab in this application is intended to use the generic term as an example of any antibody. Thus, it is understood that the modifications and chemistries described herein with reference to trastuzumab can be equally applied to any antibody or monoclonal antibody, including those specifically listed herein.

[0919] Trastuzumab is a humanized monoclonal antibody that binds to the domain IV of the extracellular 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 cancer, HER2 may send signals without mitogens arriving and binding to any receptor, making it overactive.

[0920] HER2 extends through the cell membrane, and carries signals 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 biochemical pathways. This includes the PI3K/Akt pathway and the MAPK pathway. These signals promote invasion, survival and growth of blood vessels (angiogenesis) of cells.

[0921] Cells treated with trastuzumab undergo arrest during the G1 phase of the cell cycle so there is reduced 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 arrest. Also, trastuzumab suppresses angiogenesis by both induction of antiangiogenic factors and repression of proangiogenic factors. It is thought that a contribution to the unregulated growth observed in cancer could be due to proteolytic cleavage of HER2/neu that results in the release of the extracellular domain. Trastuzumab has been shown to inhibit HER2/neu ectodomain cleavage in breast cancer cells.

[0922] VIII. Cellular Uptake of Non-Natural Amino Acids

[0923] 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 α-amino acids suggests that these compounds 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 illustrate non-limiting examples of tests which can be done on non-natural amino acids). See, e.g., the toxicity assays in, e.g., the U.S. 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 designing non-natural amino acids that are amenable to cellular uptake pathways is to provide biosynthetic pathways to create amino acids in vivo.

[0924] Typically, the non-natural amino acid produced via cellular uptake as described herein is produced in a concentration sufficient for efficient protein biosynthesis, including but not limited to, a natural cellular amount, but not to such a degree as to affect the concentration of the other amino acids or exhaust cellular resources. Typical concentrations produced in this manner are about 10 mM to about 0.05 mM.

[0925] IX. Biosynthesis of Non-Natural Amino Acids

[0926] Many biosynthetic pathways already exist in cells for the production of amino acids and other compounds. While a biosynthetic method for a particular non-natural amino acid may not exist in nature, including 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 WO 2002/085923 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 comprising the genes. The genes, when expressed in the cell, provide an enzymatic pathway to synthesize the desired compound. Examples of the types of enzymes that are optionally added are provided herein. Additional enzymes sequences are found, for example, in Genbank. 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.

[0927] A variety of methods are available for producing novel enzymes for use in biosynthetic pathways or for evolution of existing pathways. For example, recursive recombination, including but not limited to, as developed by Maxygen, Inc. (available on the world wide web at www.maxygen.com), can be used to develop novel enzymes and pathways. See, e.g., Stemmer (1994), Rapid evolution of a protein in vitro by DNA shuffling, Nature 370(4):389-391; and, Stemmer, (1994), DNA shuffling by random fragmentation and reassembly: In vitro recombination for molecular evolution, Proc. Natl. Acad. Sci. USA., 91:10747-10751. Similarly DesignPath™, 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 pathways 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.

[0928] 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 natural cellular amount, but not to such a degree as to affect the concentration 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 plasmid comprising the genes used to produce enzymes desired for a specific pathway and a non-natural amino acid is generated, in vivo selections are optionally used to further optimize the production of the non-natural amino acid for both ribosomal protein synthesis and cell growth.

[0929] X. Additional Synthetic Methodology

[0930] The non-natural amino acids described herein may be synthesized using methodologies described in the art or using the techniques described herein or by a combination thereof. As an aid, the following table provides various starting 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 herein.

TABLE-US-00001 TABLE 1 Examples of Covalent Linkages and Precursors Thereof Covalent Linkage Product Electrophile Nucleophile Carboxamides Activated esters amines/anilines Carboxamides acyl azides amines/anilines Carboxamides acyl halides amines/anilines Esters acyl halides alcohols/phenols Esters acyl nitriles alcohols/phenols Carboxamides acyl nitriles amines/anilines Imines Aldehydes amines/anilines Hydrazones aldehydes or ketones Hydrazines Oximes aldehydes 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 Thioethers Epoxides Thiols Thioethers haloacetamides Thiols Ammotriazines halotriazines amines/anilines Triazinyl ethers halotriazines alcohols/phenols Amidines imido esters amines/anilines Ureas Isocyanates amines/anilines Urethanes Isocyanates alcohols/phenols Thioureas isothiocyanates amines/anilines Thioethers Maleimides Thiols Phosphite esters phosphoramidites Alcohols Silyl ethers silyl halides Alcohols Alkyl amines sulfonate esters amines/anilines Thioethers sulfonate esters Thiols Esters sulfonate esters carboxylic acids Ethers sulfonate esters Alcohols Sulfonamides sulfonyl halides amines/anilines Sulfonate esters sulfonyl halides phenols/alcohols

[0931] In general, carbon electrophiles are susceptible to attack by complementary nucleophiles, including carbon nucleophiles, wherein an attacking nucleophile brings an electron pair to the carbon electrophile in order to form a new bond between the nucleophile and the carbon electrophile.

[0932] 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 advantage 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 generate 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.

[0933] Non-limiting examples of non-carbon nucleophiles suitable for coupling to carbon electrophiles include but are not limited to primary and secondary amines, thiols, thiolates, and thioethers, alcohols, alkoxides, azides, semicarbazides, and the like. These non-carbon nucleophiles, when used in conjunction with carbon electrophiles, typically generate heteroatom linkages (C—X—C), wherein X is a hetereoatom, including, but not limited to, oxygen, sulfur, or nitrogen.

EXAMPLES

Example 1: Synthesis of Compound 1

[0934] ##STR00181##

[0935] Compound 1-3: Tetra (ethylene glycol) 1-1 (10 g, 51.5 mmol), N-hydroxyphthalimide 1-2 (8.4 g, 51.15 mmol) and triphenylphosphine (17.6 g, 67 mmol) were dissolved in 300 mL of tetrahydrofuran followed by addition of DIAD (12.8 mL, 61.78 mmol) at 0° C. The resulting solution was stirred at room temperature overnight, and then concentrated to dryness. The residue was purified by flash column chromatography to give 5.47 g (31%) of compound 1-3.

[0936] Compound 1-4: To a solution of compound 1-3 (200 mg, 0.59 mmol) in 15 mL dichloromethane was added Dess-Martin Periodinane (300 mg, 0.71 mmol). The reaction mixture 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 separated. The organic layer was washed with saturated sodium bicarbonate, brine, dried over sodium sulfate, filtered and concentrated in vacuo. The residue was purified by flash column chromatography to give 150 mg (75%) of compound 1-4.

[0937] Compound 1-6: To a solution of monomethyldolastatin hydrochloride salt 1-5 (50 mg, 0.062 mmol) in 1 mL of DMF was added compound 1-4 (63 mg, 0.186 mmol) and 70 L 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 diluted with water and purified by HPLC to give 60 mg (80%) of compound 1-6. MS (ESI) m/z 547 [M+2H], 1092 [M+H]. 1 d.

[0938] Compound 1: Compound 1-6 (60 mg, 0.05 mmol) was dissolved in 1 mL of DMF. 32 μL of hydrazine was added. The resulting solution was stirred at ambient temperature for 1 hour. The reaction was quenched with 1N hydrochloride 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].

Example 2: Synthesis of Compound 2

[0939] ##STR00182##

[0940] 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 of Compound 3

[0941] ##STR00183##

[0942] Compound 3 was synthesized via similar synthetic route to Example 1. MS (ESI) m/z 438 [M+2H], 974 [M+H].

Example 4: Synthesis of Compound 4

[0943] Compound 4-2: To a solution of Val (OtBu)—OH.HCl 4-1 (1 g, 4.77 mmol) and bromoethanol (304.7 μL, 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 Boc.sub.2O was added to the reaction 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 sulfate and concentrated in vacuo. The residue was purified by flash column chromatography to give 0.66 g of compound 4-2.

