Compositions containing non-natural amino acid-linked dolastatin derivatives, use methods and uses thereof

Abstract

To provide 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.SOLUTION: The dolastatin analogs may have a wide range of possible functional groups, 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.SELECTED DRAWING: Figure 4

Description

Detailed Description of the Invention

[0001] [Cross reference] This application is a continuation of U.S. Provisional Application No. 61 / 491,146, filed May 27, 2011. Compositions containing non-natural amino acid-linked dolastatin derivatives, methods using same, and This application claims priority from "Use of the Invention" in accordance with the present application, the entirety of which is incorporated herein by reference. More incorporated.

[0002] BACKGROUND OF THEINVENTION Adding non-genetically encoded amino acids (i.e., "unnatural amino acids") to proteins The ability to incorporate naturally occurring functional groups (e.g., –NH at the ε position of lysine) into 2 , Sistai These provide useful alternatives to the sulfhydryl-SH of amino acids and the imino group of histidine. Some chemical functional groups are genetically encoded. It is inert to the functional groups found in the 20 common amino acids, but is compatible with unnatural amino acids. It reacts efficiently and precisely with the functional groups that can be absorbed to form stable bonds. It is known that:

[0003] Here are 20 common amino acids that are not present in proteins and are genetically encoded: It is chemically inert towards all functional groups present and can be efficiently combined with reagents containing specific functional groups. and selecting chemical functional groups that can be used to selectively react to form stable covalent bonds. This paper provides a method for implementing the

[0004] Summary of the Invention As used herein, a toxic moiety having one or more linkers is linked to an unnatural amino acid. The present invention provides a method for producing such unnatural amino acids and polypeptides. As shown.

[0005] In some embodiments of the present invention, a compound according to formula (I), or a salt thereof, is described. It has been done.

[0006] [ka]

[0007] (In the formula, Z has the structure

[0008] [ka]

[0009] R 5 H,COR 8 , alkyl having 1 to 6 carbon atoms, or thiazole; R 8 is OH or -NH-(alkylene-O) n -NH 2 and R 6 is OH or H, Ar is phenyl or pyridine; R 7 is an alkyl group having 1 to 6 carbon atoms or hydrogen; Y is hydroxylamine, methyl, aldehyde, protected aldehyde, ketone, Protected ketones, thioesters, esters, dicarbonyls, hydrazines, amidines, imines amines, diamines, azides, ketoamines, ketoalkynes, alkynes, cycloalkynes, and , enedione; L is -alkylene-, -alkylene-C(O)-, -(alkylene-O) n -Arki Rene-, -(alkylene-O) n -Alkylene-C(O)-, -(alkylene-O)n - (CH 2 ) n’ -NHC(O)-(CH 2 ) n’’ -C(Me) 2 -SS-(CH 2 ) n’’’ -NHC(O)-(alkylene-O) n’’’’ -Alkylene, -(alkylene -O) n -Alkylene-W-, -Alkylene-C(O)-W-, -(Alkylene-O) n -alkylene-U-alkylene-C(O)-, and -(alkylene-O) n -Arki is a linker selected from the group consisting of: aryl-U-alkylene; W has the structure

[0010] [ka]

[0011] U has the structure

[0012] [ka]

[0013] Or, L is absent, Y is methyl and R 5 is COR 8 and R 8 -NH(A alkylene-O) n -NH 2 where n, n', n'', n''', and n'''' are , each independently an integer of 1 or greater.) In some embodiments, R 5 is thiazole. In other embodiments, R 6 is H In some embodiments, Ar is phenyl. In terms of form, R 7 In some embodiments, n is 0 to 20, 0 to 10. , or an integer from 0 to 5.

[0014] In some embodiments, compounds according to formula (II) are described.

[0015] [ka]

[0016] In certain embodiments, L is -(alkylene-O). n -alkylene-. In one embodiment, each alkylene is -CH 2 CH 2 -, n=3, R 7 is methyl In other embodiments, L is -alkylene-. In certain embodiments, each of the above Alkylene is -CH 2 CH 2 - and R 7 is methyl or hydrogen. In embodiments, L is -(alkylene-O). n -alkylene-C(O). In this embodiment, each alkylene is -CH 2 CH 2 -, n=4, R 7 is methyl In further or additional embodiments, L is -(alkylene-O). n -(CH 2 ) n’ -NHC(O)-(CH 2 ) n’’ -C(Me) 2 -SS-(CH 2 ) n’’ ’-NHC(O)-(alkylene-O) n’’’’ -alkylene. In the form, each alkylene is -CH 2 CH 2 -, n is 1 and n' is 2 where n'' is 1, n''' is 2, n'''' is 4, and R 7 is methyl be.

[0017] In some embodiments, Y is azide. In other embodiments, Y is cyclooctyl. In certain embodiments, the cyclooctyne has the structure:

[0018] [ka]

[0019] (In the formula, Each R 19 are independently selected from alkyl having 1 to 6 carbon atoms, alkoxy having 1 to 6 carbon atoms, ester alkyl, ether, thioether, aminoalkyl, halogen, alkyl ester, aryl Esters, amides, arylamides, alkyl halides, alkylamines, alkyls Sulfonic acid, alkyl nitro, thioester, sulfonyl ester, halosulfonyl, nitro is selected from the group consisting of alkyl, alkylnitrile, and nitro; q is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11. In some embodiments of the present invention, the compound represented by formula (II), (III), (IV), (V), or The compound represented by (VI) or a salt thereof is described.

[0020] [ka] JPEG2024170587000009.jpg115169

[0021] where Z has the structure:

[0022] [ka]

[0023] R 5 H,COR 8 , alkyl having 1 to 6 carbon atoms, or thiazole; R 8 is OH, Ar is phenyl or pyridine; R 7 is an alkyl group having 1 to 6 carbon atoms or hydrogen; Y and V are hydroxylamine, methyl, aldehyde, and protected aldehyde, respectively. Dehydes, ketones, protected ketones, thioesters, esters, dicarbonyls, hydrazines Amidines, imines, diamines, azides, ketoamines, ketoalkynes, alkynes, cyclohexanes selected from the group consisting of alkynes, enediones, and L 1 , L 2 , L 3 , and L 4 each independently represents a bond, -alkylene-, -(a alkylene-O) n -Alkylene-J-, -Alkylene'-J-(Alkylene-O) n -Alkylene -, -J-(alkylene-O) n -Alkylene-, -(alkylene-O) n -Alkylene-J-( Alkylene-O) n '-Alkylene-J'-, -(Alkylene-O) n -Alkylene-J-Alkyl Alkylene'-, -W-, -Alkylene-W-, Alkylene'-J- (Alkylene-NMe)n -Alkire -W-, -J-(Alkylene-NMe) n -Alkylene-W-, -J-Alkylene-NMe-Alkylene -Alkylene'-NMe-alkylene''-W-, and -Alkylene-J-alkylene'-NMe-alkylene a linker selected from the group consisting of NMe-N-alkylene-NMe-, W has the structure

[0024] [ka]

[0025] Each J and J′ independently has the structure

[0026] [ka]

[0027] Each n and n' is independently an integer of 1 or greater.

[0028] In certain embodiments, compounds comprising formula (VII) are described.

[0029] [ka]

[0030] In one embodiment, L 1 H-(alkylene-O) n -alkylene-J-, L 2 Ha -A Alkylene '-J'-(alkylene-O) n '-Alkylene-, L 3 is -J''-(alkylene-O) n ' '-alkylene-, where alkylene is -CH 2 CH 2 - and alkylene' is -(CH 2 ) 4 - and n is 1, n' and n'' are 3; J has the structure

[0031] [ka]

[0032] J′ and J″ have the following structure:

[0033] [ka]

[0034] R 7 is methyl. In another embodiment, L 1 is -J-(alkylene-O) n -Arki Ren-Are, L 2 H-(alkylene-O) n' -alkylene-J'-alkylene'-, L 3 H-(alkylene-O) n'' -alkylene-J''-, where alkylene is -CH 2 CH 2 - and alkylene' is -(CH 2 ) 4 - where n is 1 and n' and n'' are 4. and J, J', and J'' have the following structures:

[0035] [ka]

[0036] In one embodiment, Y is azide. In another embodiment, Y is cyclooctyne. In certain embodiments, the cyclooctyne has the structure:

[0037] [ka]

[0038] (In the ceremony Each R 19 are independently selected from alkyl having 1 to 6 carbon atoms, alkoxy having 1 to 6 carbon atoms, ester alkyl, ether, thioether, aminoalkyl, halogen, alkyl ester, aryl Esters, amides, arylamides, alkyl halides, alkylamines, alkyls Sulfonic acid, alkyl nitro, thioester, sulfonyl ester, halosulfonyl, nitro is selected from the group consisting of alkyl, alkylnitrile, and nitro; q is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11. In one embodiment of the present invention, a compound according to formula (VIII) or (IX) The active metabolites, pharma- ceutically acceptable prodrugs, or solvates thereof are described. .

[0039] [ka]

[0040] (In the formula, A is optional and, when present, is lower alkylene, substituted lower alkylene, lower cycloalkylene, cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, Alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene aryl, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroaryl aryl, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted heteroaryl. is a substituted aralkylene; B is optional and, when present, is lower alkylene, substituted lower alkylene, lower alkoxy, aryl, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene , or substituted alkylene)-, -S(O) k - (where k is 1, 2, or 3) -S(O) k (Alkylene or substituted alkylene)-, -C(O)-, -C (O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(a -N(R')-, -NR'-(alkylene, or substituted alkylene)-, -N(R')-, -NR'-(alkylene, or 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 Ren)-, -N(R')C(O)O-, -S(O) k N(R')-, -N(R')C(O )N(R')-, -N(R')C(S)N(R')-, -N(R')S(O) k N(R' )-, -N(R')-N=, -C(R')=N-, -C(R')=NN(R')-, - C(R') = NN =, -C(R') 2 -N=N- and -C(R') 2 -N(R' )-N(R')-, wherein each R' is independently , H, alkyl, or substituted alkyl; R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl. can be, R 1is H, an amino protecting group, a resin, at least one amino acid, a polypeptide, or , a polynucleotide, R 2 can be any combination of OH, ester protecting groups, resin, at least one amino acid, polypeptide, or or a polynucleotide, R 3 and R 4 are each independently H, halogen, lower alkyl, or substituted lower alkyl. alkyl or R 3 and R 4 , or two R 3 The group is a cycloalkyl group. alkyl or heterocycloalkyl, Z has the structure

[0041] [ka]

[0042] R 5 H,COR 8 , alkyl having 1 to 6 carbon atoms, or thiazole; R 8 is OH, R 6 is OH or H, Ar is phenyl or pyridine; R 7 is an alkyl group having 1 to 6 carbon atoms or hydrogen; L is -alkylene-, -alkylene-C(O)-, -(alkylene-O) n -Arki Rene-, -(alkylene-O) n -Alkylene-C(O)-, -(alkylene-O) n - (CH 2 ) n’ -NHC(O)-(CH 2 ) n’’ -C(Me) 2 -SS-(CH2 ) n’’’ -NHC(O)-(alkylene-O) n’’’’ -Alkylene, -(alkylene -O) n -Alkylene-W-, -Alkylene-C(O)-W-, -(Alkylene-O) n -Alkylene-UC(O)-, and -(alkylene-O) n -Alkylene-U-A is a linker selected from the group consisting of alkylene, W has the structure

[0043] [ka]

[0044] U has the structure

[0045] [ka]

[0046] n, n', n'', n''', and n'''' are each independently an integer equal to or greater than 1. It is a number.) In some embodiments, R 1 is a polypeptide. In certain embodiments, the polypeptide The polypeptide is an antibody. In certain embodiments, the antibody is Herceptin. In another embodiment, R 2 In certain embodiments, the polypeptide The tide is an antibody. In certain embodiments, the antibody is Herceptin.

[0047] In one embodiment of the present invention, a compound represented by formula (X), (XI), (XII) or (XIII) is As indicated, compounds or salts thereof are described.

[0048] [ka] JPEG2024170587000023.jpg169169

[0049] (In the formula, A is optional and, when present, is lower alkylene, substituted lower alkylene, lower cycloalkylene, cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, Alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene aryl, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroaryl aryl, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted heteroaryl. is a substituted aralkylene; B is optional and, when present, is lower alkylene, substituted lower alkylene, lower alkoxy, aryl, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene , or substituted alkylene)-, -S(O) k - (where k is 1, 2, or 3) -S(O) k (Alkylene or substituted alkylene)-, -C(O)-, -C (O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(a -N(R')-, -NR'-(alkylene, or substituted alkylene)-, -N(R')-, -NR'-(alkylene, or 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 Ren)-, -N(R')C(O)O-, -S(O) k N(R')-, -N(R')C(O )N(R')-, -N(R')C(S)N(R')-, -N(R')S(O) k N(R' )-, -N(R')-N=, -C(R')=N-, -C(R')=NN(R')-, - C(R') = NN =, -C(R') 2 -N=N- and -C(R') 2 -N(R' )-N(R')-, wherein each R' is independently , H, alkyl, or substituted alkyl; R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl. can be, R 1 is H, an amino protecting group, a resin, at least one amino acid, a polypeptide, or , a polynucleotide, R 2 can be any combination of OH, ester protecting groups, resin, at least one amino acid, polypeptide, or or a polynucleotide, R 3 and R 4 are each independently H, halogen, lower alkyl, or substituted lower alkyl. alkyl or R 3 and R 4 , or two R 3 The group is a cycloalkyl group. alkyl or heterocycloalkyl, Z has the structure

[0050] [ka]

[0051] R 5 H,COR 8, alkyl having 1 to 6 carbon atoms, or thiazole; R 8 is OH, R 6 is OH or H, Ar is phenyl or pyridine; R 7 is an alkyl group having 1 to 6 carbon atoms or hydrogen; L 1 , L 2 , L 3 , and L 4 each independently represents a bond, alkylene, -(alkyl Ren-O) n -Alkylene-J-, -Alkylene'-J-(Alkylene-O) n -alkylene-, -J-(alkylene-O) n -Alkylene-, -(alkylene-O) n -Alkylene-J-(Al Kiren-O) n '-Alkylene-J'-, -(Alkylene-O) n -alkylene-J-alkylene' -, -W-, -Alkylene-W-, Alkylene'-J-(Alkylene-NMe) n -Alkylene-W -, -J-(alkylene-NMe) n -Alkylene-W-, -J-Alkylene-NMe-Alkylene' -NMe-alkylene''-W-, and -alkylene-J-alkylene''-NMe-alkylene'' -NMe-alkylene''-W-; W has the structure

[0052] [ka]

[0053] Each J and J′ independently has the structure

[0054] [ka]

[0055] Each n and n' is independently an integer of 1 or greater.

[0056] In some embodiments, R 1 is a polypeptide. In certain embodiments, the polypeptide The polypeptide is an antibody. In certain embodiments, the antibody is Herceptin. In another embodiment, R 2 In certain embodiments, the polypeptide The tide is an antibody. In certain embodiments, the antibody is Herceptin.