##STR00184## ##STR00185##

[0944] 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 μL, 1.9 mmol) at 0° C. The resulting solution was stirred at room temperature overnight, and then concentrated in vacuo. The residue was purified by flash column chromatography to give 0.68 g of compound 4-3.

[0945] 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 Boc.sub.2O (230 μL, 1 mmol) and DIEA (352 μL, 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 compound 4-4.

[0946] Compound 4-5: To a solution of compound Boc-Val-Dil-methylDap-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 mixture is concentrated in vacuo and extracted with ethyl acetate (100 mLX1, 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 treated with HCl/EtOAC to give compound 4-5.

[0947] Compound 4-6: To a solution of compound 4-5 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 concentrated in vacuo and extracted with ethyl acetate (100 mLX1, 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.

[0948] Compound 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.

[0949] Compound 4-8: To a solution of compound 4-7 in 1 mL 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 4-8.

[0950] Compound 4: Compound 4-8 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 1N hydrochloride solution. The reaction mixture is purified by HPLC to give Compound 4.

Example 5: Synthesis of Compound 5

[0951] 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 1N hydrochloride solution. The reaction mixture is purified by HPLC to give Compound 5.

Example 6: Synthesis of Compound 6

[0952] ##STR00186## ##STR00187##

[0953] 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, 433 mg of HATU and 581 μL 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 mLX1, 50 mL X2). The organic layer was combined 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.

[0954] 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.

[0955] 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, 314 mg of HATU and 303 μL 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 mLX1, 50 mL X2). The organic layer was combined and washed with brine, dried over sodium sulfate and concentrated in vacuo. The residue was purified by flash chromatography to give 370 mg (79%) of compound 6-4.

[0956] Compound 6-5: To a solution of compound 6-4 (170 mg) in 10 mL MeOH was added 5 eq of 1N LiOH. The reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was acidified by 1NHCl and extracted with ethyl acetate washed with brine, dried over sodium sulfate and concentrated in vacuo to give 150 mg (90%) of compound 6-5.

[0957] 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 150 mg of compound 6-6.

[0958] Compound 6-7: To a solution of compound 6-6 (50 mg, 0.062 mmol) in 1 mL of DMF was added compound 1-4 (63 mg, 0.186 mmol) and 70 μL 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 diluted with water and purified by HPLC to give 60 mg (80%) of compound 6-7.

[0959] Compound 6: Compound 6-7 (60 mg, 0.05 mmol) was dissolved in 1 mL of DMF. 32 μL of hydrazine was added. The resulting solution was stirred at ambient temperature for 1 hour. The reaction was quenched with 1N hydrochloride solution. The reaction mixture was purified by HPLC to give 33 mg (55%) of Compound 6.

Example 7: Synthesis of Compound 7

[0960] ##STR00188##

[0961] Compound 7 was synthesized via similar synthetic route to Compound 1. MS (ESI) m/z 440 [M+2H], 879 [M+H].

Example 8: Synthesis of Compound 8

[0962] ##STR00189##

[0963] Compound 8 was synthesized via similar synthetic route to Compound 1. MS (ESI) m/z 418 [M+2H], 835 [M+H].

Example 9: Synthesis of Compound 9

[0964] ##STR00190##

[0965] Compound 9-1: To a solution of compound Boc-Val-Dil-methylDap-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 mLX1, 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 treated with HCl/EtOAC to give compound 9-1.

[0966] 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 concentrated in vacuo and extracted with ethyl acetate (100 mLX1, 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.

[0967] Compound 9-3: Compound 9-2 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 9-3.

[0968] Compound 9-4: To a solution of compound 9-3 in 1 mL 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.

[0969] Compound 9: Compound 9-4 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 1N hydrochloride solution. The reaction mixture is purified by HPLC to give compound 9.

Example 10: Synthesis of Compound 10

[0970] ##STR00191##

[0971] Compound 10: Compound 9-3 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 1N hydrochloride solution. The reaction mixture is purified by HPLC to give Example 10.

Example 11: Synthesis of Compound 11

[0972] ##STR00192##

[0973] Compound 11-3: To a solution of tetra (ethylene glycol) 11-1 (40.6 mL, 235 mmol) in 100 mL of tetrahedrofuran was added 47 mg of sodium. 12 mL of tert-butylacrylate was added after sodium was dissolved. The reaction mixture was stirred at room temperature for 24 hours. The reaction mixture was concentrated in vacuo and quenched with 2 mL of 1 N HCl. The residue was suspended in brine and extracted with ethyl acetate (100 mLX1, 50 mL X2). The organic layer was combined and washed with brine, dried over sodium sulfate and concentrated in vacuo to give 6.4 g (23%) of compound 11-3.

[0974] Compound 11-5: Compound 11-3 (1.0 g, 3.12 mmol), N-hydroxyphthalimide 11-4 (611 mg, 3.744 mmol) and triphenylphosphine (1.23 g, 4.68 mmol) were dissolved in 20 mL of tetrahydrofuran followed by addition of DIAD (0.84 mL, 4.06 mmol) at 0° C. The resulting solution was stirred at room temperature overnight, and then concentrated to dryness. The residue was purified by flash column chromatography using SiliaSep Cartridges (80 g), eluting with 0-100% ethyl acetate/hexanes, to give 1.0 g (100%) of compound 11-5.

[0975] Compound 11-6: Compound 11-5 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 1.0 g of compound 11-6.

[0976] Compound 11-8: To a solution of 30 mg (0.0372 mmol) of monomethyldolastatin hydrochloride, 31 mg (0.0744 mmol) of compound 11-6 and 38.2 mg (0.082 mmol) of PyBroP in 1 mL of DMF was added 33 μL (0.186 mmol) of diisopropylethylamine. The reaction mixture was stirred at room temperature for 5 hours. The reaction mixture was purified by HPLC to give 28 mg (65%) of compound 11-8. MS (ESI) m/z 785 [M+2H], 1164[M+H].

[0977] Compound 11: Compound 11-8 (28 mg, 0.024 mmol) was dissolved in 1 mL of DMF. 23 μL (0.72 mmol) of anhydrous hydrazine was added. The resulting solution was stirred at room temperature for 1 hour. The reaction was quenched with 1N hydrochloride solution. The reaction mixture was purified by preparative HPLC, eluting with 20-70% CH3CN/H2O in 20 min at 254 nm, to give 20 mg (66%) of Compound 11. MS (ESI) m/z 518 [M+2H], 1034[M+H].

Example 12: Synthesis of Compound 12

[0978] ##STR00193## ##STR00194##

[0979] Compound 12-2: To a solution of compound 12-1 (500 mg, 0.875 mmol) in 3 mL of DMF was added 283 mg of phenylalanine hydrochloride, 433 mg of HATU and 581 μL of N-methylmorpholine.

[0980] The reaction mixture was stirred at room temperature for 4 hours. The reaction mixture was concentrated in vacuo and extracted with ethyl acetate (100 mLX1, 50 mL X2). The organic layer was combined 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 12-2.

[0981] Compound 12-3: Compound 12-2 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 12-3.

[0982] Compound 12-4: To a solution of compound 12-3 (368 mg, 0.55 mmol) in 3 mL of DMF was added 255 mg of Boc-N-methyl valine, 314 mg of HATU and 303 μL 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 mLX1, 50 mL X2). The organic layer was combined and washed with brine, dried over sodium sulfate and concentrated in vacuo. The residue was purified by flash chromatography to give 370 mg (79%) of compound 12-4.

[0983] Compound 12-5: To a solution of compound 12-4 (170 mg) in 10 mL MeOH was added 5 eq of 1N LiOH. The reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was acidified by 1NHCl and extracted with ethyl acetate washed with brine, dried over sodium sulfate and concentrated in vacuo to give 150 mg (90%) of compound 12-5.

[0984] Compound 12-6: Compound 12-5 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 150 mg of compound 12-6.

[0985] Compound 12-7: To a solution of compound 12-6 in DMF was added formylaldehyde (3 eq) and 20 eq of acetic acid, followed by addition of 2 eq of sodium cyanoborohydride. The resulting mixture was stirred at ambient temperature for 2 hours. The reaction mixture was diluted with water and purified by HPLC to give compound 12-7.

[0986] Compound 12-10: tert-Butyl 2-(2-hydroxyethoxy)ethylcarbamate (2.05 g, 10 mmol), N-hydroxyphthalimide (1.8 g, 11 mmol) and triphenylphosphine (3.67 g, 14 mmol) were dissolved in 100 mL of tetrahydrofuran followed by addition of DIAD (2.48 mL, 12 mmol) at 0° C. The resulting solution was stirred at room temperature overnight, and then concentrated to dryness. The residue was treated with 50 mL of 4N HCl/dioxane. The mixture was stirred at room temperature for 2 hours. The solvent was removed in vacuo. The residue was treated with ether, filtered, washed with ether and dried in vacuo to get 2.6 g (91%) of compound 12-10. MS (ESI) m/z 251 [M+H].

[0987] Compound 12-11: To a solution of compound 12-10 (20 mg, 0.026 mmol) in 1 mL of DMF was added 11.2 mg of compound 12-10, 15 mg of HATU and 23 μL of DIEA. The reaction mixture was stirred at room temperature for 2 hours. The reaction mixture was purified by HPLC to give 20 mg (70%) of compound 12-4. MS (ESI) m/z 490 [M+2H], 978[M+H].

[0988] Compound 12: Compound 12-11 (20 mg, 0.0183 mmol) was dissolved in 1 mL of DMF. 18 μL (0.56 mmol) of anhydrous hydrazine was added. The resulting solution was stirred at room temperature for 1 hour. The reaction was quenched with 1N hydrochloride solution. The reaction mixture was purified by preparative HPLC, eluting with 20-70% CH3CN/H2O in 20 min at 254 nm, to give 14 mg (72%) of Compound 12. MS (ESI) m/z 425 [M+2H], 848[M+H].

Example 13: Synthesis of Compound 13

[0989] ##STR00195## ##STR00196##

[0990] Compound 13-2: Tert-butyl 6-hydroxyhexanoate 13-1 (1.5 g, 1.97 mmol), N-hydroxyphthalimide (1.42 g, 8.76 mmol) and triphenylphosphine (2.82 g, 10.76 mmol) were dissolved in 50 mL of tetrahydrofuran followed by addition of DIAD (2 mL, 9.564 mmol) at 0° C. The resulting solution was stirred at room temperature overnight, and then concentrated to dryness. The residue was purified by flash column chromatography to give 2.5 g (95%) of compound 13-2.

[0991] Compound 13-3: The compound 13-2 was treated with 15 mL 4N HCl in dioxane. The reaction mixture was stirred at ambient temperature for 12 hours and concentrated to dryness in vacuo to give 900 mg (100%) of compound 13-3.

[0992] Compound 13-4: To a solution of compound 13-3 (900 mg, 3.0 mmol) in 10 mL of THF was added 397 mg of N-hydroxysuccinimide, followed by adding 669 mg of DCC. The reaction mixture was stirred at ambient temperature overnight and filtered. The filtration was concentrated and treated with 10 mL of DCM. The DCM solution was stayed at ambient temperature for 1 hour and filtered. The filtration was concentrated and purified by flash column chromatography to give 800 mg (71%) of compound 13-4.

[0993] Compound 13-6: The mixture of compound 13-4 (435 mg, 1.16 mmol) and Val-Cit-PABOH 13-5.sup.1 (400 mg, 1.054 mmol) in 12 mL of DMF was stirred at ambient temperature for 24 hours. The solvent was removed in vacuo. The residue was treated with ether, filtered and washed with ether. The solid was dried in vacuo to give 660 mg (98%) of compound 13-6.

[0994] Compound 13-7: To the solution of compound 13-6 (200 mg, 0.313 mmol) in 6 mL of DMF was added bis (p-nitrophenyl) carbonate (286 mg, 0.94 mmol), followed by addition of 110.2 μL of DIEA. The reaction mixture was stirred at ambient temperature for 5 hours and concentrated. The residue was treated with ether and filtered. The collected solid was washed with ether, 5% citric acid, water, ether and dried in vacuo to give 210 mg (83%) compound 13-7.

[0995] Compound 13-9: To a solution of monomethylauristatin hydrochloride salt 13-8 (100 mg, 0.1325 mmol) in 2 mL of DMF was added compound 13-7 (159 mg, 0.2 mmol) and 10 mg of HOBt, followed by addition of 35.2 μL of DIEA. The resulting mixture was stirred at ambient temperature for 2 days. The reaction mixture was diluted with water and purified by HPLC to give 93 mg (51%) of compound 13-9. MS (ESI) m/z 692 [M+2H], 1382 [M+H].

[0996] Compound 13: The compound 13-9 (50 mg, 0.036 mmol) was dissolved in 1 mL of DMF. 23 μL of hydrazine was added. The resulting solution was stirred at ambient temperature for 3 hours. The reaction was quenched with 1N hydrochloride solution. The reaction mixture was purified by HPLC to give 32 mg (65%) of Compound 13. MS (ESI) m/z 638.5 [M+Na+2H], 1253.3 [M+H], 1275.8 [M+Na].

Example 14: Synthesis of Compound 14

[0997] ##STR00197## ##STR00198##

[0998] Compound 14-3: To a solution of tetra (ethylene glycol) 14-1 (40.6 mL, 235 mmol) in 100 mL of tetrahedrofuran was added 47 mg of sodium. 12 mL of tert-butylacrylate was added after sodium was dissolved. The reaction mixture was stirred at room temperature for 24 hours. The reaction mixture was concentrated in vacuo and quenched with 2 mL of 1 N HCl. The residue was suspended in brine and extracted with ethyl acetate (100 mLX1, 50 mL X2). The organic layer was combined and washed with brine, dried over sodium sulfate and concentrated in vacuop to give 6.4 g (23%) of compound 14-3.

[0999] Compound 14-5: Compound 14-3 (1.0 g, 3.12 mmol), N-hydroxyphthalimide 14-4 (611 mg, 3.744 mmol) and triphenylphosphine (1.23 g, 4.68 mmol) were dissolved in 20 mL of tetrahydrofuran followed by addition of DIAD (0.84 mL, 4.06 mmol) at 0° C. The resulting solution was stirred at room temperature overnight, and then concentrated to dryness. The residue was purified by flash column chromatography using SiliaSep Cartridges (80 g), eluting with 0-100% ethyl acetate/hexanes, to give 1.0 g (100%) of compound 14-5.

[1000] Compound 14-6: Compound 14-5 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 1.0 g of compound 14-6.

[1001] Compound 14-7: To a solution of compound 6 (1.93 g, 4.68 mmol) and N-hydroxysuccinimide (646 mg, 5.616 mmol) in 20 mL of tetrahedrofuran was added 1.062 g (5.148 mmol) of DCC. The reaction mixture was stirred at room temperature overnight and filtered. The filtration was concentrated and purified by flash column chromatography using SiliaSep Cartridges (80 g), eluting with 0-100% ethyl acetate/hexanes to give 2.37 g (100%) of compound 14-7.

[1002] Compound 14-8: Compound 14-8 was made according to the literature (Bioconjugat Chem. 2002, 13 (4), 855-869.)

[1003] Compound 14-9: To a solution of compound 14-8 (200 mg, 0.527 mmol) in 2 mL of DMF was added 295 mg (0.58 mmol) of compound 14-7. The reaction mixture was stirred at room temperature overnight and concentrated in vacuo. The residue was treated with ether, filtered, washed with ether and dried in vacuo to give 402 mg (98%) of compound 14-9.

[1004] Compound 14-10: To a solution of compound 14-9 (406 mg, 0.527 mmol) and bis(p-nitrophenol) carbonate (481 mg, 1.58 mmol) in 10 mL of DMF was added 0.186 mL (1.054 mmol) of diisopropylethylamine. The reaction mixture was stirred at room temperature for 5 hours. The solvent was removed in vacuo. The residue was treated with ether, filtered, washed with ether, 5% citic acid, water, ether and dried in vacuo to give 350 mg (72%) of compound 14-10.