[0057] In certain embodiments, the compound represented by formula (I), (III), (IV), (V), or (VI) 1. A method for derivatizing a dolastatin analog comprising: The method includes contacting the dolastatin analog with a reagent of formula (XXXVII): Including, Formula (I), (III), (IV), (V) or (VI) corresponds to:

[0058] [ka] JPEG2024170587000028.jpg185169

[0059] (In the formula, Z has the structure

[0060] [ka]

[0061] R 5 H,COR 8, alkyl having 1 to 6 carbon atoms, or thiazole; R 8 is OH or -NH-(alkylene-O) n -NH 2 and R 6 is OH or H, Ar is phenyl or pyridine; R 7 is an alkyl group having 1 to 6 carbon atoms or hydrogen; Y is NH 2 -O- or methyl; L, L 1 , L 2 , L 3 , and L 4 each independently represents a bond, -alkylene-, - Alkylene-C(O)-, -(alkylene-O) n -Alkylene-, -(alkylene-O ) n -Alkylene-C(O)-, -(alkylene-O) n -(CH 2 ) n’ -NHC(O )-(CH 2 ) n’’ -C(Me) 2 -SS-(CH 2 ) n’’’ -NHC(O)-( Alkylene-O) n’’’’ -Alkylene, -(alkylene-O) n -Alkylene-W- , -alkylene-C(O)-W-, -(alkylene-O) n -Alkylene-J-, -Alkylene '-J-(alkylene-O) n -Alkylene-, -(alkylene-O) n -Alkylene-J-Al alkylene'-, -J-(alkylene-O) n -Alkylene-, -(alkylene-O) n -Alkylene -J-(alkylene-O) n'-Alkylene-J'-, -W-, -Alkylene-W-, Alkylene' -J-(alkylene-NMe) n -Alkylene-W-, and -J- (alkylene-NMe) n -Arki olefin-W-,-(alkylene-O) n -Alkylene-U-Alkylene; -J-Alkylene- NMe-alkylene'-NMe-alkylene''-W-, and -alkylene-J-alkylene'-NM e-alkylene''-NMe-alkylene''-W-; W has the structure

[0062] [ka]

[0063] U has the structure

[0064] [ka]

[0065] Each J and J′ independently has the structure

[0066] [ka]

[0067] Or, L is absent, Y is methyl and R 5 is COR 8 and R 8 Ha-NH(A alkylene-O) n -NH 2 where n, n', n'', n''', and n'''' are , each independently an integer of 1 or greater.) Formula (XXXVII) corresponds to the following, methods of which are described herein:

[0068] [ka]

[0069] (In the formula, A is optional and, when present, is lower alkylene, substituted lower alkylene, lower alkoxy, aryl, substituted lower alkenylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aryl It is Ralkyren, B is optional and, when present, is lower alkylene, substituted lower alkylene, lower alkoxy, substituted lower alkenylene, -O-, -O-(alkylene, or substituted alkylene -, -S-, -S-(alkylene or substituted alkylene)-, -S(O) k -( where k is 1, 2, or 3), -S(O) k (Alkylene or substituted alkyl -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-(a alkylene or substituted alkylene)-, -N(R')C(O)O-, -S(O) k N( R')-, -N(R')C(O)N(R')-, -N(R')C(S)N(R')-, - N(R')S(O) k N(R')-, -N(R')-N=, -C(R')=N-, -C( R')=NN(R')-, -C(R')=NN=, -C(R') 2 -N=N-, and and -C(R') 2 -N(R')-N(R')- (wherein each R' is independently H, alkyl, or substituted alkyl. Each R' is , independently H, alkyl, or substituted alkyl; K is

[0070] [ka]

[0071] and R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl. can be, R 1 is H, an amino protecting group, a resin, at least one amino acid, or a polynucleotide Chid, R 2 is an OH, an ester protecting group, a resin, at least one amino acid, or a polynucleotide It is leotide, R 3 and R 4 are each independently H, halogen, lower alkyl, or substituted lower alkyl or R 3 and R 4 , or two R 3 The group is a cycloa The aryl group may optionally form an alkyl, alkyl, or heterocycloalkyl. In some embodiments, the derivatized dolastatin analog has the formula (VIII ), (IX), (X), (XI), (XII), or (XIII): It is an amino acid that contains at least one oxime.

[0072] [ka] JPEG2024170587000036.jpg176169 JPEG2024170587000037.jpg169169

[0073] In certain embodiments, the dolastatin analog is represented by formula (XXXVII): The reagent is contacted in an aqueous solution under weakly acidic conditions.

[0074] In one embodiment of the present invention, the compound of formula (XXV), (XXVI), (XXVII), (XXV The compounds shown in formula (III), (XXIX), or (XXX) are described.

[0075] [ka] JPEG2024170587000039.jpg224169 JPEG2024170587000040.jpg103169

[0076] (In the formula, Z has the structure

[0077] [ka]

[0078] R 5 H, CO 2 H, alkyl having 1 to 6 carbon atoms, or thiazole; R 6 is OH or H, Ar is phenyl or pyridine; R 1 is H, an amino protecting group, a resin, at least one amino acid, a polypeptide, or , a polynucleotide, R 2 can be any combination of OH, ester protecting groups, resin, at least one amino acid, polypeptide, or or a polynucleotide, R 4 is H, halogen, lower alkyl, or substituted lower alkyl; R 7 is an alkyl group having 1 to 6 carbon atoms or hydrogen; L, L 1 , L 2 , L 3 , and L 4 each independently represents a bond, -alkylene-, - Alkylene-C(O)-, -alkylene-J-, -(alkylene-O) n -Alkylene- , -(alkylene-O) n -Alkylene-C(O)-, -(alkylene-O) n -J-, -( Alkylene-O) n -J-alkylene, -(alkylene-O) n -(CH 2 ) n’ -NHC (O)-(CH 2 ) n’’ -C(Me) 2 -SS-(CH 2 ) n’’’ -NHC(O) -(alkylene-O) n’’’’ -Alkylene, -(alkylene-O) n -Alkylene- W-, -alkylene-C(O)-W-, -(alkylene-O) n -Alkylene-J-, -Al alkylene'-J-(alkylene-O) n -Alkylene-, -(alkylene-O) n -Alkylene-J -Alkylene'-, -J-(alkylene-O) n -Alkylene-, -(alkylene-O) n -Al alkylene-J-(alkylene-O)n '-Alkylene-J'-, -W-, -Alkylene-W-, Alkylene Alkylene'-J-(Alkylene-NMe) n -Alkylene-W-, -J-(Alkylene-NMe) n -Arki ylene-W-, -(alkylene-O) n -Alkylene-UC(O)-, -(alkylene- O) n -Alkylene-U-alkylene;-J-alkylene-NMe-alkylene'-NMe-alkylene -alkylene'-W-, and -alkylene-J-alkylene'-NMe-alkylene'-NMe-alkenyl is a linker selected from the group consisting of: xylene'''-W-; W has the structure

[0079] [ka]

[0080] U has the structure

[0081] [ka]

[0082] Each J and J′ independently has the structure

[0083] [ka]

[0084] n and n' are each independently an integer of 1 or greater; Each R 16 are independently hydrogen, halogen, alkyl, NO 2 , CN, and substituted alkenes (A) is selected from the group consisting of In some embodiments, R 1is a polypeptide. In certain embodiments, the polypeptide The polypeptide is an antibody. In certain embodiments, the antibody is Herceptin. In another embodiment, R 2 In certain embodiments, the polypeptide The tide is an antibody. In certain embodiments, the antibody is Herceptin.

[0085] In some embodiments of the present invention, the compound represented by formula (XXXI), (XXXII), (XXXIII) ), (XXXIV), (XXXV), or (XXXVI) is described. It is being done.

[0086] [ka] JPEG2024170587000046.jpg195169 JPEG2024170587000047.jpg93169

[0087] (In the formula, Z has the structure

[0088] [ka]

[0089] R 5 H, CO 2 H, alkyl having 1 to 6 carbon atoms, or thiazole; R 6 is OH or H, Ar is phenyl or pyridine; R 1 is H, an amino protecting group, a resin, at least one amino acid, a polypeptide, or , a polynucleotide, R 2can be any combination of OH, ester protecting groups, resin, at least one amino acid, polypeptide, or or a polynucleotide, R 4 is H, halogen, lower alkyl, or substituted lower alkyl; R 7 is an alkyl group having 1 to 6 carbon atoms or hydrogen; L, L 1 , L 2 , L 3 , and L 4 each independently represents a bond, -alkylene-, - Alkylene-C(O)-, -alkylene-J-, -(alkylene-O) n -Alkylene- , -(alkylene-O) n -Alkylene-C(O)-, -(alkylene-O) n -J-, -( Alkylene-O) n -J-alkylene, -(alkylene-O) n -(CH 2 ) n’ -NHC (O)-(CH 2 ) n’’ -C(Me) 2 -SS-(CH 2 ) n’’’ -NHC(O) -(alkylene-O) n’’’’ -Alkylene, -(alkylene-O) n -Alkylene- W-, -alkylene-C(O)-W-, -(alkylene-O) n -Alkylene-J-, -Al alkylene'-J-(alkylene-O) n -Alkylene-, -(alkylene-O) n -Alkylene-J -Alkylene'-, -J-(alkylene-O) n -Alkylene-, -(alkylene-O) n -Al alkylene-J-(alkylene-O) n '-Alkylene-J'-, -W-, -Alkylene-W-, Alkylene Alkylene'-J-(Alkylene-NMe) n -Alkylene-W-, -J-(Alkylene-NMe) n -Arki ylene-W-, -(alkylene-O) n -Alkylene-UC(O)-, -(alkylene- O) n -Alkylene-U-alkylene;-J-alkylene-NMe-alkylene'-NMe-alkylene -alkylene'-W-, and -alkylene-J-alkylene'-NMe-alkylene'-NMe-alkenyl is a linker selected from the group consisting of: xylene'''-W-; W has the structure

[0090] [ka]

[0091] U has the structure

[0092] [ka]

[0093] Each J and J′ independently has the structure

[0094] [ka]

[0095] n and n' are each independently an integer of 1 or greater; D has the structure

[0096] [ka]

[0097] Each R 17are independently H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, Alkynyl, substituted alkynyl, alkoxy, substituted alkoxy, alkylalkoxy, substituted Alkylalkoxy, polyalkylene oxide, substituted polyalkylene oxide, aryl aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkaryl, substituted alkali Aralkyl, substituted aralkyl, -(alkylene or substituted alkylene)-ON (R”) 2 , -(alkylene or substituted alkylene)-C(O)SR", -(alkyl alkylene or substituted alkylene)-SS-(aryl or substituted aryl) , -C(O)R”, -C(O) 2 R” or -C(O)N(R”) 2 From the group consisting of wherein each R" is independently selected from hydrogen, alkyl, substituted alkyl, alkenyl, substituted Substituted alkenyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl , alkaryl, substituted alkaryl, aralkyl, or substituted aralkyl; each Z 1 is a bond, CR 17 R 17 ,O,S,NR',CR 17 R 17 -CR 17 R 17 , C.R. 17 R 17 -O, O-CR 17 R 17 , C.R. 17 R 17 -S, S-CR 17 R 17 , C.R. 17 R 17 -NR' or NR'-CR 17 R 17 and each R' is H, alkyl, or substituted alkyl; each Z 2 is a bond, -C(O)-, -C(S)-, or an optionally substituted alkyl group having 1 to 3 carbon atoms. alkylene, optionally substituted alkenylene having 1 to 3 carbon atoms, and optionally substituted heptylene. arylalkyl; each Z 3 is a bond, an optionally substituted alkylene having 1 to 4 carbon atoms, an optionally substituted Alkenylene having 1 to 4 carbon atoms, optionally substituted heteroalkyl, -O-, -S-, -C(O )-, -C(S)-, and -N(R')-; Each T 3 is a bond, C(R")(R"), O, or S (where T 3 But, O, or when S, R″ is not a halogen; Each R" is H, halogen, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl. It is a chloroalkyl, m and p are 0, 1, 2, or 3 (provided that at least one of m or p is one of which is not 0), M 2 teeth,

[0098] [ka]

[0099] (wherein (a) represents a bond to the B group, and (b) represents a bond to the heterocyclic group, respectively) (indicating a bond at the position of M 3 teeth,

[0100] [ka]

[0101] (wherein (a) represents a bond to the B group, and (b) represents a bond to the heterocyclic group, respectively) (indicating a bond at the position of M 4 teeth,

[0102] [ka]

[0103] where (a) represents the bond to the B group, and (b) represents the bond to the heterocyclic group, respectively. (indicating the bond at each position) Each R 19 are independently selected from alkyl having 1 to 6 carbon atoms, alkoxy having 1 to 6 carbon atoms, ester alkyl, ether, thioether, aminoalkyl, halogen, alkyl ester, aryl Esters, amides, arylamides, alkyl halides, alkylamines, alkyls Sulfonic acid, alkyl nitro, thioester, sulfonyl ester, halosulfonyl, nitro is selected from the group consisting of alkyl, alkylnitrile, and nitro; q is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11; Each R 16 are independently hydrogen, halogen, alkyl, NO 2 , CN, and substituted alkenes (A) is selected from the group consisting of In some embodiments, R 1 is a polypeptide. In certain embodiments, the polypeptide The polypeptide is an antibody. In certain embodiments, the antibody is Herceptin. In another embodiment, R 2 In certain embodiments, the polypeptide The tide is an antibody. In certain embodiments, the antibody is Herceptin.

[0104] In some embodiments, compounds according to formula (XXXI-A) are described.

[0105] [ka]

[0106] In one embodiment, any of the above compounds may be used in combination with a pharma- ceutically acceptable carrier, excipient, or describes a pharmaceutical composition comprising a binder.

[0107] In further or alternative embodiments, detecting the presence of a polypeptide in a patient. The method includes the steps of: and administering to the subject the heterocycle-containing non-natural amino acid polypeptide. The method of the present invention is to alter the immunogenicity of a peptide relative to its cognate natural amino acid.

[0108] The methods and compositions described herein include the methodology, protocols, cell lines, It is understood that the formulas, compositions, and reagents are not limited and may vary. The terminology used herein is for the purpose of describing particular embodiments only and is defined in the appended claims. This does not limit the scope of the methods and compositions described herein, which are limited only by the It is understood that this is not the case.

[0109] As used in this specification and the appended claims, terms described in the singular This includes cases where the term is used in the plural, regardless of whether it is first or previously mentioned, but the singular is used if the context dictates otherwise. This does not apply when it is clear that the scope of the present invention refers only to the scope described in the above.

[0110] Unless otherwise defined, all technical and scientific terms used herein are to which the present invention pertains. It is intended to have the same meaning as that commonly understood by a person skilled in the art. Methods, apparatus, and materials for use in practicing or testing the inventions described herein Any methods, apparatus, and materials similar or equivalent to those described herein are also contemplated. Methods, devices, and materials can be used, but the following describes preferred methods, devices, and materials. .

[0111] The publications and patent documents cited herein are for illustrative purposes only and are not intended to be limiting unless otherwise specified. This document describes the constructs and methodologies. The above publications and patent documents are hereby incorporated by reference in their entireties. It is noted that the publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Any statements herein are not intended to be construed as a representation that the inventors described herein have made any such claims within the scope of their disclosures. If the applicant is not recognized as having the status of an inventor who made an invention prior to the invention because of prior art or for other reasons, This does not endorse the idea that such a thing is possible.

[0112] The term "aldol-based linkage" or "mixed aldol-based linkage" refers to a The enolate of a carbonyl compound may be the same or different from the enolate of another carbonyl compound. / Enols and β-hydroxycarbonyl compounds (β-hydroxycarbonyl compounds) by acid- or base-catalyzed condensation This refers to the production of a diol (i.e., an aldol).

[0113] As used herein, the term "affinity label" refers to a label that can be reversibly or or irreversibly binds to and modifies or destroys it, or forms a compound with it. As an example, an affinity label may be an enzyme and its substrate, or an antibody and and antigens thereof.

[0114] The terms "alkoxy," "alkylamino," and "alkylthio" (or thioalkenyl) koxy) is used in its conventional sense and refers to a bond between an oxygen atom, an amino group, or a sulfur atom and a molecule. Each refers to a chain of alkyl groups.

[0115] Unless otherwise stated, the term "alkyl," either alone or as part of another molecule, refers to a fully saturated , monounsaturated, or polyunsaturated and has a specified number of carbon atoms (i.e., C 1 ~C 10 means 1 to 10 carbons) and may include divalent and polyvalent groups. It refers to linear, branched, or cyclic hydrocarbon groups, or combinations thereof, that are saturated with Examples of hydride radicals are methyl, ethyl, n-propyl, isopropyl, n-butyl, t -butyl, isobutyl, sec-butyl, cyclohexyl, (cyclohexyl)methyl, and cyclopropylmethyl, as well as, for example, n-pentyl, n-hexyl, n-heptyl. and homologs and isomers such as n-octyl. An unsaturated alkyl group is one that has one or more double or triple bonds. Examples of alkyl groups include vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-( butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl , 1-propynyl, 3-propynyl, 3-butynyl, higher homologs, and higher isomers. The term "alkyl" is also used herein, unless otherwise specified. Derivatives of alkyl groups, such as "heteroalkyl", "haloalkyl", and "Alkyl" is intended to include all radicals, such as "alkyl" and "homoalkyl."

[0116] The term "alkylene" alone or as part of another molecule means (-CH 2 -) n (n is 1~ Divalent radicals derived from alkanes such as those exemplified by: By way of example, such a group is -CH 2 CH 2 - and -CH 2 CH 2 C H 2 CH 2 -, etc., "Lower alkyl" or "lower alkylene" generally refers to a group having eight or fewer carbon atoms. The term "alkylene" also refers to any alkyl or alkylene group that is longer than 1 or 2 unless otherwise specified. Unless otherwise specified, it is intended to include groups described herein as "heteroalkylene." do.

[0117] The term "amino acid" refers to natural and unnatural amino acids, as well as amino acids that function similarly to the natural amino acids. This refers to amino acid analogs and amino acid mimetics that are naturally encoded amino acids. The amino acids are made up of 20 common amino acids (alanine, arginine, asparagine, Glycine, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine , leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine), pyrrolidine, and selenocysteine. Amino acid analogues refer to compounds whose basic chemical structure is identical to that of natural amino acids. Examples of the α-carbon bonded to hydrogen, carboxyl groups, amino groups, and R groups are These amino acid analogs have the same basic chemical structure as natural amino acids, but with modified R groups (e.g., norleucine) or modified peptide backbone Non-limiting examples of amino acid analogs include homoserine, norleucine, Methionine, methionine sulfoxide, and methionine methylsulfonium.

[0118] In this specification, amino acids may be referred to by their names or by the IUPAC -IUB Biochemical Nomenclature C It may also be indicated by a three-letter or one-letter abbreviation recommended by the EPA. Nucleotides are also referred to by their commonly accepted single-letter codes. There is a saying.