[1005] Compound 14-11: To a solution of 50 mg (0.062 mmol) of monomethyldolastatin hydrochloride, 87.2 mg (0.093 mmol) of compound 14-10 and 4.7 mg (0.031 mmol) of HOBt in 1 mL of DMF was added 22 μL (0.124 mmol) of diisopropylethylamine. The reaction mixture was stirred at room temperature for 16 hours. The reaction mixture was purified by HPLC to give 41 mg (42%) of compound 14-11. MS (ESI) m/z 785 [M+2H].

[1006] Compound 14: Compound 14-11 (41 mg, 0.026 mmol) was dissolved in 1 mL of DMF. 17 μL (0.52 mmol) of anhydrous hydrazine was added. The resulting solution was stirred at room temperature for 1 hour. The reaction was quenched with 1N hydrochloride solution. The reaction mixture was purified by preparative HPLC, eluting with 20-70% CH3CN/H2O in 20 min at 254 nm, to give 22 mg (58%) of compound 14. MS (ESI) m/z 720 [M+2H].

Example 15: Synthesis of Compound 15

[1007] ##STR00199## ##STR00200##

[1008] Compound 15-2: To a solution of 50 mg (0.062 mmol) of monomethyldolastatin hydrochloride, 75 mg (0.093 mmol) of compound 13-7 and 4.7 mg (0.031 mmol) of HOBt in 1 mL of DMF was added 22 μL (0.124 mmol) of diisopropylethylamine. The reaction mixture was stirred at room temperature for 16 hours. The reaction mixture was purified by HPLC to give 41 mg (42%) of compound 15-2. MS (ESI) m/z 718 [M+2H], 1435 [M+H].

[1009] Compound 15-2: Compound 15-2 (41 mg, 0.026 mmol) was dissolved in 1 mL of DMF. 17 μL (0.52 mmol) of anhydrous hydrazine was added. The resulting solution was stirred at room temperature for 1 hour. The reaction was quenched with 1N hydrochloride solution. The reaction mixture was purified by preparative HPLC, eluting with 20-70% CH3CN/H2O in 20 min at 254 nm, to give 22 mg (58%) of example 15. MS (ESI) m/z 653 [M+2H], 1305 [M+H].

Example 16: Synthesis of Compound 16

[1010] ##STR00201## ##STR00202## ##STR00203##

[1011] Compound 16-3: To a solution of ethylene glycol 16-1 (13.1 mL, 235 mmol) in 100 mL of tetrahedrofuran was added 47 mg of sodium. 12 mL of tert-butylacrylate was added after sodium was dissolved. The reaction mixture was stirred at room temperature for 24 hours. The reaction mixture was concentrated in vacuo and quenched with 2 mL of 1 N HCl. The residue was suspended in brine and extracted with ethyl acetate (100 mLX1, 50 mL X2). The organic layer was combined and washed with brine, dried over sodium sulfate and concentrated in vacuo. The residue was purified by flash column chromatography to give 5.2 g (24%) of compound 16-3.

[1012] Compound 16-5: Compound 16-3 (2.0 g, 10.5 mmol), N-hydroxyphthalimide (2.05 g, 12.6 mmol) and triphenylphosphine (3.58 g, 13.65 mmol) were dissolved in 50 mL of tetrahydrofuran followed by addition of DIAD (3.26 mL, 15.75 mmol) at 0° C. The resulting solution was stirred at room temperature overnight, and then concentrated to dryness. The residue was purified by flash column chromatography to give compound 16-5.

[1013] Compound 16-6: Compound 16-5 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 compound 16-6.

[1014] Compound 16-7: To a solution of compound 16-6 (5.16 mmol) and N-hydroxysuccinimide (722 mg, 6.7 mmol) in 20 mL of tetrahedrofuran was added 1.28 g (6.2 mmol) of DCC. The reaction mixture was stirred at room temperature overnight and filtered. The filtration was concentrated and purified by flash column chromatography to give 500 mg of compound 16-7.

[1015] Compound 16-8: Compound 16-8 was made according to the literature (Bioconjugat Chem. 2002, 13 (4), 855-869.)

[1016] Compound 16-9: To a solution of compound 16-8 (5.0 g, 8.3 mmol) and bis(p-nitrophenol) carbonate (7.6 g, 25 mmol) in 100 mL of DMF was added 2.92 mL (16.6 mmol) of diisopropylethylamine. The reaction mixture was stirred at room temperature for 16 hours. The solvent was removed in vacuo. The residue was treated with ether, filtered, washed with ether, 5% citic acid, water, ether and dried in vacuo to give 5.0 g (81%) of compound 16-9.

[1017] Compound 16-10: To a solution of 1.0 g (1.24 mmol) of monomethyldolastatin hydrochloride, 1.42 g (1.8575 mmol) of compound 16-9 and 95 mg (0.62 mmol) of HOBt in 10 mL of DMF was added 437 μL (2.48 mmol) of diisopropylethylamine. The reaction mixture was stirred at room temperature for 16 hours. The reaction mixture was purified by HPLC to give 1.0 g (58%) of compound 16-10. MS (ESI) m/z 700 [M+2H], 1398 [M+H].

[1018] Compound 16-11: To a solution of compound 16-10 (1.0 g, 0.715 mmol) in 15 mL of tetrahedrofuran was added 5 mL (48 mmol) of diethylamine. The reaction mixture was stirred at room temperature for 1.5 hours and concentrated in vacuo. The residue was dissolved in 20 mL of DCM, treated with 200 mL of ether and filtered, washed with ether and dried in vacuo to give 860 mg of compound 16-11. MS (ESI) m/z 589 [M+2H], 1176 [M+H].

[1019] Compound 16: To a solution of 50 mg (0.0425 mmol) of compound 16-11 in 1 mL of DMF was added 32 mg (0.085 mmol) of compound 16-7. The reaction mixture was stirred at room temperature for 16 hours. The HPLC and MS showed reaction done. 27.2 μL (0.85 mmol) of anhydrous hydrazine was added to the reaction mixture. The reaction was done in 2 hours. The reaction mixture was acidified with 1N HCl and purified by HPLC to give 40 mg (66%) of compound 16. MS (ESI) m/z 654 [M+2H], 1307[M+H].

Example 17: Synthesis of Compound 17

[1020] ##STR00204## ##STR00205##

[1021] Compound 17-2: To a solution of compound 17-1 (1.0 g, 4.52 mmol) and N-hydroxysuccinimide (572 mg, 4.97 mmol) in 20 mL of tetrahedrofuran was added 1.12 g (5.424 mmol) of DCC. The reaction mixture was stirred at room temperature overnight and filtered. The filtration was concentrated to give compound 17-2.

[1022] Compound 17: To a solution of 50 mg (0.0425 mmol) of compound 16-11 in 1 mL of DMF was added 41 mg (0.1275 mmol) of compound 17-2. The reaction mixture was stirred at room temperature for 16 hours. The HPLC and MS showed reaction done. 20 μL (0.625 mmol) of anhydrous hydrazine was added to the reaction mixture. The reaction was done in 2 hours. The reaction mixture was acidified with 1N HCl and purified by HPLC to give 35 mg (60%) of compound 17. MS (ESI) m/z 625 [M+2H], 1249[M+H].

Example 18: Synthesis of Compound 18

[1023] ##STR00206## ##STR00207##

[1024] Compound 18-1: To a solution of compound 6-6, mg (0.062 mmol) of compound 14-10 and HOBt in 1 mL of DMF was added diisopropylethylamine. The reaction mixture was stirred at room temperature for 16 hours. The reaction mixture was purified by HPLC to give compound 18-1.

[1025] Compound 18: Compound 18-1 was dissolved in 1 mL of DMF. Anhydrous hydrazine was added. The resulting solution was stirred at room temperature for 1 hour. The reaction was quenched with 1N hydrochloride solution. The reaction mixture was purified by preparative HPLC, eluting with 20-70% CH3CN/H2O in 20 min at 254 nm, to give compound 18.

Example 19: Synthesis of Compound 19

[1026] ##STR00208## ##STR00209## ##STR00210##

[1027] Compound 19-2: tert-Butyl 2-(2-hydroxyethoxy)ethylcarbamate 13 (2.05 g, 10 mmol), N-hydroxyphthalimide (1.8 g, 11 mmol) and triphenylphosphine (3.67 g, 14 mmol) were dissolved in 100 mL of tetrahydrofuran followed by addition of DIAD (2.48 mL, 12 mmol) at 0° C. The resulting solution was stirred at room temperature overnight, and then concentrated to dryness. The residue was treated with 50 mL of 4N HCl/dioxane. The mixture was stirred at room temperature for 2 hours. The solvent was removed in vacuo. The residue was treated with ether, filtered, washed with ether and dried in vacuo to get 2.6 g (91%) of compound 19-2. MS (ESI) m/z 251 [M+H].

[1028] Compound 19-3: To the mixture of compound 19-2 (315 mg, 1.1 mmol), Boc-Lys(Boc)-OH (365 mg, 1 mmol), EDC (382 mg, 2 mmol) and HOBt (306 mg, 2 mmol) in 10 mL of DCM was added 1.056 mL (6 mmol) of diisopropylethylamine. The reaction mixture was stirred at room temperature for 3 hours and extracted with ethyl acetate, washed with 5% citric acid, saturate sodium bicarbonate, brine, dried over sodium sulfate, filtered and concentrated in vacuo. The residue was purified flash column chromatography using SiliaSep Cartridges (40 g), eluting with 0-100% ethyl acetate/hexanes, to give 405 mg (70%) of compound 19-3.

[1029] Compound 19-4: Compound 19-3 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 315 mg (98%) of compound 19-4. MS (ESI) m/z 379 [M+H].

[1030] Compound 19-5: To a solution of compound 14-3 (322 mg, 1 mmol) in 20 mL dichloromethane was added Dess-Martin Periodinane (636 mg, 1.5 mmol). The reaction mixture was stirred at room temperature for 3 hours. The reaction was quenched with a solution of sodium thiosulfate (1.4 g, 8.85 mmol) in 15 mL of saturated sodium bicarbonate. The mixture was separated. The organic layer was washed with saturated sodium bicarbonate, brine, dried over sodium sulfate, filtered and concentrated in vacuo. The residue was purified by flash column chromatography using SiliaSep Cartridges (40 g), eluting with 0-100% ethyl acetate/hexanes to give 170 mg (53%) of compound 19-5.

[1031] Compound 19-6: To a solution of monomethyldolastatin hydrochloride 1.0 g (1.24 mmol) in 20 mL of DMF was added 1.19 g (3.72 mmol) of compound 17 followed by 1.4 mL (24.8 mmol) of acetic acid and 156 mg (2.48 mmol) of sodium cyanoborohydride. The resulting mixture was stirred at room temperature for 2 hours. The solvent was removed in vacuo. The residue was adjusted to pH 8 by sodium bicarbonate and extracted with DCM, washed with brine, dried over sodium sulfate, filtered and concentrated in vacuo. The residue was purified by flash column chromatography using SiliaSep Cartridges (40 g), eluting with 0-5% methanol/DCM to give 680 mg (51%) of compound 19-6. MS (ESI) m/z 538 [M+2H], 1075 [M+H].

[1032] Compound 19-7: To a solution of compound 19-6 (680 mg, 0.632 mmol) in 5 mL of DCM was added 20 mL of 4N HCl/dioxane. The reaction mixture was stirred at room temperature for 2 hours and concentrated in vacuo. The residue was treated with ether, filtered, washed with ether and dried in vacuo to give 660 mg (98%) of compound 19-7. MS (ESI) m/z 510 [M+2H], 1019 [M+H].

[1033] Compound 19-8: To a solution of compound 19-7 (280 mg, 0.257 mmol), compound 19-4 (38 mg, 0.0857 mmol) and N-methylmorpholine (0.283 mL, 2.57 mmol) in 5 mL of N-methylmpyrrolidinone was added 98 mg (0.257 mmol) of HATU. The reaction mixture was stirred at room temperature for 1 hour. The reaction mixture was purified by HPLC to give 160 mg (71%) of compound 19-8. MS (ESI) m/z 596 [M+4H], 794[M+3H], 1191 [M+2H].

[1034] Compound 19: Compound 19-8 (160 mg, 0.0613 mmol) was dissolved in 1.5 mL of DMF. 20 μL (0.613 mmol) of anhydrous hydrazine was added. The resulting solution was stirred at room temperature for 1 hour. The reaction was quenched with 1N hydrochloride solution. The reaction mixture was purified by preparative HPLC, eluting with 20-70% CH3CN/H2O in 20 min at 254 nm, to give 120 mg (75%) of compound 19. MS (ESI) m/z 451[M+5H], 563[M+4H], 751 [M+3H], 1126 [M+2H].

Example 20: Synthesis of Compound 20

[1035] ##STR00211## ##STR00212## ##STR00213##

[1036] Compound 20-2: To a solution of tetra (ethylene glycol) 20-1 (8.0 g, 41.2 mmol) in 100 mL of tetrahedrofuran was added 1.65 g of sodium hydride at 0° C. The reaction mixture was stirred at room temperature for 30 min. 6.21 g of TBS-Cl was added to this solution. The reaction mixture was stirred at room temperature overnight. The reaction mixture was concentrated in vacuo and quenched with 2 mL of 1 N HCl. The residue was suspended in brine and extracted with ethyl acetate (100 mLX1, 50 mL X2). The organic layer was combined and washed with brine, dried over sodium sulfate and concentrated in vacuo. The residue was purified by flash column chromatography to give 5.7 g of compound 20-2.

[1037] Compound 20-3: To a solution of compound 20-2 (500 mg, 1.62 mmol) in 30 mL dichloromethane was added Dess-Martin Periodinane (1.03 g, 2.43 mmol). The reaction mixture was stirred at room temperature for 3 hours. The reaction was quenched with a solution of sodium thiosulfate (1.4 g, 8.85 mmol) in 15 mL of saturated sodium bicarbonate. The mixture was separated. The organic layer was washed with saturated sodium bicarbonate, brine, dried over sodium sulfate, filtered and concentrated in vacuo. The residue was purified by flash column chromatography to give 400 mg of compound 20-3.

[1038] Compound 20-4: To a solution of monomethyldolastatin hydrochloride 213 mg (0.263 mmol) in 4 mL of DMF was added 245 mg (0.75 mmol) of compound 20-3 followed by 0.303 mL (5 mmol) of acetic acid and 34 mg (0.5 mmol) of sodium cyanoborohydride. The resulting mixture was stirred at room temperature for 2 hours. The solvent was removed in vacuo. 3 mL of 60% acetonitrile was added, followed by 0.2 mL of HF. Pyridine at 0° C. The resulting solution was stirred at room temperature for 2 hours. The organic solvent was removed in vacuo. The residue was adjusted to pH 8 by sodium bicarbonate and extracted with DCM, washed with brine, dried over sodium sulfate, filtered and concentrated in vacuo. The residue was purified by flash column chromatography to give 160 mg of compound 20-4. MS (ESI) m/z 474 [M+2H], 947[M+H].

[1039] Compound 20-5: To a solution of compound 20-4 (50 mg, 0.062 mmol) in 4 mL of DCM was added 0.3 mL of phosgene/toluene at 0° C. The reaction mixture was stirred at 0° C. for 3 hours and concentrated in vacuo for next step without purification.

[1040] Compound 20-6: To a solution of compound 19-4 (7.6 mg, 0.017 mmol) and compound 20-5 (0.062 mmol) was added 25 μL of diisopropylethylamine. The reaction mixture was stirred at room temperature for 1 hour. The reaction mixture was purified by HPLC, eluting with 20-70% CH3CN/H2O in 20 min at 254 nm, to give 33 mg of compound 20-6. MS (ESI) m/z 582[M+4H], 775[M+3H], 1163 [M+2H].