[0119] "Amino terminal modification group" refers to any molecule capable of being attached to a terminal amine group. As such, such terminal amine groups are useful in the preparation of polymers (polypeptides, polynucleotides, and The terminal modification group may be located at the end of a polysaccharide (including, but not limited to, a polysaccharide). The polypeptides include, but are not limited to, various water-soluble polymers, peptides, or proteins. In one example, the terminal modification group includes polyethylene glycol or serum albumin. End modification groups may be used to modify the therapeutic properties of the polymer. These include, but are not limited to, increasing the serum half-life of

[0120] "Antibody fragment" refers to an antibody in any form other than the full-length form. These include antibodies that are smaller elements present in full-length antibodies, as well as antibodies that have been modified. The antibody fragments include Fv, Fc, Fab, (Fab')2, single chain Fv (scFv), diabody, Triabodies, tetrabodies, bifunctional hybrid antibodies, CDR1, CDR2, CDR 3. CDR combination, variable region, framework region, constant region, heavy chain, light chain, variable These include, but are not limited to, antibody domains, alternative scaffold non-antibody molecules, and bispecific antibodies. No (Maynard & Georgiou, 2000, Annu. Rev. Biomed. Eng. 2:339-76; Hudson, 1998, Curr. Opin. Biotechnol. 9:395-402). Other functional substructures are linked by peptide linkers. A single-chain Fv ( scFv) (Sz Hu et al., 1996, Cancer Research, 56, 3055-3061). These small (Mr 25,000) proteins are antigenic in a single polypeptide. Since the specificity and affinity for the target antigen are maintained, it is possible to construct larger, more antigen-specific molecules. Except where otherwise noted, Any description or claim using the term "antigen" (singular or plural) may be construed as including "antigenic fragments" (singular or plural). This specifically includes the plural forms.

[0121] As used herein, "antibody-drug conjugates" or "ADCs" refer to A “body-drug conjugate” is a drug that is covalently linked to one or more biologically active molecules. The term "biologically active molecule" refers to an antibody molecule or fragment thereof that is It may be attached to the antibody via other covalent bonds.

[0122] As used herein, the term "aromatic" or "aryl" refers to a bonded pi-electron system. It refers to a closed ring structure having at least one ring, including carbocyclic aryl groups and heterocyclic aryl groups. Heteroaryl groups (or "heteroaryl groups" or "heteroaromatic groups") A carbocyclic or heterocyclic aromatic group contains from about 5 to about 20 ring atoms. The above terms refer to monocyclic rings or polycyclic fused rings (i.e., adjacent rings) that are covalently linked. Aromatic groups include cyclic groups sharing a pair of carbon atoms. Aromatic groups can be unsubstituted. can also be substituted aromatic groups. Non-limiting examples of "aromatic" or "aryl" groups are , phenyl, 1-naphthyl, 2-naphthyl, 4-biphenyl, anthracenyl, and furanyl. Substituents for each of the above-mentioned aryl and heteroaryl ring systems include phenanthracenyl. is selected from the group of acceptable substituents described herein.

[0123] Briefly, the term "aryl" is intended to mean any of the other terms (aryloxy, arylthiox, etc.) When used in combination with alkyl, alkyl groups, including but not limited to alkyl, aryl ... It includes both aryl and heteroaryl rings as defined above. The term "aralkyl" or "alkaryl" refers to an alkyl group (e.g., benzyl, phenethyl, and pyridylmethyl) to which an aryl group is bonded The alkyl group is intended to include radicals having a carbon atom (methyl) and a methyl group (methylphenyl). a heteroatom (such as, but not limited to, an oxygen atom) Examples of such aryl groups include phenoxymethyl, 2-pyridyl, lysyloxymethyl, and 3-(1-naphthyloxy)propyl. Not limited.

[0124] As used herein, the term "arylene" refers to a divalent aryl radical. Non-limiting examples of "arylene" include phenylene, pyridinylene, pyrimidinylene, and thiophene. Arylene group substituents are selected from the permissible substituents described herein. is selected from the group.

[0125] "Bifunctional polymers," also called "bifunctional linkers," are polymers that react specifically with other moieties. It refers to a polymer that contains two functional groups capable of forming a covalent or non-covalent bond with each other. Such moieties may be the side chain groups of natural or unnatural amino acids or the side chains of natural or unnatural amino acids. The side chain groups of the peptides containing bifunctional linkers or The other moieties that can be attached to the polymer can be the same or different moieties. Thus, a bifunctional linker has a functional group reactive to a group on a first peptide and a functional group reactive to a group on a second peptide. and another functional group reactive to the group of the first peptide, thereby The complex comprises a functional linker and the second peptide. Numerous techniques and binding molecules for binding to tides are known. No. 188,256, U.S. Pat. No. 4,671,958, U.S. Pat. No. 4,659,839 No. 4,414,148, U.S. Pat. No. 4,699,784, U.S. Pat. See U.S. Patent Nos. 4,569,789 and 680,338. and US Pat. No. 5,993,333, the entire contents of which are incorporated herein by reference. Also called "multifunctional linkers," these contain two or more functional groups that can react with other moieties. Such moieties refer to polymers that form covalent or non-covalent bonds. contains a side chain group of an unnatural amino acid or a side chain group of a peptide containing a natural or unnatural amino acid. Bifunctional (including but not limited to the side chain groups of amino acids) The functional or multifunctional polymer may be of any desired length or molecular weight, A molecule or molecules that bind to a compound and the molecule or compound to which it binds The spacing may be selected to provide a certain desired gap or configuration.

[0126] As used herein, the term "bioavailability" refers to the ability of a substance or its activity to The rate at which the active moiety is delivered from the drug formulation and becomes available at the site of action or in the systemic circulation and Increased bioavailability refers to the degree to which a substance or its active moieties become Increasing the rate and extent to which a dosage form is delivered and becomes available at the site of action or in the systemic circulation By way of example, the increased bioavailability may be achieved by the addition of other substances or other active moieties. This may be indicated by increased blood levels of the substance or an active portion thereof compared to A non-limiting example of a method for evaluating the increase in bioavailability is provided in Example 2. The method can be used to determine the bioavailability of any polypeptide. An evaluation may be performed.

[0127] As used herein, the terms "biologically active molecule," "biologically active moiety," or "biologically active compound" refer to a compound that is The "activator" is an organism (virus, bacteria, bacteriophage, transposon, prion) Biology (including, but not limited to, organisms, insects, fungi, plants, animals, and humans) can affect the physical or biochemical properties of a system, pathway, molecule, or interaction In particular, as used herein, a biologically active molecule refers to any substance that is capable of being administered to humans. or for the diagnosis, cure, mitigation, treatment, or prevention of disease in animals or other animals; Any substance that enhances the physical or mental well-being of a human or animal, Examples of bioactive molecules include, but are not limited to, peptides, proteins, enzymes, small molecule drugs, herbicides, and the like. hard drugs, soft drugs, prodrugs, carbohydrates, inorganic atoms or molecules, dyes, fats Substances, nucleosides, radionuclides, oligonucleotides, toxins, cells, viruses, liposomes The methods and methods described herein include, but are not limited to, nanoparticles, microparticles, and micelles. The types of biologically active molecules suitable for use in combination with the compositions include drugs, prodrugs, radioactive Nuclides, contrast agents, polymers, antibiotics, bactericides, antiviral agents, anti-inflammatory agents, antitumor agents, cardiovascular vascular agonists, anxiolytics, hormones, growth factors, steroids, and bacterial toxins. Including, but not limited to:

[0128] "Modulating a biological activity" means increasing or decreasing the reactivity of a polypeptide. To modify the selectivity of a polypeptide, or to improve or enhance the substrate selectivity of a polypeptide. The modified biological activity assay can be used to determine the biological activity of the non-natural polypeptide. This can be done by comparing the biological activity of the native polypeptide with that of the native polypeptide.

[0129] As used herein, the term "biomaterial" refers to a biologically derived material. The biologically derived materials may be produced using bioreactors and / or recombinant methods and techniques. This includes, but is not limited to, materials obtained from

[0130] As used herein, the term "biophysical probe" refers to a probe that detects changes in the structure of a molecule. Such molecules include probes capable of detecting or observing "Biophysical probes" can be used to detect, but are not limited to, proteins and other large An example of a biophysical probe is a spin label. These include, but are not limited to, identifiers, fluorescent probes, and photoactivatable groups.

[0131] As used herein, the term "biosynthetic" refers to a translation system (cellular or non-cellular translation system). Any method utilizing a nucleic acid sequence encoding a polynucleotide, a codon, a tRNA, and a ribonucleotide sequence, As an example, the present invention relates to a method of producing a medicament for use in a pharmaceutical composition comprising the steps of: The methods and techniques described in the specification and in non-limiting Example 20 ("Polymers Containing Unnatural Amino Acids"). The non-natural amino acid polypeptide can be produced by the in vivo synthesis of non-natural amino acids. In addition, non-natural amino acid polypeptides may be prepared by "biosynthetic incorporation" of non-natural amino acids. Methods for selecting useful unnatural amino acids that can be "biosynthetically incorporated" into a compound include, but are not limited to, As described in Example 20.

[0132] As used herein, the term "biotin analog" (or "biotin mimetic" The antibody, also called the “antibody” (Avidin-Avidin / Streptavidin-Avidin complex), binds with high affinity to avidin and / or streptavidin. The ligand is any molecule other than biotin that binds to the ligand.

[0133] As used herein, the term "carbonyl" refers to -C(O)-, -S(O)-, -S(O) 2 -, -C(S)-, and -C(S)-. Examples of such compounds include, but are not limited to, one ketone group, at least one aldehyde group, , at least one ester group, at least one carboxylic acid group, and / or at least Each of these carbonyl groups contains one thioester group. These include aldehydes, carboxylic acids, esters, and thioesters. Such groups may be part of a linear, branched, or cyclic molecule.

[0134] The term "carboxy terminal modification group" refers to any molecule that can be attached to a terminal carboxy group. As an example, such a terminal carboxy group may be used in the synthesis of a polymer (polypeptide, polynucleotide, etc.). (including, but not limited to, nucleotides, and polysaccharides) The terminal modification groups may include a variety of water-soluble polymers, peptides, or proteins. By way of example, the terminal modification group may be, but is not limited to, polyethylene glycol or includes serum albumin. End-modification groups may be used to modify the therapeutic properties of the polymer. Such modifications include, but are not limited to, extending the serum half-life of the peptide.

[0135] As used herein, the term "chemically cleavable group" refers to a group that is "chemically unstable." It is also called a radical initiator, and is sensitive to acids, bases, oxidizing agents, reducing agents, chemical initiators, or radical initiators. Refers to a group that is destroyed or cleaved when exposed to light.

[0136] As used herein, the term "chemiluminescent group" refers to a group that is a luminescent molecule that is a product of a chemical reaction that does not involve heating. An example is luminol (5-amino-2,3-dihydro-1 ,4-phthalazinedione) reacts with hydrogen peroxide (H 2 O 2 ) The reaction product (3-aminophthalate (3-APA: 3-aminophthalate).

[0137] As used herein, the term "chromophore" refers to a chromophore that is capable of emitting visible, ultraviolet, or infrared light. Refers to molecules that absorb light of linear wavelengths.

[0138] As used herein, the term "cofactor" refers to an atom or molecule that is essential for the action of a macromolecule. A cofactor is an inorganic ion, coenzyme, protein, or other molecule required for enzyme activity. Factors include, but are not limited to, heme of hemoglobin, chlorophyll Examples of metal ions include magnesium, and metal ions found in proteins.

[0139] As used herein, "cofolding" refers to the interaction of two or more molecules with each other. At least two molecules were used to identify unfolded or improperly folded Refolding converts a bound molecule into a properly folded molecule. As an example, "cofolding" refers to the process, reaction, or method of and detecting unfolded or The improperly folded polypeptide is converted to a properly folded polypeptide. Such polypeptides may be converted to polypeptides containing natural amino acids and / or It may contain at least one unnatural amino acid.

[0140] As used herein, a "comparison window" refers to the region in which two sequences are optimally aligned. After the sequence is generated, it is used to compare the sequence to a reference sequence consisting of the same number of consecutive positions. A segment consisting of any number of consecutive positions. Such consecutive positions may range from about 20 to about 600. Consecutive units (containing about 50 to about 200 sequence units, about 100 to about 150 sequence units) As an example, the group consisting of such a structure is, but is not limited to, The strings include polypeptides and polypeptides containing unnatural amino acids, and are The positions include, but are not limited to, natural and unnatural amino acids. By way of example, such a sequence may be a polynucleotide sequence consisting of nucleotides corresponding to successive units. Methods for aligning sequences for comparison are well known in the art. Optimal alignment of sequences for comparison was performed using the local homology algorithm (Smith and Waterman (1970) Adv. Appl. Math. 2:482c), the homology alignment algorithm (Needleman and Wun sch (1970) J. Mol. Biol. 48:443), similarity search method (Pearson and Lipman (1988) Proc. Nat'l. Acad. Sci. USA 85:2444), and the implementation of these algorithms by computers (G AP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genet ics Computer Group, 575 Science Dr., Madison, WI), or manual alignment and Visual inspection (e.g., Ausubel et al., Current Protocols in Molecular Biology (1995 sup The method can be implemented by, but is not limited to, the following techniques.

[0141] As an example, the percent sequence identity and percent sequence similarity can be determined using Possible algorithms are described in Altschul et al. (1997) Nuc. Acids Res. 25:3389-3402 and A The BLAST algorithms described in Iltschul et al. (1990) J. Mol. Biol. 215:403-410 were used. The algorithm and BLAST 2.0 algorithm are used to perform BLAST analysis. The software is provided by the National Center for Biotechnology Information. Publicly available through the Center for Biotechnology Information The BLAST algorithm variables W, T, and X determine the alignment. The sensitivity and speed of the BLASTN program (for nucleotide sequences) are determined. ,As the initial settings, word length (W) is 11, expectation value (E) is 10, M = 5, N = -4, and A comparison of both strands is used. For amino acid sequences, the BLASTP program uses The default settings are word length (W) 3, expectation (E) 10, and BLOSUM62 score. Ring matrix (Henikoff and Henikoff (1992) Proc. Natl. Acad. Sci. USA 89:109 15) Alignment (B) 50, expectation (E) 10, M=5, N=-4, and both strides The BLAST algorithm generally uses a "low complexity" This is done with filtering turned off.

[0142] The BLAST algorithm also performs a statistical analysis of the similarity between two sequences (e.g., Ka (See Rlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5787). One measure of similarity provided by the ST algorithm is the degree to which two nucleotide sequences or is the minimum sum probability (P( For example, the minimum sum probability when the test nucleic acid is compared with the reference nucleic acid is less than about 0.2. , less than about 0.01, or less than about 0.001, the nucleic acid is similar to the reference sequence. It is believed that there are.

[0143] The term "conservatively modified variants" refers to both natural and unnatural amino acids, both natural and For a particular nucleic acid sequence, "Conservatively modified variants" refer to variants that contain identical or essentially identical natural and unnatural amino acids. Alternatively, the natural and non-natural nucleic acids may be natural and and where the amino acid sequence is not encoded by a non-naturally occurring amino acid sequence, "conservatively modified variants" are by definition As an example, due to the degeneracy of the genetic code, there are many sequences that have the same function. Any particular protein is encoded by a number of nucleic acids. For example, the codons GCA, GCC The codons, GCG, and GCU, all code for the amino acid alanine. By modifying the encoded polypeptide at all positions where an alanine is specified by The codons can be altered to any of the corresponding codons described. The variant is a "silent variant," which is one type of conservatively modified variant. Thus, by way of example, all natural or non-natural polypeptides herein are encoded by A naturally occurring or non-naturally occurring nucleic acid sequence includes all possible silent variations in naturally occurring or non-naturally occurring nucleic acids. The description also includes the differences between the codons (AUG, AGG, and BGG) of natural or non-natural nucleic acids. and TGG, with AUG being the only codon that normally codes for methionine. (TGG is the only codon that normally codes for tryptophan) It will be appreciated that it is possible to produce functionally identical molecules. Each silent variation of naturally occurring and non-naturally occurring nucleic acids which encode naturally occurring and non-naturally occurring polypeptides is , which are implicit in each sequence described.

[0144] In terms of amino acid sequences, it is possible to identify a single amino acid or a small percentage of the amino acids in the encoded sequence. The modification, addition, or deletion of amino acids in a nucleic acid, peptide, polypeptide, or tag Individual substitutions, deletions, or additions to a protein sequence are considered to be modifications that result in the deletion of amino acids. Deletion, addition, or substitution of natural or unnatural amino acids with chemically similar amino acids. Conservatively modified variants are those which provide functionally similar amino acids. Substitution tables are well known in the art. Such conservatively modified variants The polymorphic variants, interspecies homologs, and alleles of the methods and compositions described herein are This includes, in addition to and does not exclude, the following:

[0145] Conservative substitution tables providing functionally similar amino acids are well known to those of skill in the art. The following eight groups contain amino acids that are conservative substitutions for each other: There are: (1) Alanine (A), Glycine (G); (2) aspartic acid (D), glutamic acid (E); (3) Asparagine (N), Glutamine (Q); (4) arginine (R), lysine (K); (5) isoleucine (I), leucine (L), methionine (M), valine (V); (6) phenylalanine (F), tyrosine (Y), tryptophan (W); (7) serine (S), threonine (T); and (8) Cysteine ​​(C), Methionine (M) (See, e.g., Creighton, Proteins: Structure and Molecular Properties (WH Freeman & Co.; 2nd edition (December 1993).

[0146] The terms "cycloalkyl" and "heterocycloalkyl", alone or in combination with other terms, When used in combination, unless otherwise specified, the terms "alkyl" and "heteroalkyl" are used interchangeably. Thus, cycloalkyl or heterocycloalkyl are each intended to mean a cyclic form of includes saturated, partially saturated, and fully saturated ring bonds. For the ring, a heteroatom may occupy the position at which the heterocycle is attached to the remainder of the molecule. Heteroatoms may include, but are not limited to, oxygen, nitrogen, or sulfur. Examples of cycloalkyl are cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3 -cyclohexenyl, and cycloheptyl, and the like. Examples of cycloalkyl are 1-(1,2,5,6-tetrahydropyridyl), 1-piperidinyl, dinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl, 3-morpholinyl, Tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothienyl 2-yl, tetrahydrothien-3-yl, 1-piperazinyl, and 2-piperazinyl Additionally, the above term includes polycyclic structures (bicyclic or tricyclic). Similarly, the term "heterocycloalkyl" includes, but is not limited to, the ring structures of "Xylene," alone or as part of another molecule, is a dicyclic group derived from a heterocycloalkyl. The term "cycloalkylene" refers to a cycloalkyl group, either alone or as part of another molecule. , a divalent radical derived from cycloalkyl.

[0147] As used herein, the term "cyclodextrin" refers to at least one ring-formed It also refers to a ring-shaped carbohydrate made up of 6 to 8 glucose molecules. The outer part of the ring is water-soluble. The central portion of the ring contains a relatively non-polar group that can accommodate small molecules. It is a recess of sexuality.

[0148] As used herein, the term "cytotoxic" refers to a compound that is damaging to cells. Point.

[0149] As used herein, a "modifier" refers to any agent that causes reversible unfolding of a polymer. The term "denaturant" refers to any compound or material that induces the reversible unfolding of a protein. The merits of a denaturant depend on both the characteristics and the concentration of the particular denaturant. By way of example, denaturants include chaotropes, detergents, organics, water-miscible solvents, etc. The lipids include, but are not limited to, lipid mediators, phospholipids, or combinations thereof. Non-limiting examples of amines include urea, guanidine, and sodium thiocyanate. Non-limiting examples of detergents include sodium dodecyl sulfate or polyoxyethylene Strong detergents such as ether (e.g., Tween or Triton detergents), Sar kosyl, mild non-ionic detergents (e.g., digitonin), N-2,3-(diisopropyl ether), Weak cationic cleaners such as (oxy)-propyl-N,N,N-trimethylammonium detergents, weak ionic detergents (e.g., sodium cholate or sodium deoxycholate) ammonium), or zwitterionic detergents (sulfobetaine (amphoteric detergent), 3-(3-chloro Amidopropyl)dimethylammonio-1-propane sulfate (CHAPS), and and 3-(3-chloroamidopropyl)dimethylammonio-2-hydroxy-1-propane including, but not limited to, benzodiazepines (CHAPSO) Non-limiting examples of organic water-miscible solvents include acetonitrile, lower alkanols (especially ethanol or isopropanol, etc. 2 ~C 4 alkanol), or lower alkanol Lactone diols (C such as ethylene glycol) 2 ~C 4 alkanediols), The present invention is not limited to these. In addition, the above-mentioned non-limiting examples of organic water-miscible solvents may be used as denaturants. Non-limiting examples of phospholipids include phosphatidylethanolamine and phosphatidylcholine. Natural phospholipids such as phosphatidylserine, and phosphatidylinositol, or dihexanoylphosphatidylcholine or diheptanoylphosphatidylcholine This includes, but is not limited to, any synthetic phospholipid derivative or variant.

[0150] As used herein, the term "desired functional group" is selected from the following: refers to any group that can be used to Conductors; Photocrosslinkers; Cytotoxic compounds; Drugs; Affinity labels; Photoaffinity labels photoaffinity label; reactive compound; resin; second protein analog, polypeptide peptide analogs or polypeptide analogs; antibodies or antibody fragments; metal chelators ;Cofactors;Fatty acids;Carbohydrates;Polynucleotides;DNA;RNA;Antisense polynucleotides Nucleotides;Polysaccharides;Water-soluble dendrimers;Cyclodextrins;Biocompatible materials;Nano Particles;Spin labels;Fluorescent probes;Metal-containing moieties;Radioactive moieties;Novel functional groups;Other molecules a group that interacts covalently or noncovalently with; a photocaged moiety; an actinic radiation-activated moiety; a ligand photoisomerizable moiety; biotin; biotin analogue; biotin analogue; heavy atom-incorporated Chemically cleavable groups; Photocleavable groups; Extended side chains; Carbon-bonded sugars; redox active substances; aminothio acids; toxic moieties; isotopically labeled moieties; biophysical pro Fluorescent groups; Phosphorescent groups; Chemiluminescent groups; Electron-dense groups; Magnetic groups; Intercalating groups; Chromophores; Energy transfer transfer agents; biologically active agents (wherein biologically active agents include agents having therapeutic activity); and the non-natural amino acid polypeptide or modified non-natural amino acid can have a therapeutic agent attached thereto. The therapeutic agent can act as a means to deliver the therapeutic or co-therapeutic agent to a desired location in the organism. ; detectable label; small molecule; inhibitory ribonucleic acid; radioactive nucleotide; neutron capture agent ;Biotin derivatives;Quantum dots;Nanotransmitters;Radioactive transmitters;Antibody enzymes;Activated Complex activators; viruses; adjuvants; aggrecan; allergan; angiostatin; Antihormones;Antioxidants;Aptamers;Guiding RNA;Saponins;Shuttling vectors;Macromolecules; mimotopes; receptors; reverse micelles; and any combination thereof.

[0151] As used herein, the term "diamine" refers to a compound having at least two amine functional groups. The above groups / molecules include hydrazine groups, amidine groups, imine groups, 1,1- Diamine groups, 1,2-diamine groups, 1,3-diamine groups, and 1,4-diamine groups. In addition, such groups may be linear, branched, or cyclic. It may be part of a molecule.

[0152] As used herein, the term "detectable label" refers to a label that can be detected by analytical techniques (fluorescence methods, chemiluminescence, methods, electron spin resonance methods, ultraviolet-visible absorbance spectroscopic methods, mass spectrometry methods, nuclear magnetic resonance using techniques including, but not limited to, magnetic resonance, electrochemical, and This refers to a mark that is observable by a human eye.

[0153] The term "dicarbonyl" refers to -C(O)-, -S(O)-, -S(O) 2 - and -C (S)-, wherein the moiety is selected from the group consisting of: ,2-dicarbonyl group, 1,3-dicarbonyl group, 1,4-dicarbonyl group, and a few At least one ketone group, at least one aldehyde group, and at least one ester group , at least one carboxylic acid group, and / or at least one thioester group. Such dicarbonyl groups include, but are not limited to, groups such as diketones, ketones, The compounds include aldehyde, keto acid, keto ester, and ketothioester groups. Such groups may be part of a linear, branched, or cyclic molecule. The two moieties of the phenyl group may be the same or different from each other, and one of them may be, for example, Contains substituents that form esters, ketones, aldehydes, thioesters, or amides. It's okay to be.

[0154] As used herein, the term "medicine" refers to a drug that is used to prevent, diagnose, alleviate, or treat a disease or condition. Refers to any substance used in treatment or healing.

[0155] As used herein, the term "dye" refers to a soluble color-forming agent that contains a chromophore.

[0156] As used herein, the term "effective amount" refers to an amount that is sufficient to treat one or more of the diseases or conditions being treated. This refers to that amount of an administered drug or compound sufficient to relieve to some extent the symptoms of , reducing and / or reducing the signs, symptoms, or causes of disease, or other desired alterations in a biological system. In one example, the administered drug or compound may be a natural amino acid. Polypeptides, non-natural amino acid polypeptides, modified natural amino acid polypeptides, or Such polypeptides include, but are not limited to, modified non-amino acid polypeptides. Natural amino acid polypeptides, non-natural amino acid polypeptides, modified natural amino acid polypeptides or compositions comprising modified non-natural amino acid polypeptides for prophylactic, enhancing, and and / or therapeutic treatment. may be determined using techniques such as a dose escalation study.

[0157] As used herein, the term "electron dense group" refers to a group that, when irradiated with an electron beam, This refers to groups that cause electron scattering in the cations of ammonium molybdate, ammonium subnitrate, etc. Sumasu Cadmium Iodide, 99%, Carbohydrazide, Ferric Chloride Hexahydrate, Hexamethion Lentetramine, 98.5%, Indium trichloride anhydrous, Lanthanum nitrate, Lead acetate trihydrate, Lead citrate trihydrate, lead nitrate, periodic acid, phosphomolybdic acid, phosphotungstic acid, phenanthroline Potassium ricyanide, potassium ferrocyanide, ruthenium red, silver nitrate, silver proteinate (Ag assay: 8.0-8.5%) "Strong", tetraphenylporphyrin Silver tetraphenylprophin (S-TPPS), sodium chloroaurate, tungsten Sodium arginate, thallium nitrate, thiosemicarbazide (TSC), uranyl acetate, These include, but are not limited to, uranyl nitrate, and vanadyl sulfate.

[0158] As used herein, the term "energy transfer agent" refers to an agent that provides energy to other molecules. This refers to a molecule that either donates or receives energy from other molecules. FRET is a method of transferring excited state energy from a fluorescent donor molecule to a non-excited acceptor molecule. This non-excited acceptor molecule then transfers the donated energy to a longer wavelength. This is a process of dipole-dipole coupling that results in fluorescence emission at .

[0159] The terms "enhance" or "enhance" refer to an increase or shortening in potency or duration of a desired effect. For example, "improving" the effect of a therapeutic agent means Increase the effectiveness or duration of effect of a therapeutic agent acting during the treatment of a disease or condition; As used herein, an "enhancing effective amount" refers to the ability to improve or prolong the life of a disease, disorder, or refers to an amount adequate to enhance the effectiveness of a therapeutic agent in treating a disease state. In some cases, an amount effective for such uses will depend on the severity and course of the disease, disorder, or condition, the severity ... This decision will depend on the treatment, the patient's health condition and response to medication, and the treating physician's judgment. It becomes.

[0160] As used herein, the term "eukaryote" refers to the phylogenetic eukaryotic domain. Organisms in the domain Eukarya are animals (mammals, insects, reptiles, etc.). insects, birds, etc.), ciliates, plants (monocotyledons, dicotyledons, etc.) (including, but not limited to, leafy plants, and algae), fungi, yeasts, flagellates, microsporidia, and protists, but are not limited to.

[0161] As used herein, the term "fatty acid" refers to a fatty acid having a hydrocarbon side chain of C 6 or more Refers to a long-chain carboxylic acid.

[0162] As used herein, the term "fluorescent probe" refers to a probe that emits a photon when excited. It refers to a fluorescent molecule that emits light.

[0163] As used herein, the terms "functional group," "active moiety," "activating group," "leaving group," "reactive group," "active moiety," "active group ... "Group," "Reactive Site," "Chemically Reactive Group," and "Chemically Reactive Moiety" refer to the site at which a chemical reaction occurs. Refers to a part or unit of a molecule. These terms have some synonymy in the chemical arts. As used herein, it refers to a molecule that provides a certain function or activity and reacts with other molecules. It is used as a term to refer to the part of a molecule that

[0164] The term "halogen" is inclusive of fluorine, chlorine, iodine and bromine.

[0165] As used herein, the term "haloacyl" refers to a halogen moiety (-C(O)CH 3 , -C(O)CF 3 , and -C(O)CH 2 OCH 3 Including, but not limited to, It refers to an acyl group that contains an acyl group (not including an acyl group).

[0166] As used herein, the term "haloalkyl" refers to a halogen moiety (-CF 3 and -CH 2 CF 3 and the like.

[0167] As used herein, the term "heteroalkyl" refers to an alkyl group and an O, N, Si and at least one heteroatom selected from the group consisting of nitrogen The nitrogen and sulfur atoms may be optionally oxidized and the nitrogen heteroatoms may be optionally quaternized. refers to straight-chain, branched-chain, or cyclic hydrocarbon radicals, or combinations thereof. The heteroatoms O, N, S, and Si may be at any interior position of a heteroalkyl group or at any alkyl group. The group may be located at the position where it is attached to the remainder of the molecule. 2 -CH 2 -O-CH 3 , -CH 2 -CH 2 -NH-CH 3 , -CH 2 -CH 2 -N(CH 3 )-C H 3 , -CH 2 -S-CH 2 -CH3 , -CH 2 -CH 2 , -S(O)-CH 3 , -CH 2 -CH 2 -S(O) 2 -CH 3 , -CH=CH-O-CH 3 , -Si(CH 3 ) 3 , - CH 2 -CH=N-OCH 3 , and -CH=CH-N(CH 3 )-CH 3 It encompasses In addition, up to two heteroatoms (for example, -CH 2 -NH-OCH 3 and -CH 2 -O-Si(CH 3 ) 3 etc.) may be consecutive.

[0168] The term "heterocycle-based bond" or "heterocycle bond" refers to a bond in which a dicarbonyl group is bonded to a diamine The resulting reaction product is a heteroaryl group. The resulting heterocycle is a heterocycle containing a aryl or heterocycloalkyl group. The group is a chemical bond between a non-natural amino acid or a non-natural amino acid polypeptide and another functional group. In one embodiment, the heterocyclic bond is a nitrogen-containing heterocyclic bond, for example , pyrazole bond, pyrrole bond, indole bond, benzodiazepine bond, and pyrazole bond. Contains pyrazolone bonds.

[0169] Similarly, the term "heteroalkylene" refers to heteroalkyl (-CH 2 -CH 2 -S-CH 2 -CH 2- and -CH 2 -S-CH 2 -CH 2 -NH-CH 2 - is an example of (including, but not limited to, the following): are the same or different heteroatoms (alkyleneoxy, alkylenedioxy, alkylene These include, but are not limited to, amino, alkylenediamino, and aminooxyalkylene. In addition, alkylene linking groups and For heteroalkylene and heteroalkylene linking groups, the direction of the linking group is the same as the direction of the formula showing the linking group. As an example, the formula -C(O) 2 R'- is -C(O) 2 R' -and-R'C(O) 2 - indicates both.

[0170] As used herein, the term "heteroaryl" or "heteroaromatic" refers to a heteroaryl group that is It refers to an aryl group containing at least one heteroatom selected from C, O, and S. In addition, the nitrogen atom and sulfur atom of the aryl group may be appropriately oxidized. The carbon atoms may be optionally quaternized. Heteroaryl groups may be substituted or unsubstituted. Heteroaryl groups can also be attached to the remainder of the molecule through a heteroatom. Non-limiting examples of the heteroaryl groups include 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, -Pyrrolyl, 3-pyrazolyl, 2-imidazolyl, 4-imidazolyl, pyrazinyl, 2- Oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5-oxazolyl , 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thiazole Enyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl , 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl, 1-isopropyl Isoquinolyl, 5-isoquinolyl, 2-quinoxalinyl, 5-quinoxalinyl, 3-quinolyl quinolyl, and 6-quinolyl.

[0171] As used herein, the term "homoalkyl" refers to an alkyl group that is a hydrocarbon group. Point.

[0172] As used herein, the term "identical" refers to two or more identical sequences or subsequences. Additionally, as used herein, the term "substantially identical" refers to a comparison The measurements are compared and compared using algorithms or by manual alignment and visual inspection. When aligned to maximize correspondence within a comparison window or specified region, Refers to a sequence of two or more sequences that have a certain percentage of consecutive units that are identical. The identity of consecutive units across a particular region is approximately 60%, 65%, 70%, or 75%; In the case of about 80%, about 85%, about 90%, or about 95%, the two or more sequences are "real Such a percentage of identity may be "qualitatively identical" to the identity of two or more sequences. The percent identity is measured based on the sequence identity of at least about 75-100 sequences. the entire area of ​​about 50 consecutive units in length, or an unspecified area of ​​about 50 consecutive units in length. In some cases, the term may be used throughout the entire sequence. In one example, two or more polypeptide sequences may have identical amino acid residues. In contrast, if the amino acid residues in a particular region are identical, the polypeptide sequences are identical. Monogamy is about 60%, 65%, 70%, 75%, 80%, 85%, 90%, or In about 95% of cases, the two or more polypeptide sequences are "substantially identical." Identity is measured over a region of at least about 75 to about 100 amino acids in length, about 50 amino acids in length, or over a region of at least about 100 amino acids in length. The amino acid sequence of the polypeptide may be present throughout the entire region of the polypeptide, or, in some cases, throughout the entire polypeptide sequence. Further, by way of example, for two or more polynucleotide sequences, a nucleic acid Where residues are identical, the polynucleotide sequences are identical. The identity of the nucleic acid residues in the %, about 90%, or about 95% of the two or more nucleotide sequences are "real" The identity is "qualitatively identical" to the above-mentioned sequence. the entire region, the entire region of about 50 nucleic acids in length, or in certain cases the nucleotide sequence It can be present in the entire array of columns.