[1041] Compound 20: Compound 20-6 (33 mg, 0.014 mmol) was dissolved in 1 mL of DMF. 14 μL (0.43 mmol) of anhydrous hydrazine was added. The resulting solution was stirred at room temperature for 1 hour. The reaction was quenched with 1N hydrochloride solution. The reaction mixture was purified by preparative HPLC, eluting with 20-70% CH3CN/H2O in 20 min at 254 nm, to give 10 mg of compound 20. MS (ESI) m/z 549[M+4H], 732[M+3H], 1098[M+2H].

Example 21: Synthesis of Compound 21

[1042] ##STR00214## ##STR00215## ##STR00216##

[1043] Compound 21-2: The mixture of N, N′-Dimethylene diamine 21-1 (5 mL, 46.5 mmol) and tert-butyl acrylate 13 mL. (116 mmol) was heated at 85° C. for 1 hour. Another 13 mL (116 mmol) of tert-butyl acrylate was added. The reaction mixture was continually heated at 85° C. for 1 hour and stirred at room temperature overnight. The reaction mixture was concentrated in vacuo. The residue was diluted with hexanes and purified by flash column chromatography using SiliaSep Cartridges (120 g), eluting with 0-5% methanol/DCM to give 10.1 g (62%) of compound 21-2. MS (ESI) m/z 345 [M+H].

[1044] Compound 21-3: To a solution of compound 21-2 (5.0 g, 14.5 mmol) in 50 mL of DCM was added 40 mL of 4N HCl/dioxane. The reaction mixture was stirred at room temperature for 2 days and concentrated in vacuo. The residue was treated with ether, filtered, washed with ether and dried in vacuo to give 4.3 g (97%) of compound 21-3.

[1045] Compound 21-4: To a solution of 166 mg (0.544 mmol) of compound 21-3 and 0.15 mL of N-methylmorpholine in 10 mL of N-methylpyrrolidinone was added 160 mg of compound 16-11, followed by 0.068 mL (0.408 mmol) of DECP. The reaction mixture was stirred at room temperature for 1 hour. The reaction mixture was purified by preparative HPLC, eluting with 35-70% CH3CN/H2O in 20 min at 254 nm, to give 100 mg (50%) of compound 21-4. MS (ESI) m/z 464[M+3H], 696 [M+2H], 1391 [M+H].

[1046] Compound 21-5: To a solution of compound 19-4 (11 mg, 0.025 mmol), compound 21-4 (115 mg, 0.077 mmol) and N-methylmorpholine (0.028 mL, 0.25 mmol) in 1.5 mL of N-methylmpyrrolidinone was added 29.3 mg (0.077 mmol) of HATU. The reaction mixture was stirred at room temperature for 1 hour. The reaction mixture was purified by HPLC, eluting with 20-70% CH3CN/H2O in 20 min at 254 nm, to give 60 mg (67%) of compound 21-5. MS (ESI) m/z 625 [M+5H], 781 [M+4H], 1041 [M+3H].

[1047] Compound 21: Compound 21-5 (60 mg, 0.014 mmol) was dissolved in 1 mL of DMF. 7 μL (0.21 mmol) of anhydrous hydrazine was added. The resulting solution was stirred at room temperature for 1 hour. The reaction was quenched with 1N hydrochloride solution. The reaction mixture was purified by preparative HPLC, eluting with 20-70% CH3CN/H2O in 20 min at 254 nm, to give 29 mg (58%) of compound 21. MS (ESI) m/z 599[M+5H], 749[M+4H], 998 [M+3H].

Example 22: Synthesis of Compound 22

[1048] ##STR00217##

[1049] Compound 22-1: To a solution of compound 19-4 (7.6 mg, 0.017 mmol), compound 12-7 (40 mg, 0.051 mmol) and DIEA (0.030 mL, 0.17 mmol) in 2 mL of DMF was added 32 mg (0.085 mmol) of HATU. The reaction mixture was stirred at room temperature for 2 hour. The reaction mixture was purified by HPLC, eluting with 20-70% CH3CN/H2O in 20 min at 254 nm, to give 24 mg (68%) of compound 22-1. MS (ESI) m/z 612 [M+3H], 917 [M+2H], 1834[M+H].

[1050] Compound 22: Compound 22-1 (24 mg, 0.012 mmol) was dissolved in 1 mL of DMF. 12 μL (0.36 mmol) of anhydrous hydrazine was added. The resulting solution was stirred at room temperature for 1 hour. The reaction was quenched with 1N hydrochloride solution. The reaction mixture was purified by preparative HPLC, eluting with 20-70% CH3CN/H2O in 20 min at 254 nm, to give 15 mg (58%) of Example 21. MS (ESI) m/z 569[M+3H], 852[M+2H], 1726[M+2H].

Example 23: Synthesis of Compound 23

[1051] ##STR00218## ##STR00219## ##STR00220##

[1052] Compound 23-1: To a solution of compound 14-3 (4.0 g, 12.4 mmol) and 6.6 mL (37.2 mmol) of DIEA in 50 mL of DCM was added 3.31 g of tolunenesulfonyl chloride at 0° C. The reaction mixture was stirred at room temperature for 2 days. The reaction mixture was extracted with ethyl acetate. The organic layer was combined and washed with 5% citric acid, water, brine, dried over sodium sulfate and concentrated in vacuo. The residue was purified by flash column chromatography to give 3.5 g of compound 23-1.

[1053] Compound 23-2: To a solution of compound 23-1 (3.5 g, 7.34 mmol) in 20 mL DMF was added solium azide (1.44 g, 22.02 mmol). The reaction mixture was stirred at 50° C. for 2 days. The reaction mixture was extracted with ethyl acetate. The organic layer was washed with water, brine, dried over sodium sulfate, filtered and concentrated in vacuo. The residue was purified by flash column chromatography to give 2.1 g of compound 23-2.

[1054] Compound 23-3: To a solution of compound 23-2 (2.1 g, 6.05 mmol) in 50 mL MeOH was added 400 mg (10%) of Pd—C. The resulting mixture was stirred at room temperature under 1 atm H.sub.2 for 24 hours. The reaction mixture was filtered and concentrated in vacuo to give 2.1 g of compound 23-3. MS (ESI) m/z 322[M+H].

[1055] Compound 23-4: To a solution of compound 23-3 (33 mg, 0.102 mmol), compound 12-7 (40 mg, 0.051 mmol) and 54 μL of diisopropylethylamine in 1 mL of DMF was added 38 mg of HATU. The reaction mixture was stirred at room temperature for 4 hours. The reaction mixture was purified by HPLC to give 52 mg of compound 23-4. MS (ESI) m/z 525[M+2H], 1049[M+H].

[1056] Compound 23-5: Compound 23-4 (52 mg, 0.045 mmol) was dissolved in 5 mL 4N HCl/Dioxane. The reaction mixture was stirred at room temperature for 2 hours and concentrated in vacuo to give 52 mg (100%) of compound 23-5. MS (ESI) m/z 497[M+2H], 993[M+H].

[1057] Compound 23-6: To a solution of compound 19-4 (7.6 mg, 0.017 mmol), compound 23-6 (52 mg, 0.051 mmol) and DIEA (0.030 mL, 0.17 mmol) in 2 mL of DMF was added 32 mg (0.085 mmol) of HATU. The reaction mixture was stirred at room temperature for 2 hour. The reaction mixture was purified by HPLC, eluting with 20-70% CH3CN/H2O in 20 min at 254 nm, to give 26 mg (61%) of compound 23-6. MS (ESI) m/z 583[M+4H], 777[M+3H], 1165[M+2H].

[1058] Compound 23: Compound 23-6 (26 mg, 0.01 mmol) was dissolved in 1 mL of DMF. 10 μL (0.31 mmol) of anhydrous hydrazine was added. The resulting solution was stirred at room temperature for 1 hour. The reaction was quenched with 1N hydrochloride solution. The reaction mixture was purified by preparative HPLC, eluting with 20-70% CH3CN/H2O in 20 min at 254 nm, to give 10 mg (40%) of Example 23. MS (ESI) m/z 550[M+4H], 733[M+3H], 1100[M+2H].

TABLE-US-00002 TABLE 1 Structures of Compounds 1-23 Example Structure  1 [00221]embedded image  2 [00222]embedded image  3 [00223]embedded image  4 [00224]embedded image  5 [00225]embedded image  6 [00226]embedded image  7 [00227]embedded image  8 [00228]embedded image  9 [00229]embedded image 10 [00230]embedded image 11 [00231]embedded image 12 [00232]embedded image 13 [00233]embedded image 14 [00234]embedded image 15 [00235]embedded image 16 [00236]embedded image 17 [00237]embedded image 18 [00238]embedded image 19 [00239]embedded image 20 [00240]embedded image 21 [00241]embedded image 22 [00242]embedded image 23 [00243]embedded image

Example 24: Analysis of HER-Tox Binding to HER2 Receptor

[1059] Her2-Fc was immobilized on CM5 chip to a density of ˜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. 30 sec pulse of 20 mM HCl was used for regeneration. The Bivalent Analyte model was utilize to fit the data (FIG. 1). Analysis of the data indicates that HerTox HA121-NC2D: Kd˜60 pM (chi2=4) and HerTox HA121-NC1D: Kd 300 pM (chi2=14).

Example 25: Transient Transfection

[1060] CHO—S culture is seeded at 0.75×10̂6/mL approximately 16 hours pre-transfection in FreeStyle Cho medium. Cells are ready to transfect the next day when the cell count has reached 1.4-1.6×10̂6/mL. When cells reach target count, 400 mM pAF stock is added to a 1.4 mM final culture concentration. PEI/DNA complex is prepared as described: DNA (1.42 ug/1×10̂6 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 mixture and swirled. The flasks are transferred to a 32° C. incubator. At day 6 post-transfection, a western blot analysis is performed. At day 7 post-transfection, the supernatant is harvested.

Example 26: Anti-Her2 Variant Expression Test

[1061] 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 Fc ELISA. (FIGS. 2 and 3)

Example 27: In Vitro Inhibition of Proliferation Assay

[1062] At day 1, the cells were seeded. The media was aspirated and the T-225 culture flasks of cells was rinsed with 30 mL PBS −/. PBS was aspirated and 6 mL of 0.25% Trypsin-EDTA was added to each flask. The flasks were incubated at 37° C., 5% CO2 for 2-5 minutes. Adherent cells were dislodged by hitting flask and trypsin was neutralized by adding 14 mL culture medium. The cell suspension was mixed and transferred to a 50 mL conical 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 hemacytometer. Cells were seeded at appropriate cell densities into 96-well flat-bottom, clear plates and incubated overnight at 37° C., 5% CO.sub.2 to allow cells to attach. Plating volume was 80 uL/well. 80 uL/well of culture medium was utilized as the “no cell” control.

[1063] Cell plating density examples include: [1064] BT474 (high Her2)—20,000 cells/well in F12k/DMEM (50/50), 10% FBS, P/S [1065] MDA-MB-468 (Her2 negative)—6,000 cells/well in F12k/DMEM (50/50), 10% FBS, P/S [1066] HCC1954—5,000 cells/well in RPMI 1640, 10% FBS, P/S [1067] SKOV-3-6,000 cells/well in RPMI 1640, 10% FBS, P/S [1068] HT29—20,000 cells/well in McCoy's 5A, 10% FBS, P/S

[1069] At day 2, the test samples were added to the cells. The test samples were diluted in culture medium to a 9× stock concentration in Column 2 of a round-bottom 96-well plate. 3× serial dilutions were made from Column 2 to Column 11 with a multi-channel pipettor. 10 uL/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 90 uL/well. 10 uL/well of culture medium was added to avoid edge effects for sample wells. Media Control wells were included in the inner wells where no sample was added (just 10 uL/well of culture medium) to be used in proliferation calculations. The plates were incubated at 37° C., 5% CO.sub.2 for 72 hrs.

[1070] At day 5, proliferation readout occurred. To detect cell proliferation, 10 uL/well of WST-8 reagent was added (Cell Counting Kit-8, cat# CK04-20, Dojindo Labs) to all wells. The plate was incubated for 4 hrs at 37° C., 5% CO.sub.2. The plates were measured for absorbance at OD 450 nm. 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, standard deviation, and % CV of each sample's duplicate % Media Control values; plot each samples average % Media Control value against sample concentration; calculate IC50 values using 4-parameter logistic fit regression analysis to determine potency of test samples.

[1071] FIG. 4 and FIG. 5 illustrate results from the in vitro proliferation assay with dolastatin linker derivatives and breast cancer line HCC1954, HER2+++. FIGS. 6 and 7 illustrate results from the in vitro proliferation assay with dolastatin linker derivatives and ovarian cancer line SKOV-3, HER2+++. FIGS. 8 and 9 illustrate results from the in vitro proliferation assay with dolastatin linker derivatives and breast cancer line MDS-MB-468, HER2 negative.

TABLE-US-00003 TABLE 2 HER-Tox Proliferation Assay Summary: IC.sub.50 Values [nM] after 72 hr drug treatment Data Set I Experiment Date Sep. 13, Sep. 4, 2010 Sep. 4, 2010 Sep. 4, 2010 Sep. 4, 2010 Sep. 4, 2010 Sep. 4, 2010 Sep. 13, 2010 Sep. 13, 2010 2010 HER2 exp. (literature/in house) +++/+++ +++/+++ ?/+++ +++/++++ +++/+++ +++/++++ +++/+++ +++/+++ ? In vivo sensitivity to Herceptin + + − ? − ? + − ? MDA-MB- Sample BT474 HCC1954 LS513 NCI-N87 SKOV-3 ZR-75-30 BT474 SKOV-3 175 Dolastatin 0.1 0.06 0.2 >30 0.2 >30 0.1 0.1 no fit NC-D1 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- 0.2 0.1 >10 >10 (0.04)* >10 0.2 no fit 0.3 NC-D1 Mab HA121- 1.0 0.3 >10 >10 0.3 >10 0.4 no fit 0.7 NC-D2 Mab HA121- PHC-D2 Fab K136pAF >10 >10 >10 >10 >10 >10 Fab K136-NC- 1.8 0.5 >10 >10 2.5 >10 D1 Data Set II Experiment Date Sep. 24, 2010 Sep. 24, 2010 Oct. 8, 2010 Oct. 8, 2010 Oct. 8, 2010 HER2 exp. (literature/in house) ++/? +++/+++ +++/+++ +++/+++ −/? In vivo sensitivity to Herceptin unlikely + + − ? Sample HT29 BT474 HCC1954 SKOV-3 MDA-MB-468 Dolastatin 4   2.5 0.04 0.2 <0.01 NC-D1 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- >10  1 0.2 1.3 >30 NC-D1 Mab HA121- >10    0.8 0.03 (1.3)* >30 NC-D2 Mab HA121-  5* 2 0.1 0.3 (5.8)* PHC-D2 Fab K136pAF Fab K136-NC-D1

TABLE-US-00004 TABLE 3 In vitro Cellular Data ADC, IC50, nM EGFR Small Molecule, IC50, nM C225-HC- C225-NC- Cancer Cell Line KRAS BRAF expression MMD NC-D-1 C-D-1 NC-D-2 C225-NC-D-1 D-1 D-2 Skin A431 wt wt +++ 0.1 8.22 16.54 0.09 0.12 0.19 Colon Colo 205 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 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 Lung A549* mut wt ++ 0.19 6.44 39.82 39.82 >100 >100 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 28: In Vivo Anti-Tumor Efficacy of her2-ADCs in HCC1954 (Human Breast Carcinoma) Xenograft Animal Model

[1072] HCC1954 (human breast carcinoma) cells were obtained from American Type Culture Collection (Manassas, Va.) and cultured in RPMI+10% FBS, 37° C., 5% CO.sub.2 until 80% confluent. Cells were harvested by trypsinization and suspended in PBS at 1×10.sup.8 cells/mL.