[0173] For sequence comparison, usually one sequence acts as a reference sequence, to which test sequences are compared. When using a comparison algorithm, the test and reference sequences are input into a computer; If necessary, the coordinates of the subarray are specified, and the variables of the array algorithm program are specified. Alternatively, you can use the initial settings variables in the sequence algorithm program. Replacement variables can also be specified. The sequence comparison algorithm then The percent sequence identity of the test sequence to the sequence is calculated based on the program parameters. do.

[0174] As used herein, the term "immunogenicity" refers to an antibody response to administration of a therapeutic agent. Quantitative analytical methods and methods for detecting anti-non-natural amino acid polypeptide antibodies in biological fluids and qualitative analysis methods to obtain immunogenicity for therapeutic non-natural amino acid polypeptides These assays include radioimmunoassay (RIA), enzyme-linked immunosorbent assay (EL These include immunoassay (ISA), luminescence immunoassay (LIA), and fluorescence immunoassay (FIA). Detection of immunogenicity to therapeutic non-natural amino acid polypeptides can be used to detect therapeutic The antibody response following administration of a therapeutic non-natural amino acid polypeptide is compared with that following administration of a therapeutic natural amino acid polypeptide. This includes comparing the antibody responses following administration of the tidoside.

[0175] As used herein, the term "intercalating agent" is also referred to as an "intercalating group" and refers to an intercalating agent that inserts an intercalating agent into the intramolecular space. or a chemical substance capable of inserting into the intermolecular space. It may be a DNA molecule. It is a molecule that inserts between the stacked bases of double-stranded DNA. It is also possible.

[0176] As used herein, the term "isolated" refers to separating an element of interest from elements other than the element of interest. The term "isolated" refers to removing or separating material from a substance. Isolated material can be dry or semi-dry, but It may also be in a solution (including, but not limited to, an aqueous solution). Alternatively, the composition may be formulated with additional pharma- ceutically acceptable carriers and / or excipients. Purity and homogeneity can be determined by analytical chemistry techniques (e.g., Polyacrylamide gel electrophoresis or high performance liquid chromatography, but In addition, the element of interest may be isolated and the determination may be performed using a method such as the following: As used herein, such an element is referred to as substantially purified when it is the predominant species present in a preparation. As used herein, the term "purified" means at least pure. 85% purity, at least 90% purity, at least 95% purity, or at least 99% purity By way of example, a nucleic acid or protein may refer to a target element that is not associated with the nucleic acid or protein in its natural state. If a substance does not contain at least some of the cellular components that comprise it, it is considered "isolated." or concentrated to levels higher than those obtainable in in vitro or in vivo production. In one embodiment, the nucleic acid or protein is "isolated." Genes are classified into open reading frames that flank genes and code for proteins other than the gene of interest. It is isolated when it is separated from the

[0177] As used herein, the term "label" refers to a compound that is incorporated into a compound and is easily detectable. The physical distribution of said substance may be detected and / or monitored.

[0178] As used herein, the term "bond" refers to the bond between a functional group of a linker and another molecule. A bond or chemical moiety that occurs as a result of a chemical reaction. Such bonds are called covalent bonds and Such chemical moieties may include, but are not limited to, non-covalent bonds. Esters, carbonates, imine phosphate esters, hydrazones, acetals, orthoesters These include, but are not limited to, ethers, peptide bonds, and oligonucleotide linkages. A hydrolytically stable bond is one that exhibits substantial stability in water and at useful pH values ​​(e.g., physiological conditions). Denotes bonds that do not react with water for extended periods or indefinitely. Hydrolytically unstable or decomposable The bond is decomposable in water or in aqueous solutions (including, for example, blood). An enzyme-labile or enzyme-degradable bond means that the bond can be broken down by one or more enzymes. As an example, PEG and related polymers are Degradable within the molecular backbone or within the bond between the polymer backbone and one or more of the polymer's terminal functional groups Such degradable linkages may include PEG carboxylic acid or activated PEG. An ester bond formed by reaction of a carboxylic acid with an alcohol group on a bioactive agent In addition, such an ester group generally has a structure that is stable under physiological conditions. The other hydrolytically degradable bonds are the carbohydrate moieties, which hydrolyze under reduced pressure to release the bioactive agent. nate bonds; imine bonds formed from the reaction of amines and aldehydes; Phosphate ester bonds formed by reacting with phosphate groups; hydrazides and aldehydes hydrazone bonds, which are the reaction products of aldehydes and alcohols; cetal bonds; orthoester bonds, which are the reaction products of formates and alcohols; amines groups (including, but not limited to, amine groups located at the termini of polymers such as PEG) and and peptide bonds formed by the carboxyl groups of peptides; and phosphoramidites groups (including but not limited to phosphoramidite groups at the ends of the polymer) and oligo It includes an oligonucleotide linkage formed from the 5' hydroxyl group of a nucleotide. Not limited to:

[0179] As used herein, the term "medium" or "culture medium" refers to a medium for the growth and and recovery, and / or expression and / or secretion of products by such cells. Such a "medium" or "culture medium" refers to any culture medium in which the host Cells (e.g., bacterial host cells, yeast host cells, insect host cells, plant host cells, eukaryotic host cells, Host cells, mammalian host cells, CHO cells, prokaryotic host cells, E. coli host cells, or or Pseudomonas host cells), and may retain or contain the cell contents. Examples of suitable carriers include, but are not limited to, liquids, solids, semi-solids, or fixed supports that can be dissolved in water. Such a "medium" or "culture medium" is a liquid in which the host cells are cultured and the polypeptide is secreted. The media or culture medium used (either before or after the growth phase) is included. In addition, examples of such a "medium" or "culture solution" include, but are not limited to, In one example, the polypeptide is produced intracellularly and the host cell is lysed or destroyed. A buffer or reagent containing a host cell lysate that is disrupted to release the polypeptide. These include, but are not limited to:

[0180] As used herein, the term "metabolite" includes, by way of example only, naturally occurring amino acid polypeptides. polypeptides, non-natural amino acid polypeptides, modified natural amino acid polypeptides, refers to derivatives of compounds such as modified non-natural amino acid polypeptides, and examples include natural Natural amino acid polypeptides, non-natural amino acid polypeptides, modified natural amino acid polypeptides When a compound such as a peptide or modified non-natural amino acid polypeptide is metabolized The term "pharmacologically active metabolite" or "active metabolite" is an example As the polypeptides, natural amino acid polypeptides, non-natural amino acid polypeptides, modified natural amino acid polypeptides, The biology of compounds such as non-natural amino acid polypeptides or modified non-natural amino acid polypeptides. Examples of the functional derivatives include natural amino acid polypeptides and non-natural amino acid polypeptides. Peptides, modified natural amino acid polypeptides, or modified non-natural amino acid polypeptides. They are produced when compounds such as peptides are metabolized.

[0181] As used herein, the term "metabolized" refers to the process by which a particular substance is metabolized by an organism. This refers to the entire process of change. Such processes include hydrolysis and enzyme-catalyzed reactions. For further details on metabolism, see The Ph.D. See armacological Basis of Therapeutics, 9th Edition, McGraw-Hill (1996). As the polypeptides, natural amino acid polypeptides, non-natural amino acid polypeptides, modified natural amino acid polypeptides, Metabolites of non-natural amino acid polypeptides, or modified non-natural amino acid polypeptides, are Amino acid polypeptides, non-natural amino acid polypeptides, modified natural amino acid polypeptides and administering the modified non-natural amino acid polypeptide to a host and extracting the polypeptide from the host. By analysis of the sample, or by determining whether natural amino acid polypeptides, non-natural amino acid polypeptides , a modified natural amino acid polypeptide, or a modified non-natural amino acid polypeptide. In vitro incubation with hepatocytes and analysis of the resulting compounds Therefore, it can be identified.

[0182] As used herein, the term "metal chelator" refers to a metal chelator that chelates a metal ion. These molecules form complexes with two metal ions, one of which is a central metal ion. The above coordinate bonds can be formed to form a ring structure.

[0183] As used herein, the term "metal-containing moiety" refers to a metal ion, atom, Such moieties include cisplatin, chelated Metal ions (nickel, iron, platinum, etc.) and metal nanoparticles (nickel, iron, platinum etc.), but are not limited to these.

[0184] As used herein, the term "moiety incorporating a heavy atom" generally refers to a It refers to a group that incorporates ions of atoms heavier than carbon. Such ions or atoms include , silicon, tungsten, gold, lead, and uranium. .

[0185] As used herein, the term "modified" refers to a natural amino acid, a non-natural amino acid, The presence of alterations to amino acids, natural amino acid polypeptides, or non-natural amino acid polypeptides. Such changes or modifications may include natural amino acids, non-natural amino acids, natural amino acid polynucleotides, and the like. By post-synthetic modification of peptides or non-natural amino acid polypeptides or to naturally occurring amino acids an acid, a non-natural amino acid, a natural amino acid polypeptide, or a non-natural amino acid polypeptide The modified or unmodified forms are obtained by co- or post-translational modification. The natural amino acids, non-natural amino acids, natural amino acid polypeptides, or non-natural The naturally occurring amino acid polypeptides are optionally modified, i.e., the naturally occurring amino acids described are an acid, a non-natural amino acid, a natural amino acid polypeptide, or a non-natural amino acid polypeptide It means that it may or may not be modified.

[0186] As used herein, the term "modified serum half-life" refers to its unmodified form Refers to a positive or negative change in the circulating half-life of a modified biologically active molecule compared to the original half-life. As an example, modified biologically active molecules include natural amino acids, non-natural amino acids, and amino acid, natural amino acid polypeptide, or non-natural amino acid polypeptide, By way of example and not limitation, serum half-life is a measure of the efficacy and safety of a biologically active molecule or modified Taking blood samples at various time points after administration of the selected biologically active molecule. The concentration of the molecule in each of the samples is measured by determining the concentration of the molecule in each of the samples. By correlation with serum concentration, the serum half-life can be calculated. For example, increasing the blood half-life may allow for improved dosing regimens or reduce toxicity. Such serum enhancement can be at least about 2-fold, at least about 3-fold, fold, at least about 5-fold, or at least about 10-fold. Examples of such methods include, but are not limited to, those given in Example 33. This method can be used with any polysaccharide. It can be used to evaluate the half-life of a peptide in the blood.

[0187] As used herein, the term "modulated therapeutic half-life" refers to its unmodified form A positive or negative change in the half-life of a therapeutically effective dose of the modified biologically active molecule compared to As an example, modified biologically active molecules include the natural amino acid , non-natural amino acids, natural amino acid polypeptides, or non-natural amino acid polypeptides. By way of example, the therapeutic half-life can be determined by measuring the time course of various by measuring the pharmacokinetic and / or pharmacological properties of the molecule at different time points. The enhanced therapeutic half-life may be determined by determining whether a particularly beneficial dosing regimen or a particularly beneficial The total dose can be increased or reduced to avoid undesirable effects. The increased therapeutic half-life may result in increased efficacy, increased targeting of the modified molecule to its target. or reduced binding, enhancement or attenuation of other elements or mechanisms of action of the unmodified molecule. or, for example, increased or decreased molecular breakdown by enzymes such as proteases. An example of a method for evaluating the enhancement of therapeutic half-life is given in Example 33. This method may be used to assess the therapeutic half-life of any polypeptide. can.

[0188] As used herein, the term "nanoparticle" refers to a nanoparticle having a particle size of about 500 nm or less. This refers to particles of approximately 1 nm in size.

[0189] As used herein, the term "near stoichiometric" refers to a chemical reaction. This refers to a molar ratio of the compounds present being from about 0.75 to about 1.5.

[0190] As used herein, the term "non-eukaryotic organism" refers to a non-eukaryotic organism. Examples of non-eukaryotic organisms include eubacteria (Escherichia coli, Thermus truncatula, rmus thermophilus, or Bacillus stearother mophilus, Pseudomonas fluorescens, Pseudom Onas aeruginosa, Pseudomonas putida, etc. (phylogenetic domains), or Archaea (Methan ococcus jannaschii, Methanobacterium ther moautotrophicum, Archaeoglobus fulgidus, P yrococcus furiosus, Pyrococcus horikoshii , Aeuropyrum pernix, or Halobacterium (e.g. NRC- for Haloferax volcanii and Halobacterium spp. 1) and others) (or phylogenetic region). This can be done.

[0191] An "unnatural amino acid" is an amino acid that is not one of the 20 common amino acids, such as pyrrolyl lysine or refers to an amino acid that is not selenocysteine. Synonymous with the term "unnatural amino acid" Other terms that may be used include "non-naturally encoded amino acid," "non-naturally "amino acids," "non-naturally occurring amino acids," and their various hyphenated forms The term "unnatural amino acid" includes hyphenated and non-hyphenated forms. , naturally encoded amino acids (the 20 common amino acids or pyrrolysine and selenosilyl These naturally occur through modifications of the ribozymes (including, but not limited to, ribozymes) and their translation. These include amino acids that are not incorporated into the growing polypeptide chain by the Examples of naturally occurring amino acids that are not naturally encoded include, but are not limited to: N-acetylglucosaminyl-L-serine, N-acetylglucosaminyl-L- These include, but are not limited to, threonine, and O-phosphotyrosine. The term "unnatural amino acid" refers to an amino acid that does not occur in nature and is obtained synthetically. These include amino acids that are naturally occurring or that are obtained by modification of non-natural amino acids. However, the present invention is not limited to the above.

[0192] As used herein, the term "nucleic acid" refers to deoxyribonucleotides, deoxyribonucleotides, xylribonucleosides, ribonucleosides, or ribonucleotides, and any one of them It refers to a polymer in single-stranded or double-stranded form. Examples of such nucleic acids and nucleic acid polymers include As such, (i) a nucleic acid that has similar binding properties to a reference nucleic acid and is a naturally occurring nucleotide (ii) analogues of natural nucleotides that are metabolized in a manner similar to oligonucleotides; Peptide analogs (PNA (peptide nucleic acid)), DNA used in antisense technology Analogs such as phosphorothioates and phosphoramidates are also included. (iii) conservatively modified variants of these (including degenerate codon substitutions) (including, but not limited to, the complementary sequences and sequences explicitly indicated) In one example, degenerate codon substitutions can be performed using one or more selected The third position of the selected (or all) codons is a mixed base and / or a deoxyribonucleic acid This can be achieved by generating sequences in which the nucleotide residues are replaced with nucleotide residues (Batzer et al., Nucleotide Sequence Analysis, 1997). ic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985) ; Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

[0193] As used herein, an "oxidizing agent" refers to a chemical compound capable of removing electrons from a compound that is oxidized. Examples of oxidizing agents include oxidized glutathione, cystine, and cysteine. oxidized dithiothreitol, oxidized erythreitol and oxygen, A wide variety of oxidizing agents are suitable for use in the methods and compositions described herein. This is preferable.

[0194] As used herein, the term "pharmaceutical acceptable" refers to a compound that is biologically This refers to salts, carriers, or diluents that do not interfere with the activity or properties and are relatively non-toxic. The present invention is not limited to the above, i.e., without or without causing undesirable biological effects. It can be administered to an individual without interacting with any of the components of the composition in a manner that would degrade them. This refers to a substance that

[0195] As used herein, the term "photoaffinity label" refers to a label that is In the case of a label, the label refers to a label with a group that forms a bond with a molecule for which the label has affinity. Such bonds may be covalent or non-covalent.

[0196] As used herein, the term "photocaged moiety" refers to a moiety that is photocaged upon illumination with a particular wavelength. refers to a group that can covalently or noncovalently bond to other ions or molecules.

[0197] As used herein, the term "photocleavable group" refers to a group that is cleaved when exposed to light. This refers to a group that can be

[0198] As used herein, the term "photocrosslinker" refers to a compound that reacts with light to form two Two or more functional groups that form covalent or non-covalent bonds with one or more monomeric molecules or polymers It refers to a compound containing

[0199] As used herein, the term "photoisomerizable moiety" refers to a moiety that is capable of being cleaved when exposed to light. Refers to a group that changes from one isomeric form to another.