[1073] Female, SCID-beige mice, 5-8 weeks old, were obtained from Charles River Laboratories. HCC1954 cells (human, breast carcinoma, ATCC, # CRL-2338) were mixed 1:1 with Matrigel (BD Biosciences, Bedford Mass.) and injected subcutaneously into the mice. When tumors reached an average size of 100-200) mm.sup.3, mice were sorted into groups of 9-10 mice each. Caliper measurements were taken twice weekly until the end of the study. To estimate tumor volume, two orthogonal diameters were measured with calipers and the values entered into the formula, (L×W×W)/2=V, (where W=the shortest diameter, L=the longest diameter and V=volume), to obtain an estimated volume. The tumor volume was converted to tumor weight in the Excel data file by assuming 1 mm.sup.3=1 mg. Endpoint was based on a study design of tumor growth inhibition (TGI). When the mean tumor volume of the control group reached approx. 1,000 mm.sup.3 all mice were euthanized or day 28, whichever came first.

[1074] 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-NCD1 (Ab:Drug ratio=1:2, non-cleavable linker), and Her2-HS122/LK145-HCD1 (Ab:Drug ratio=1:4, cleavable linker) See FIG. 16.

Example 29: In Vivo Studies of her2-Dolastatin Linked Derivative

[1075] HCC1954 cells were utilized for this study with 10.sup.7 cells/mouse in Matrigel, SC in the right flank. Mice were SCID-bg female 4-8 weeks. Grouping was performed at day 5 after cell implantation (tumors ˜100 mm.sup.3): 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 mm.sup.3 or 60 days). (FIG. 10) Paclitaxel 25 mg/kg, IV, qod×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 (cleavable linker) were tested. (FIGS. 11 and 12)

TABLE-US-00005 TABLE 4 Calculation of T/C (Treated/Control) for the HCC1954 study at day 28 Median Tumor Treatment Regimen 1 Volume Group n Agent mg/kg Route Schedule (mm3) T/C .sup. 1.sup.# 10 vehicle — iv qd × 1 486 — 2 10 trastuzumab 3.3 iv qd × 1 405 0.833 3 10 trastuzumab 10 iv qd × 1 446 0.918 4 10 trastuzumab 20 iv qd × 1 385 0.792 5 10 Her-HS122-NC1D-002 3.3 iv qd × 1 40 0.082 6 10 Her-HS122-NC1D-002 10 iv qd × 1 14 0.029 7 10 Her-HS122-NC1D-002 20 iv qd × 1 18 0.037 8 10 Her-HS122/LK145-HC1D-001 3.3 iv qd × 1 40 0.082 9 10 Her-HS122/LK145-HC1D-001 10 iv qd × 1 25 0.051 10  10 Her-HS122/LK145-HC1D-001 20 iv qd × 1 18 0.037 11  10 paclitaxel 25 iv qod × 5 18 0.037

Example 30: Pharmacokinetic Studies

[1076] Assay was performed that detected antibody binding to ErbB2 receptor. (FIG. 13) Assay was performed that detected at least two dolastatins linked to an antibody (FIG. 14).

[1077] FIG. 15.

Example 31: Treatment for Breast Cancer

[1078] Human Clinical Trial of the Safety and/or Efficacy of Trastuzumab-Linked Dolastatin Derivative for Breast Cancer Therapy

[1079] Objective: To compare the safety and pharmacokinetics of administered composition comprising trastuzumab-linked dolastatin derivative.

[1080] 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 dolastatin derivative prior to the study entry. Patients must not have received treatment for their cancer within 2 weeks of beginning the trial. Treatments include the use of chemotherapy, hematopoietic growth factors, and biologic 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.

[1081] Phase I: Patients receive i.v. trastuzumab-linked dolastatin 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 derivative until the maximum tolerated dose (MTD) for trastuzumab-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 (Aug. 9, 2006).

[1082] Phase II: Patients receive trastuzumab-linked dolastatin derivative as in phase I at the MTD determined in phase I. Treatment repeats every 4 weeks for 2-6 courses in the absence of disease progression or unacceptable toxicity. After completion of 2 courses of study therapy, patients who achieve a complete or partial response may receive an additional 4 courses. Patients who maintain stable disease for more than 2 months after completion of 6 courses of study therapy may receive an additional 6 courses at the time of disease progression, provided they meet original eligibility criteria.

[1083] 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 shipped on dry ice.

[1084] Pharmacokinetics: Patients undergo plasma/serum sample collection for pharmacokinetic evaluation before beginning treatment and at days 1, 8, and 15. Pharmacokinetic parameters are calculated by model independent methods on a Digital Equipment Corporation VAX 8600 computer system using the latest version of the BIOAVL software. The following pharmacokinetics parameters are determined: peak serum concentration (C.sub.max); time to peak serum concentration (t.sub.max); area under the concentration-time curve (AUC) from time zero to the last blood sampling time (AUC.sub.0-72) calculated with the use of the linear trapezoidal rule; and terminal elimination half-life (t.sub.1/2), computed from the elimination rate constant. The elimination rate constant is estimated by linear regression of consecutive data points in the terminal linear region of the log-linear concentration-time plot. The mean, standard deviation (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.

[1085] Patient Response to combination therapy: Patient response is assessed 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 similar 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/TherasseRECISTJNCI.pdf). Patients also undergo cancer/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 32: Treatment for Breast Cancer

[1086] Human Clinical Trial of the Safety and Efficacy of Trastuzumab-Linked Dolastatin Derivative for Breast Cancer Therapy

[1087] Objective: Compare the efficacy and toxicity of trastuzumab-linked dolastatin derivative alone followed at disease progression by combination trastuzumab and paclitaxel vs first-line combination trastuzumab and paclitaxel in women with HER2-overexpressing metastatic breast cancer.

[1088] Study Design: This study is a randomized, multicenter study. Patients are stratified according to degree of HER2/neu-overexpression (2+vs 3+), prior anthracycline-containing adjuvant 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 progression, 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.

[1089] Treatment continues in both arms in the absence of disease progression or unacceptable toxicity. Quality of life is assessed at baseline 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 33: Treatment for Bladder Cancer

[1090] Objective: Determine the acute toxicity of paclitaxel and radiotherapy with or without a dolastatin derivative described herein in patients who have undergone prior transurethral bladder resection for muscle-invasive transitional cell carcinoma of the bladder.

[1091] Disease Characteristics: 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, N0, 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 provided 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 transurethral 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.

[1092] Study Design: This study is a non-randomized, multicenter study. Patients are assigned to 1 of 2 treatment groups according to HER2/neu status (HER2/neu 2+ or 3+ staining [group 1] vs HER2/neu 0 or 1+ staining [group 2]).

[1093] Group 1: Patients receive paclitaxel IV over 1 hour on days 1, 8, 15, 22, 29, 36, and 43 and a dolastatin derivative described herein via IV over 90 minutes on day 1 and then over 30 minutes on days 8, 15, 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 toxicity.

[1094] Group 2: Patients receive paclitaxel and undergo radiotherapy 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 34: Treatment for Ovarian Cancer

[1095] Human Clinical Trial of the Safety and Efficacy of aDolastatin Derivative described herein for Ovarian Cancer Therapy

[1096] Objective: Evaluate the safety and efficacy of a four week once weekly IV dosage of composition comprising a dolastatin derivative described herein in women with HER2-overexpressing ovarian cancer.

[1097] 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 derivative will be administered by IV infusion on Study Days 1, 8, 15, and 22. Urine samples will be taken on days 1 and 22.

[1098] Blood Sampling Serial blood is drawn by direct vein puncture before and after administration of the 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 aliquots. All serum samples are stored at −20° C. Serum samples are shipped on dry ice.

[1099] Treatment continues in the absence of disease progression or unacceptable toxicity. Quality of life is assessed at baseline and day 1 of courses 2, 3, 4, 5, 6, 8, 10, and 12. Patients 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 function (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.