[0200] As used herein, the term "polyalkylene glycol" refers to linear or refers to a branched polymeric polyether polyol. Examples of glycols include polyethylene glycol, polypropylene glycol, and polybutylene glycol. Examples of suitable amines include, but are not limited to, amines and their derivatives. For example, see the catalogue "Polyethylene Glycol and De" by Shearwater Corporation. A list of commercial suppliers, such as “Catalog of Alternatives for Biomedical Applications” (2001) In one example, such a high molecular weight polyether polyol is about 0. .1 kDa to about 100 kDa. As the high molecular weight polyether polyol, between about 100 Da and about 100,000 Da or more The molecular weight of the polymer may be, but is not limited to, about 100 Da to about 100 Da. Between about 100,000 Da, for example, about 100,000 Da, about 95,000 Da, about 9,000 Da 0Da, about 85000Da, about 80000Da, about 75000Da, about 70000Da, Approximately 65,000 Da, approximately 60,000 Da, approximately 55,000 Da, approximately 50,000 Da, approximately 450 00Da, about 40000Da, about 35000Da, about 30000Da, about 25000Da , about 20000Da, about 15000Da, about 10000Da, about 9000Da, about 800 0Da, approx. 7000Da, approx. 6000Da, approx. 5000Da, approx. 4000Da, approx. 300 0Da, approx. 2000Da, approx. 1000Da, approx. 900Da, approx. 800Da, approx. 700Da , about 600 Da, about 500 Da, 400 Da, about 300 Da, about 200 Da, and about 1 In some embodiments, the polymer The molecular weight of the polypeptide is between about 100 Da and about 50,000 Da. In some embodiments, In some embodiments, the molecular weight of the polymer is between about 100 Da and about 40,000 Da. In some embodiments, the molecular weight of the polymer is between about 1,000 Da and about 40,000 Da. In some embodiments, the molecular weight of the polymer is from about 2,000 Da to about 50,000 Da. In some embodiments, the molecular weight of the polymer is between about 5000 Da and about 40 In some embodiments, the molecular weight of the polymer is between about 10,000 Da. In some embodiments, the poly(ethylene glycol) is between about 40,000 Da and about 40,000 Da. Recall) molecules are branched polymers. The molecular weight of branched PEG is about 1000. Between Da and about 100000 Da, about 100000 Da, about 95000 Da, about 9000 0Da, about 85000Da, about 80000Da, about 75000Da, about 70000Da, Approximately 65,000 Da, approximately 60,000 Da, approximately 55,000 Da, approximately 50,000 Da, approximately 450 00Da, about 40000Da, about 35000Da, about 30000Da, about 25000Da , about 20000Da, about 15000Da, about 10000Da, about 9000Da, about 800 0Da, approx. 7000Da, approx. 6000Da, approx. 5000Da, approx. 4000Da, approx. 300 These include, but are not limited to, 0 Da, about 2000 Da, and about 1000 Da. In some embodiments, the molecular weight of the branched PEG is from about 1000 Da to about 50 Da. In some embodiments, the molecular weight of the branched PEG is between about 1,000 Da. In some embodiments, the molecular weight of the branched chain is between about 0,000 Da and about 40,000 Da. The molecular weight of the PEG is between about 5,000 Da and about 40,000 Da. In the present invention, the molecular weight of the branched PEG is between about 5,000 Da and about 20,000 Da. In another embodiment, the molecular weight of the branched PEG is from about 2,000 Da to about 50,000 Da. Between Da.

[0201] As used herein, the term "polymer" refers to a polymer made up of repeating subunits. Such molecules include polypeptides, polynucleotides, or polysaccharides. , or polyalkylene glycols.

[0202] The terms "polypeptide," "peptide," and "protein" are used herein. are used interchangeably to refer to a polymer of amino acid residues, i.e., a polypeptide The descriptions covered apply equally to peptide descriptions and protein descriptions, and vice versa. The term refers to naturally occurring amino acid polymers and to those having one or more amino acid residues. The term "polypeptide" refers to an amino acid polymer in which the amino acid group is a non-natural amino acid. The terms "peptide," "peptide" and "protein" refer to any length, including full-length proteins. The term "protein" refers to a chain of amino acids, such as proteins, in which the amino acid residues are bound by covalent peptide bonds. are joined by

[0203] The term "post-translational modification" refers to the modification of an amino acid sequence after it has been translationally incorporated into a polypeptide chain. This refers to any modification of a natural or unnatural amino acid that occurs in an acid. Co-translational modification in vitro (e.g., cell-free translation systems), This includes in vivo and in vitro post-translational modifications, However, the present invention is not limited to the above.

[0204] As used herein, "prodrug" or "pharmaceutical acceptable prodrug" refers to a compound that is The term "drug" refers to a substance that is converted into the parent drug in vivo or in vitro; A substance that does not interfere with the biological activity or properties of a drug and is relatively non-toxic, i.e. without causing undesirable biological effects or deteriorating any of the components of the composition in which it is contained. It refers to a substance that is administered to an individual without undergoing any disruptive interactions. A drug precursor is a drug that undergoes any process following administration to a subject and subsequent absorption. are converted through a metabolic pathway (e.g., by a metabolic pathway) to an active or more active species. Some prodrugs reduce the activity and / or modify the drug to be soluble or otherwise The prodrug has a chemical group present that confers the properties of When modified from the prodrug and / or the active drug is generated. They are converted to active drugs by enzymatic or non-enzymatic reactions in the body. The drug is capable of enhancing solubility, specifically targeting delivery to specific cells, tissues, organs, or ligands. This leads to improved physicochemical properties such as improved molecular weight and thus improved therapeutic value of the drug. The advantages of such prodrugs include (i) ease of administration compared to the parent drug; (ii) Unlike the parent drug, the prodrug can be absorbed and utilized by the body when administered orally; and (iii) compared to the parent drug, the prodrug is also more soluble in pharmaceutical compositions. Prodrugs are pharmacologically ineffective substitutes for active drugs. Prodrugs include derivatives that are active or less active. Prodrugs are compounds that have physicochemical, biopharmaceutical properties, or by manipulating the properties of drugs, such as their pharmacokinetic properties, to reach the intended site of action. The purpose is to regulate the amount of a drug or biologically active molecule. For example, as esters ("prodrugs"), aqueous solubility adversely affects mobility. It is administered to facilitate transport across cell membranes and is then metabolically hydrolyzed to carbohydrates. The unnatural amino acid polypeptide becomes an ester of carboxylic acid (active substance) and has beneficial water solubility in cells. Prodrugs are used to deliver drugs to site-specific tissues. They are designed as reversible drug derivatives to be used as modulators to enhance drug delivery.

[0205] As used herein, the term "prophylactically effective amount" refers to a dose that is effective to treat one or more symptoms of a disease. At least one such compound is administered prophylactically to a patient who is likely to suffer to some degree from the condition or disorder being treated. Each of the polypeptides comprises at least one non-natural amino acid or at least one modified non-natural amino acid. In such prophylactic applications, the amount refers to the amount of the composition containing the acid polypeptide. The efficacy of the drug depends on the patient's health and weight, etc. Routine experimentation (e.g., clinical trials of dose escalation) should be performed. Determination of such prophylactically effective amounts is within the skill of those in the art, including, but not limited to, clinical trials. is considered to be well within the range.

[0206] As used herein, the term "protected" refers to a compound that is chemically unreactive under certain reaction conditions. Protective groups refer to the presence of a "protecting group" or moiety that prevents reaction of a reactive functional group. The amount of reactive groups present in the amine group varies depending on the type of reactive group present in the amine group. When the depicted group is an amine or hydrazide, the protecting group is tert-butyloxycarbonyl. t-Boc and 9-fluorenylmethoxycarbonyl (Fmoc) and (ii) when the chemically reactive group is a thiol, the protecting group is an o (iii) the chemically reactive group is butanoic acid or When is a carboxylic acid, such as propionic acid, or a hydroxyl group, the protecting group is may be an alkyl group such as phenyl or methyl, ethyl or tert-butyl. .

[0207] In one example, the blocking / protecting group is

[0208] [ka]

[0209] may be selected from:

[0210] In addition, protecting groups include photolabile groups such as Nvoc and MeVoc, as well as other groups that are well known in the art. These include, but are not limited to, other protecting groups known in the art. Other protecting groups are listed by reference. Greene and Wuts, Protective Groups in Organic Synthesis, incorporated herein by reference in its entirety. hesis, 3rd Ed., John Wiley & Sons, New York, NY, 1999.

[0211] As used herein, the term "radioactive moiety" includes alpha particles, beta particles, It refers to groups that have atomic nuclei that spontaneously emit radiation, such as alpha particles, gamma particles, or alpha particles. A particle is a helium nucleus, a beta particle is an electron, and a gamma particle is a high-energy photon.

[0212] As used herein, the term "reactive compound" refers to a compound that reacts with another atom under appropriate conditions. It refers to a compound that reacts with a molecule or compound.

[0213] The term "recombinant host cell" also referred to as "host cell" refers to the method used for insertion. (e.g., direct uptake, transduction, f-mating, or recombinant host cells) Regardless of the known method used to generate the cells, the exogenous polynucleotide By way of example, an exogenous polynucleotide refers to a cell that contains an exogenous polynucleotide that is integrated into the host genome. It may be a non-integrating vector (e.g., a plasmid) or it may be integrated into the host genome. obtain.

[0214] As used herein, the term "oxidation-reduction active substance" refers to a substance that converts another molecule into an acid. Redox-active substances are molecules that are either oxidized or reduced, and thus redox-active substances are reduced or oxidized. Examples of redox-active substances are ferrocene, quinone, Ru 2+ / 3+ composite Body, Co 2+ / 3+ Complex, and Os 2+ / 3+ Complexes include, but are not limited to: It won't be done.

[0215] As used herein, the term "reducing agent" refers to a compound that adds electrons to a reduced compound. An example of a reducing agent is dithiothreitol. Dithioerythritol (DTT), 2-mercaptoethanol, dithioerythritol, cysteine, These include steamine (2-aminoethanethiol) and reduced glutathione. As an example, the reducing agent may be a compound that maintains a sulfhydryl group in a reduced state. It is used to reduce intramolecular or intermolecular disulfide bonds.

[0216] As used herein, "refolding" refers to the process of refolding an improperly folded protein. from a stretched or unfolded state to a native or properly folded conformation. Refolding refers to any process, reaction, or method that transforms a molecule into a different conformation. By folding disulfide-containing polypeptides, The protein can be transformed from a bound or unfolded state to a native conformation with respect to disulfide bonds. or transforms it into a properly folded conformation. The polypeptide containing the amino acid sequence includes a natural amino acid polypeptide or a non-natural amino acid polypeptide. Lipeptides include:

[0217] As used herein, the term "resin" refers to high molecular weight insoluble polymer beads. As an example, these beads are used as supports for solid-phase peptide synthesis or Or they can be used as a site for attachment of molecules prior to purification.

[0218] As used herein, the term "monosaccharide" refers to a series of carbohydrates, including sugars, monosaccharides, and the like. These include, but are not limited to, sugars, oligosaccharides, and polysaccharides.

[0219] As used herein, the term "safety" or "safety" refers to the degree to which a drug is administered. This refers to possible side effects related to the administration of a drug, relative to the number of times it is administered. A drug that produces few or no side effects at most has excellent safety profile. It can be said that it has the safety. An example of the method for evaluating safety is given in Example 26. This method can be used to assess the safety of any polypeptide. .

[0220] As used herein, "selectively hybridizes to" or "to The phrase "specifically hybridizes to" refers to a sequence that is specifically hybridized to a complex mixture (such as a whole cell or are present in the library (including, but not limited to, DNA or RNA) Specific nucleotide sequences under stringent hybridization conditions are This refers to the binding of a molecule, duplexing of a molecule, or hybridization of a molecule with a nucleic acid sequence.

[0221] As used herein, the term "spin label" refers to a molecule that is capable of being labeled by electron spin resonance spectroscopy. An atom or atoms that exhibit an unpaired electron spin that can be detected by scopy and attached to another molecule Spin-labeled molecules include a group (i.e., a stable paramagnetic group). The nitrile radical and nitroxide, and single or double spin-labeled These include, but are not limited to:

[0222] As used herein, the term "stoichiometric" refers to a chemical reaction. It refers to a molar ratio of the compounds of about 0.9 to about 1.1.

[0223] As used herein, the term "stoichiometric" refers to a change or addition of reaction conditions. A chemical reaction that is stoichiometric or nearly stoichiometric depending on the presence or absence of a substance. Changes in include, but are not limited to, an increase in temperature or a change in pH. Such additives include, but are not limited to, accelerators.

[0224] The term "stringent hybridization conditions" refers to conditions with low ionic strength and DNA, RNA, PNA, or other nucleic acid mimetics, or The term refers to the hybridization of sequences of a single molecule or a combination thereof. Under sensitive conditions, the probe reacts with a complex mixture of nucleic acids, including but not limited to whole cells. , DNA library, or RNA library) hybridize to the subsequence but not to other sequences in the complex mixture. Stringent conditions are sequence-dependent and may be more stringent under different circumstances. For example, the longer the sequence, the more specific the hybridization conditions required. Stringent hybridization conditions are defined as: (i) a defined ionic Approximately 5-10°C lower than the thermal melting temperature (Tm) of the specific sequence at that strength and pH. and (ii) a salt concentration of about 0.01 M to about 1.0 M at about pH 7.0 to about pH 8.3. and the temperature is preferably 10 to 50 nucleotides. for long probes (including but not limited to) at least about 30°C; (including probes longer than 50 nucleotides) at least about 60° C. (iii) adding a destabilizing agent, such as, but not limited to, formamide; (iv) Incubate at 42°C in 50% formamide, 5x SSC, and 1% SDS. or incubate in 5x SSC, 1% SDS at 65°C. The mixture was incubated at 65° C. for about 5 minutes to about 120 minutes in 0.2×SSC and about 0.1% SDS. Examples of suitable methods include, but are not limited to, washing the sample for 10 minutes. For the detection of specific hybridization, a positive signal is considered to be above background. The hybridization of the two bands is at least twice as high as that of the wild type. Extensive guidance on nucleic acid hybridization is provided by, but not limited to, Tijssen, Laboratory Techniques. chniques in Biochemistry and Molecular Biology--Hybridization with Nucleic Probe s, “Overview of principles of hybridization and the strategy of nucleic acids as This is shown in "says" (1993).

[0225] As used herein, the term "subject" refers to the object of treatment, observation or experiment. By way of example, the subject may include, but is not limited to, mammals such as humans. Not done.

[0226] As used herein, the term "substantially purified" refers to the product of a subject prior to purification. It contains substantially or predominantly another element that is incidental to or interacts with the element that constitutes it. As an example, a preparation of the ingredient of interest may be less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 5% %, less than about 4%, less than about 3%, less than about 2%, or less than about 1% of contaminating elements The subject matter is said to be "substantially purified" when it is "substantially The target elements that are "refined to about 70%, about 75%, about 80%, about 85%, about 90% , about 95%, about 96%, about 97%, about 98%, about 99% or more pure. In one example, a natural amino acid polypeptide or a non-natural amino acid polypeptide can be The present invention relates to a method for producing a polypeptide comprising the steps of: In one example, the preparation is purified from the host cell in the case where it is produced commercially. as) less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, Contains less than 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% contaminants A preparation of a natural amino acid polypeptide or a non-natural amino acid polypeptide is referred to as " By way of example, a naturally occurring amino acid polypeptide or a non-naturally occurring polypeptide may be used. When an amino acid polypeptide is recombinantly produced by a host cell, it is a naturally occurring amino acid polypeptide. The peptide or non-natural amino acid polypeptide comprises about 30%, about 25%, or about 30% of the dry weight of the cells. About 20%, about 15%, about 10%, about 5%, about 4%, about 3%, about 2%, or about 1%, or In one embodiment, the natural amino acid polypeptide or the non-natural amino acid polypeptide is present in a proportion less than or equal to the natural amino acid polypeptide. When a natural amino acid polypeptide is produced recombinantly by a host cell, it is referred to as a natural amino acid polypeptide. The peptide or non-natural amino acid polypeptide is about 5 g / L, about 4 g / L, or Approx. 3g / L, Approx. 2g / L, Approx. 1g / L, Approx. 750mg / L, Approx. 500mg / L, Approx. 250 mg / L, about 100mg / L, about 50mg / L, about 10mg / L, or about 1mg / L As an example, a "substantially purified" naturally occurring amino acid sequence may be present in the culture medium at or below this concentration. The amino acid polypeptide or non-natural amino acid polypeptide can be purified by a suitable method (e.g., SDS Examples of such techniques include PAGE analysis, RP-HPLC, SEC and capillary electrophoresis. When determined by the method including, but not limited to, about 30%, about 35%, about 40%, about 45%, approx. 50%, approx. 55%, approx. 60%, approx. 65%, approx. 70%, approx. 75%, approx. 80%, approx. It has a purity of 85%, about 90%, about 95%, about 99% or more.

[0227] The term "substituents", also referred to as "non-interfering substituents", refers to groups that do not substitute for other groups in a molecule. These groups include halo, C 1 ~C 10 Alkyl, C 2 ~C 10 Alkenyl, C 2 ~ 10 Alkynyl, C 1 ~C 10 Alkoxy, C 5 ~C 1 2 Aralkyl, C 3 ~C 12 Cycloalkyl, C 4 ~C 12 Cycloalkenyl, phenyl , substituted phenyl, toluolyl, xylenyl, biphenyl, C 2 ~C 12 Al Coxyalkyl, C 5 ~C 12 Alkoxyaryl, C 5 ~C 12 Aryloxyalkyl Lu, C 7 ~C 12 Oxyaryl, C 1 ~C 6 Alkylsulfinyl, C 1 ~C 10 Al Chilsulfonyl, -(CH2 ) m -O-(C 1 ~C 10 alkyl) (where m is 1 or 8), aryl, substituted aryl, substituted alkoxy, fluoroalkyl, heterocycle Radicals, substituted heterocyclic radicals, nitroalkyl, -NO 2 , -CN, -NRC(O) -(C 1 ~C 10 alkyl), -C(O)-(C 1 ~C 10 Alkyl), C 2 ~C 10 a Alkylthioalkyl, -C(O)O-(C 1 ~C 10 alkyl), -O H, -SO 2 , =S, -COOH, -NR 2 , carbonyl, -C(O)-(C 1 ~C 10 Alkyl)-CF 3 , -C(O)-CF 3 , -C(O)NR 2 , -(C 1 ~C 10 Ally ru)-S-(C 6 ~C 10 aryl), -C(O)-(C 6 ~C 10 Aryl), -(C H 2 ) m -O-(CH 2 ) m -O-(C 1 ~C 10 alkyl) (wherein each m is 1 to 8), -C(O)NR 2 , -C(S)NR 2 , -SO 2 NR 2 , -NRC( O)NR 2 , -NRC(S)NR 2and salts thereof, but are not limited to these. Each of the R groups in the above list can be H, alkyl or substituted alkyl. aryl or substituted aryl, or alkaryl. When substituents are specified by conventional chemical formulas written from left to right, they are , equally encompassing chemically identical substituents resulting from writing the structure from right to left. (e.g., -CH 2 O-OCH 2 - is equivalent to

[0228] As an example, alkyl radicals and heteroalkyl radicals (alkylene, alkenyl, aryl, heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl, heterocyclo These groups are referred to as alkyl, cycloalkenyl and heterocycloalkenyl. The substituents for the aryl group are -OR, =O, =NR, =N-OR, -NR 2 , -SR, -Halogen, -SiR 3 , -OC(O)R, -C(O)R, -CO 2 R, -CONR 2 , -OC(O)NR 2 , -NRC(O)R, -NRC(O)NR 2 , -NR(O) 2 R, - NR-C(NR 2 ) = NR, -S(O)R, -S(O) 2 R, -S(O) 2 NR 2 , -N RSO 2 R, -CN and -NO 2 These include, but are not limited to, the above. Each R group in the list can be hydrogen, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heteroalkyl, or unsubstituted aryl (including aryl substituted with 1 to 3 halogen atoms, substituted or unsubstituted alkyl, alkoxy or thioalkoxy; groups, or aralkyl groups. When bonded to an atom, they may be joined with the nitrogen atom to form a 5-, 6-, or 7-membered ring. For example, -NR 2 Contains 1-pyrrolidinyl and 4-morpholinyl This is intended to include, but is not limited to,

[0229] By way of example, the substituents for aryl and heteroaryl groups can range from 0 to 1000 aryl groups. In the range of the total number of vacant valences in the system, -OR, =O, =NR, =N- OR, -NR 2 , -SR, -Halogen, -SiR 3 , -OC(O)R, -C(O)R, - CO 2 R, -CONR 2 , -OC(O)NR 2 , -NRC(O)R, -NRC(O)NR 2 , -NR(O) 2 R, -NR-C(NR 2 ) = NR, -S(O)R, -S(O) 2 R, -S(O) 2 NR 2 , -NRSO 2 R, -CN, -NO 2 , -R, -N 3 , -CH(Ph ) 2 , Fluoro(C 1 ~C 4 ) alkoxy, and fluoro (C 1 ~C 4 )Alkyl Examples of R groups include, but are not limited to, can be hydrogen, alkyl, heteroalkyl, aryl, or heteroaryl; Not limited to these.

[0230] As used herein, the term "therapeutically effective amount" refers to a therapeutically effective amount that is effective to treat an already existing disease, illness, or condition. For an unwell patient, one or more symptoms of the disease, disorder or illness to be treated To treat or at least partially arrest or somewhat alleviate the above at least one non-natural amino acid polypeptide and / or at least one The term "non-natural amino acid modified polypeptide" refers to the amount of a composition that contains one or more modified non-natural amino acid polypeptides. The effectiveness of the treatment depends on the severity and course of the condition (disease, disorder or illness, previous treatments, the patient's overall health, These factors include, but are not limited to, the patient's overall health and response to medication, and the judgment of the treating physician. By way of example, the therapeutically effective amount may be determined by routine experimentation (dose escalation). The present invention is determined by various methods, including, but not limited to, clinical trials.

[0231] As used herein, the term "thioalkoxy" refers to a molecule substituted or unsubstituted through an oxygen atom. It refers to a sulfur-containing alkyl group bonded to

[0232] The term "thermal melting temperature" or Tm refers to the temperature at which a given molecule is melted (at a defined ionic strength, pH and concentration of nucleic acid). At equilibrium, 50% of the probes complementary to the target hybridize to the target sequence. This is the temperature.

[0233] As used herein, the term "toxic moiety" or "toxic group" refers to a compound that is capable of damaging, impairing, or Toxic moieties include auristatins, DNA Minor groove binders, DNA minor groove alkylators, enediynes, lexitropsin, zuokarma Isin, taxanes, puromycin, dolastatins, maytansinoids, vinca alkaloids Lloyd, AFP, MMAF, MMAE, AEB, AEVB, Auristatin E, Paclitaxel doxorubicin, docetaxel, CC-1065, SN-38, topotecan, morpholinodoxol Bicine, rhizoxin, cyanomorpholinodoxorubicin, dolastatin-10, echinoma Isin, Combretatstatin, Calicheamicin cin), maytansine, DM-1, netropsin, podophyllotoxin (e.g., etoposide cytoside, teniposide, etc.), bacactin and its derivatives, antitubulin substances, cryptofungal cryptophysin, combretastatin, auristatin E, vincristine, Vinblastine, vindesine, vinorelbine, VP-16, camptothecin epothilone A, epothilone B, nocodazole, colchicine, colcimid , estramustine, cemadotin, discodermolide, maytansine, eleutherobin , mechlorethamine, cyclophosphamide, melphalan, carmustine, lomustine , Semustine, Streptozotocin, Chlorozotocin, Uracil Mustard, Chlormethine , ifosfamide, chlorambucil, pipobroman, triethylenemelamine, triethylene anti-inflammatory drugs, such as antiophosphoramine, busulfan, dacarbazine, and temozolomide, itarabine (ytarabine), cytosine arabinoside, fluorouracil, floxiridine, 6-thiouracil Oguanine, 6-mercaptopurine, pentostatin, 5-fluorouracil, methotrexate Xate, 10-propargyl-5,8-dideazafolic acid, 5,8-dideazatetrahydro Folic acid, leucovorin, fludarabine phosphate, pentostatin, gemcitabine Bin, Ara-C, Paclitaxel, Docetaxel, Deoxycoformycin, Mitoma Isin-C, L-asparaginase, azathioprine, brequinar, antibiotics (e.g. , anthracyclines, gentamicin, cephalothin, vancomycin, telavancin , daptomycin, azithromycin, erythromycin, rocithromycin mycin), furazolidone, amoxicillin, ampicillin, carbenicillin, flucloxil Saccharin, methicillin, penicillin, ciprofloxacin, moxifloxacin, ofloxacin Doxycycline, minocycline, oxytetracycline, tetracycline phosphate, streptomycin, rifabutin, ethambutol, rifaximin, etc.) Antiviral drugs (e.g., abacavir, acyclovir, ampligen, cidofovir, dela Virudine, Didanosine, Efavirenz, Entecavir, Fosfonet, Gansik Lovir, Ibacitabine, Immunovir, Idoxuridine, Inosine, Lopinavir, Methisa Zon, Nexavir, Nevirapine, Oseltamivir, Penciclovir, Stavudine, Trif liridine, Truvada, Valacyclovir, Zanamivir, etc.), Daunorubicin hydrochloride, Daunorubicin nomycin, rubidomycin, cerubicin, idarubicin, doxorubicin, epirubicin morpholino and morpholino derivatives, phenoxyzombis cyclopeptides (e.g., dactinomycin syn), basic glycopeptides (e.g., bleomycin), anthraquinone glycosides (e.g., , plicamycin, mithramycin), anthracenediones (e.g., mitoxantrone ), azirinopyrroloindolediones (e.g., mitomycin), macrocyclic immunosuppressants (e.g., cyclosporine, FK-506, tacrolimus, prograf, rapamycin, etc.) etc.), navelbene, CPT-11, anastrazole, letrazole, Pecitabine, reloxafine, cyclophosphamide, ifosamide, doxorubicin Roloxafine, allocorchicine, halichondrin B, colchicine, colchicine derivatives, Maytansine, rhizoxacin, paclitaxel, paclitaxel derivatives, docetaxel, Ocolchicine, tritylcysteine, vinblastine sulfate, vincristine sulfate, cisplatin platin, carboplatin, hydroxyurea, N-methylhydrazine, epidophyllotoxin These include cefotaxime, procarbazine, mitoxantrone, leucovorin, and tegafur. "Taxane" includes, but is not limited to, any active taxane derivative or protozoan. In addition to prodrugs, paclitaxel is also included.

[0234] As used herein, the terms "treat," "treating," or "treatment" The term includes the words "to alleviate, relieve, or ameliorate the symptoms of a disease or illness, Preventing the symptoms, ameliorating or preventing the underlying metabolic causes of the symptoms, treating the disease or illness suppressing the development of a disease or illness, for example, suppressing the progression of the development of a disease or illness, To ease the mind, to reverse illness or disease, or to correct abnormalities caused by illness or disease. "Treat" includes relieving or arresting the symptoms of a disease or illness. The terms "treating" or "treatment" include prophylactic and / or therapeutic These include, but are not limited to, treatment.

[0235] As used herein, the term "water-soluble polymer" refers to a polymer that is soluble in an aqueous solvent. It refers to any polymer. Such water-soluble polymers include polyethylene glycol, Polyethylene glycol propionaldehyde, mono C 1 ~C 10 Alkoxy derivatives Kuhamono C 1 ~C 10 Aryloxy derivatives (U.S. Pat. No. 5,349,313, which is incorporated herein by reference) (described in US Pat. No. 5,252,714), monomethoxypolyethylene glycol, Polyvinylpyrrolidone, polyvinyl alcohol, polyamino acids, maleic divinyl ether anhydride, N-(2-hydroxypropyl)-methacrylamide, dextran, dextran Dextran derivatives (including dextran sulfide), polypropylene glycol, polypropylene Propylene oxide / ethylene oxide copolymer, polyoxyethylated polyol, HEPA Phosphorus, heparin fragments, polysaccharides, oligosaccharides, glycans, cellulose and cellulose derivatives ( These include, but are not limited to, methylcellulose and carboxymethylcellulose. (not included), serum albumin, starch and starch derivatives, polypeptides, polyalkylenes polyalkylene glycols and their derivatives, copolymers of polyalkylene glycols and their derivatives Derivatives, polyvinyl ethyl ether, and alpha-beta-poly[(2-hydroxy Diethyl-DL-aspartamide, etc., or mixtures thereof. As an example, the water-soluble polymer and the natural amino acid polypeptide may be Binding to a polypeptide or a non-natural polypeptide results in changes, such as improved aqueous solubility. prolonged or modified serum half-life, prolonged or modified serum half-life compared to the unmodified form Improved therapeutic half-life, improved bioavailability, regulated biological activity, extended Improved circulation time, modified immunogenicity, modified physical association properties (e.g., aggregation and and multimerization), altered receptor binding, one or more Altered binding to the binding partner of the receptor, as well as altered receptor dimerization or multimerization. In addition, the water-soluble polymer may be substituted by itself. It may or may not have biological activity.

[0236] Unless otherwise specified, mass spectrometry, NMR, HPLC, protein chemistry, Traditional methods of biochemistry, recombinant DNA technology and pharmacology are used.

[0237] The compounds described herein (non-natural amino acids, non-natural amino acid polypeptides, modified Non-natural amino acid polypeptides and reagents for producing the above-mentioned compounds. Compounds that are isotopically labeled (but are not limited to) include isotopically labeled compounds. The compounds are the same compounds as those depicted by the various formulas and structures described herein, but in one The above atoms have atomic masses or masses different from those found in nature. Examples of isotopes that may be incorporated into the compounds of the present invention are: These include isotopes of hydrogen, carbon, nitrogen, oxygen, fluorine, and chlorine (e.g., 2 H , 3 H, 13 C. 14 C.15 N, 18 O. 17 O. 35 S, 18 F, 36 Cl) is mentioned Certain isotopically labeled compounds described herein, such as compounds that incorporate radioactive isotopes (e.g. , 3 H and 14 C) isotopically labeled compounds that incorporate drugs and / or substrates It is useful in tissue distribution assays. 2 Isotopes such as H Substitution with , results in greater metabolic stability (e.g., extended in vivo half-life or low These may provide certain therapeutic benefits resulting from reduced dosage requirements.

[0238] Some of the compounds described herein (non-natural amino acids, non-natural amino acid polypeptides, Modified non-natural amino acid polypeptides and reagents for producing the above-mentioned compounds are provided. (including, but not limited to, chiral carbon atoms) have an asymmetric carbon atom and therefore are enantiomeric. They can exist as diastereomers or diastereomers. Compounds can be separated based on their physicochemical differences, e.g., by chromatography and / or filtration. They can be separated into their individual diastereomers by a method known as lactal crystallization. Enantiomers can be separated by reaction with a suitable optically active compound (e.g., alcohol). By converting the mixture of enantiomers into a mixture of diastereomers, In this case, the diastereomers can be separated and the individual diastereomers can be converted to the corresponding pure form. Convert to enantiomers (e.g., by hydrolysis). Diastereomers, enantiomers, All such isomers, including mixtures thereof, are intended to be included in any of the compositions described herein. It is considered as a part.

[0239] In additional or further embodiments, the compounds described herein (non-naturally occurring Amino acids, non-natural amino acid polypeptides, modified non-natural amino acid polypeptides, and and reagents for producing the above-mentioned compounds) are In additional or further embodiments, the present invention is used in the form of a rag. The compounds described in the document (non-natural amino acids, non-natural amino acid polypeptides, modified non-natural amino acids, The present invention also includes, but is not limited to, amino acid polypeptides and reagents for producing the above-mentioned compounds. (not limited to) are metabolized when administered to an organism that needs to produce a metabolic product, and then The generated metabolites are utilized to produce a desired effect, including a desired therapeutic effect. In further or additional embodiments, unnatural amino acids and and "modified or unmodified" non-natural amino acid polypeptide active metabolic products.

[0240] The methods and formulations described herein include, but are not limited to, unnatural amino acids, unnatural amino acid polypeptides, and N-oxides, crystalline forms (also known as polymorphs) of modified non-natural amino acid polypeptides. In certain embodiments, non-naturally occurring Amino acids, non-natural amino acid polypeptides, and modified non-natural amino acid polypeptides All tautomers of the unnatural amino acids described herein may exist as tautomers. , non-natural amino acid polypeptides, and modified non-natural amino acid polypeptides. Additionally, the non-natural amino acids, non-natural amino acid polypeptides, and the modified non-natural amino acid polypeptides are dissolved in a pharma- ceutically acceptable solvent, e.g., water and and ethanol) in unsolvated and solvated forms. The unnatural amino acids, unnatural amino acid polypeptides, and modified Solvated forms of the modified non-natural amino acid polypeptides are also described herein. It is considered that there is.

[0241] Some of the compounds described herein (non-natural amino acids, non-natural amino acid polypeptides, Modified non-natural amino acid polypeptides and reagents for producing the above-mentioned compounds are provided. (including but not limited to) may exist in several tautomeric forms. All such tautomeric forms are considered as part of the compositions described herein. Also, for example, any of the compounds described herein (non-natural amino acids, non-natural amino acid polysaccharides, etc.) can be used. To generate peptides, modified non-natural amino acid polypeptides, and compounds described above. All enol-keto forms of the reagents described herein include, but are not limited to, are considered part of the compositions described herein.

[0242] Some of the compounds described herein (non-natural amino acids, non-natural amino acid polypeptides, To generate any of the modified non-natural amino acid polypeptides and compounds described above. The reagents (including, but not limited to, the following) are acidic and have a pharma- ceutically acceptable cation. Some of the compounds described herein (non-natural amino acids, non-natural ions) may form salts. Natural amino acid polypeptides, modified non-natural amino acid polypeptides, and the above-mentioned compounds The reagents used to generate the product (including, but not limited to, the above) are basic and Thus, salts may be formed with pharma- ceutically acceptable anions. All such salts, including disalts, are contemplated herein. All salts are within the scope of the compositions described herein and may be prepared by conventional methods. For example, salts can be present in aqueous, non-aqueous, or partially aqueous media. The salt can be prepared by contacting an acidic substance with a basic substance. The extract is recovered using at least one of the following techniques: filtration, precipitation with a non-solvent, and then Alternatively, the extract may be purified by filtration, evaporation of the solvent, or, in the case of aqueous solutions, lyophilization.

[0243] Pharmaceutically acceptable salts of the non-natural amino acid polypeptides described herein are the same as or similar to the parent non-natural amino acid. The acidic protons present in amino acid polypeptides are converted to metal ions (e.g., alkali metal ions). ions, alkaline earth ions, aluminum ions, or organic base equivalents In addition, the salt forms described herein may be produced when the aryl group is replaced with any of the aryl groups. The non-natural amino acid polypeptides described above can be prepared using salts of the starting materials or intermediates. The non-natural amino acid polypeptides described herein can be prepared in the free base form of the polypeptides described herein. The non-natural amino acid polypeptide is reacted with a pharma- ceutically acceptable inorganic or organic acid. By preparing a pharma- ceutically acceptable acid addition salt (a type of pharma- ceutically acceptable salt), Alternatively, the non-natural amino acid polypeptides described herein can be in the free acid form. The non-natural amino acid polypeptides described herein can be dissolved in a pharma- ceutically acceptable inorganic base or By reacting with an organic base, a pharma- ceutically acceptable base addition salt (pharma- ceutically acceptable It can be prepared as a type of salt.

[0244] Types of pharma- ceutically acceptable salts include: (1) hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, and phosphoric acid salts. , or acid addition salts produced with inorganic acids such as acetic acid, propionic acid, and hexanoic acid , cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid , malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3-(4-hydroxy (dibenzoyl)benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, 2-Naphthalenesulfonic acid, 4-methylbicyclo-[2.2.2]oct-2-ene-1 -Carboxylic acid, glucoheptonic acid, 4,4'-methylenebis-(3-hydroxy-2 -1-ene-1-carboxylic acid), 3-phenylpropionic acid, trimethylacetic acid, tert-butylacetic acid Acid, lauryl sulfate, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, (2) Acid addition salts formed with organic acids such as stearic acid, muconic acid, etc.; The acidic protons present in the complex are metal ions (e.g., alkali metal ions, alkaline earth ions, either aluminum ions or organic base coordinates Acceptable organic bases include, but are not limited to, salts formed when the compound is reacted with an organic solvent. Examples include ethanolamine, diethanolamine, triethanolamine, and tromethamine. Acceptable inorganic bases include water, ethyl amine, ethyl ketone ... Aluminum oxide, calcium hydroxide, potassium hydroxide, sodium carbonate, sodium hydroxide Examples include Um, etc.

[0245] The corresponding counter ions of the pharma- ceutically acceptable salts of the non-natural amino acid polypeptides can be prepared in a variety of ways. methods (ion exchange chromatography, ion chromatography, capillary electrophoresis , inductively coupled plasma, atomic absorption spectroscopy, mass spectrometry, or any combination thereof. The analysis and identification can be performed using a number of techniques, including but not limited to the following: The therapeutic activity of pharma- ceutically acceptable salts of such non-natural amino acid polypeptides is described in the Examples. The test can be performed using the techniques and methods described in 87 to 91.

[0246] It will be appreciated that reference to a salt may also include solvent addition forms or crystal forms thereof, particularly solvates or Solvates include stoichiometric or non-stoichiometric amounts of a solvent. Often produced during the process of crystallization from pharma- ceutically acceptable solvents such as water and ethanol. When the solvent is water, a hydrate is formed, and when the solvent is alcohol, an alcohol is formed. Polymorphs contain different crystal packing arrangements of the same component composition of a compound. Polymorphs have different X-ray diffraction patterns, infrared spectra, melting points, density, hardness, crystal shape, and optical properties. It has chemical and electrical properties, stability, and solubility. Various factors, such as temperature and storage temperature, result in one predominant crystalline form.

[0247] Polymorphs and / or solvates of pharma-ceutically acceptable salts of non-natural amino acid polypeptides The characterization of the chemistry of the samples has been carried out using a variety of techniques, including thermal analysis, X-ray diffraction, spectroscopy, vapor sorption, and microscopy. These can be screened and characterized using methods such as, but not limited to, thermal analysis. The analytical methods include thermochemical decomposition or thermophysical treatments (including, but not limited to, polymorphic transformations) ) and such methods are used to analyze the relationships between polymorphic phases, measure weight loss, and examine glass transition temperatures. These methods include differential scanning calorimetry (DSC), DSC), Modulated Differential Scanning Calorimetry (MDCS), Thermogravimetric Analysis (TGA), and Thermogravimetric Infrared Examples of X-ray diffraction methods include, but are not limited to, single crystal analysis (TG / IR). These include, but are not limited to, crystal powder diffractometers and synchrotron radiation sources. The various spectroscopic techniques used include Raman, FTIR, UVIS, and NMR (liquid phase). and solid-state). Various microscopy techniques include, but are not limited to: Polarized light microscopy, scanning electron microscopy (SEM) with energy dispersive X-ray analysis (EDX) ), environmental scanning electron microscopy using EDX (in a gas or water vapor atmosphere); These include, but are not limited to, IR microscopy, and Raman microscopy.

[0248] BRIEF DESCRIPTION OF THE DRAWINGS The novel features of the invention are set forth with particularity in the appended claims. For further details and advantages of the present invention, see the following detailed description which sets forth illustrative embodiments, in which the principles of the invention are utilized. This will be better understood with reference to the accompanying drawings, in which:

[0249] FIG. 1 shows the binding of Her-tox to the Her2 receptor.

[0250] FIG. 2 shows the expression of Anti-Her2 mutants determined by ELISA analysis. .

[0251] FIG. 3 shows the expression of Anti-Her2 mutants determined by ELISA analysis. .

[0252] [Figure 4] Cell proliferation assay using HCC1954 breast cancer cell line and dolastatin linker derivatives FIG.

[0253] [Figure 5] Cell proliferation assay using HCC1954 breast cancer cell line and dolastatin linker derivatives FIG. 1 shows the analysis of the assay.

[0254] [Figure 6] Cell proliferation assay using SKOV-3 ovarian cancer cell line and dolastatin linker derivatives FIG. 1 shows the analysis of the assay.

[0255] [Figure 7] Cell proliferation using SKOV-3 ovarian cancer cell line and trastuzumab-tox conjugate FIG. 2: Analysis of the assay.

[0256] [Figure 8] MDA-MB-468 breast cancer cell line and cells using dolastatin linker derivatives FIG. 1 shows the analysis of proliferation assays.

[0257] [Figure 9] MDA-MB-468 breast cancer cell line and cytotoxicity using trastuzumab-tox conjugate FIG. 1 shows analysis of cell proliferation assay.

[0258] [Figure 10] Various trastuzumab-conjugated dolastatin derivatives were administered once into the cardiac cavity (3.3 Measurement of tumor volume after administration of 100 mg / kg, 10 mg / kg, or 20 mg / kg (unit: mm 3 ) FIG.

[0259] [Figure 11] Concentrations of various trastuzumab-bound dolastatin derivatives in SD rat serum FIG. 1 shows an assay format used to measure

[0260] [Figure 12] Serum concentrations (units) of various trastuzumab-bound dolastatin derivatives after a single intravenous injection : ng / mL).

[0261] [Figure 13] Serum concentrations (units) of various trastuzumab-bound dolastatin derivatives after a single intravenous injection : ng / mL. This assay measures antibody binding to the ErbB2 receptor. Detect.

[0262] [Figure 14] Serum concentrations (units) of various trastuzumab-bound dolastatin derivatives after a single intravenous injection The in vivo stability measurement was performed on trastuzumab (rastuzumab). At least two bound dolastatin derivatives are detected.

[0263] [Figure 15] Body weight and FIG. 1 shows changes in tumor volume.

[0264] [Figure 16] Established tumors of HCC1954 in SCID-bg mic mice Trastuzumab, Her2-HS122-NCD1, and Her2-HS122 / LK Figure 1 shows the antitumor effect of 145-HCD1. Mice were given a single intravenous injection on day 1 (indicated by the arrow). Data points represent group means of tumor volumes and error bars represent standard error.

[0265] [Figure 17] Various types of Dras in the MDA361DYT2 Breast(2+) xenograft model FIG. 1 shows the antitumor effects of tatin linker derivatives.

[0266] [Figure 18] Various types of Dras in the MDA361DYT2 Breast(2+) xenograft model FIG. 1 shows the antitumor effects of tatin linker derivatives.

[0267] Detailed Description of the Invention Although preferred embodiments of the present invention have been shown and described herein, these embodiments It will be apparent to those skilled in the art that the above is provided by way of example only. Numerous changes, modifications, and substitutions will occur to those skilled in the art without departing from the invention. It is to be understood that various alternatives to the embodiments of the invention described herein may be employed in practice. It should be understood that the following claims define the scope of the invention and that within the scope of these claims Methods and structures, and their equivalents, are intended to be covered hereby. .

[0268] (I. Introduction) Recently, protein targeting has become possible, allowing one to overcome many of the limitations associated with site-specific modification of proteins. A completely new technology in the field of chemical science has been reported. Specifically, new components have been The endonucleotic Escherichia coli (E. coli) (e.g., L. Wang, et al., (2001), Science 292:498-500), and the eukaryotic yeast Sacchromyces cerevisiae)(S. cerevisiae)(e.g. J. Chin et al., Science 301:964-7 (2003) These components are added to the protein synthesis machinery of the This allows for the incorporation of non-genetically encoded amino acids into proteins. Many new amino acids with novel chemical, physical or biological properties (photoaffinity Amino acids labeled with a thiol label, photoisomerizable amino acids, keto amino acids, and Using this methodology, the amber codon TAG was detected in the 144 It is efficient and has high fidelity for proteins in E. coli and yeast. For example, JW Chin et al., (2002), Journal of the American College of Hemical Society 124:9026-9027 (incorporated by reference); JW Chin, & P. G. Schultz, (2002), ChemBioChem 3(11):1135-1137 (incorporated by reference ); JW 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). These studies It is chemically unsuitable for all of the functional groups found in the 20 commonly encoded amino acids. are active and can be used to react efficiently and selectively to form stable covalent bonds It is now possible to selectively and routinely introduce chemical functional groups not found in proteins. It proves something.

[0269] (II. Overview) At one level, at least one carbonyl, dicarbonyl, oxime, hydroxyl Silylamines, aldehydes, protected aldehydes, ketones, protected ketones, thioesters Ter, ester, dicarbonyl, hydrazine, azide, amidine, imine, diamine, ketone amines, ketoalkynes, alkynes, cycloalkynes, or enediones Tools (methods, compositions, techniques) for making and using tin linker derivatives or analogs At another level, at least one unnatural amino acid is Acids, or oximes, aromatic amines, heterocycles (e.g., indoles, quinoxalines, fluoresceins, etc.) Dolastatins containing unnatural amino acids modified with phenazine, pyrazole, triazole, etc. Tools (methods, compositions, techniques) for making and using linker derivatives or analogs ) are described herein.

[0270] Such dolastatin linker derivatives containing unnatural amino acids may contain additional functionality. The functionality may include polymers, water-soluble polymers, polyethylene glycol, etc. Derivatives, second proteins, polypeptides or polypeptide analogs, antibodies or antibodies fragments, and any combination thereof. It should be noted that the various functionalities described above may be classified as members of one functionality as members of another functionality. This is not intended to imply that overlaps are not possible. Indeed, overlaps may occur in certain circumstances. By way of example only, water soluble polymers range from derivatives of polyethylene glycol. Overlaps with conductors, but the overlap is not complete, so both functionalities are listed above. .

[0271] In some embodiments, carbonyls, dicarbonyls, oximes, hydroxylamines , aldehydes, protected aldehydes, ketones, protected ketones, thioesters, esters Terephthalates, dicarbonyls, hydrazines, azides, amidines, imines, diamines, ketoamines, Toxic group linker derivatization including ketoalkyne, alkyne, cycloalkyne, or enedione In some embodiments, the toxic group derivative is In another embodiment, the linker comprises at least one non-naturally occurring Amino acids, or oximes, aromatic amines, heterocycles (e.g., indoles, quinoxalates, Toxic compounds containing unnatural amino acids modified with amines (e.g., phenazines, pyrazoles, triazoles, etc.) The present invention provides tools (methods, compositions, techniques) for making and using derivatives or analogs of the . It's stated in the fine print.

[0272] In some embodiments, such toxic group derivatives comprising unnatural amino acids further include The functional groups may include polymers, water-soluble polymers, polyethylene glycols, etc. Derivatives of Recall, Second Proteins, Polypeptides or Polypeptide Analogs, Antibodies or antibody fragments, and any combination thereof. In a particular embodiment, the toxic group derivative is a dolastatin or an auridine. In one particular embodiment, the toxic group derivative is dolastatin-10. It should be noted that the various above functionalities may be used in conjunction with one another. It is not intended to imply that classification is not possible. Indeed, it may be important in certain circumstances. By way of example only, water soluble polymers range from polyethylene glycol Although it overlaps with its derivatives, this overlap is not complete, so both functionalities may not be present in the There are.

[0273] In one embodiment of the present invention, the preparation of a toxic moiety having a linker is described, These linkers reduce the toxicity of the moiety in vivo, while retaining the pharmacological activity of the toxic moiety. In some embodiments, the toxicity of the attached toxic group is such that, when administered to an animal or human, While retaining pharmacological activity, compared to free toxic groups or toxic group derivatives containing labile bonds, In some embodiments, the attached toxic group (e.g., dolastatin) is reduced or eliminated. Even when the dose of the dolastatin derivatives (linker derivatives, dolastatin derivatives linked to unnatural amino acids) was increased, In some embodiments, the toxic moiety can be attached to the cytotoxic moiety and administered to animals or humans in a highly safe manner. The resulting unnatural amino acid polypeptides (e.g., dolastatin derivatives) are useful in vivo and in vivo. In some embodiments, the non-natural amino acid linked to the toxic moiety provides stability in vitro. Non-acid polypeptides (e.g., dolastatin-10 derivatives) are effective and highly toxic. It is less toxic than other groups (e.g., dolastatin-10).

[0274] III. Dolastatin Linker Derivatives At one level, at least one unnatural amino acid or a carbonyl, dicarbonyl, The dora, which contains unnatural amino acids modified with nyl, oxime, or hydroxylamine groups, Tools (methods, compositions) for making and using statin linker derivatives or analogs Such dolastatin ligands that include unnatural amino acids are described herein. The anchor derivatives may contain further functionalities such as polymers, water-soluble Polymers, polyethylene glycol derivatives, polypeptides or polypeptide analogs The second protein includes a second protein, an antibody or antibody fragment, and any combination thereof. However, the various above functionalities may be members of a certain functionality. is not intended to suggest that it cannot be classified as a member of a different functionality. In fact, there is overlap depending on the particular situation. As just one example, water-soluble polymers fall into the category of The range of the ethylene glycol derivatives overlaps with that of the polyethylene glycol derivatives, but this overlap is not complete. Therefore, both functionalities are listed above.

[0275] In some embodiments, the dolastatin ligands are modified using these methods, compositions, and techniques. Methods for selecting and designing anchor derivatives are described herein. Statin linker derivatives, just as one example, have been used in high-throughput screening methods. As part of a method for the design, synthesis, characterisation and / or detection of a large number of polypeptides They may be newly designed based on the researcher's interests. Suitable dolastatin linker derivatives are those that are compatible with known or partially characterized polypeptides. As just one example, dolastatins have received great acclaim in the scientific community. It is the subject of much research, and new compounds may be designed based on the dolastatin structure. The principles for selecting which amino acids to substitute and / or modify are described herein, respectively. The choice of which modifications to employ is also described herein and experimentally. It can be used according to the needs of the person or end user. Manipulating the therapeutic efficacy of polypeptides and improving their safety profile. and modulating the pharmacokinetics, pharmacology, and / or pharmacodynamics of the polypeptide. Modulation of pharmacological and / or potency includes, but is not limited to, To name just a few, improving water solubility and bioavailability. In addition, it is possible to increase the half-life in the blood, increase the therapeutic half-life, and modulate immunogenicity. These include modulating biological activity or increasing circulation time. Additionally, such modifications can, by way of example only, provide additional functionality to the polypeptide. These modifications include incorporating antibodies, and any combination of the above mentioned modifications.

[0276] Also, those having or containing oxime, carbonyl, or hydroxylamine groups Described herein are dolastatin linker derivatives that can be modified to This aspect includes the steps of preparing, purifying, characterizing, and using such dolastatin linker derivatives. The present invention also includes methods for using the same.

[0277] The dolastatin linker derivative may be 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 9 or 10 or more carbonyl or dicarbonyl groups, oxime groups, hydroxyl groups, The dolastatin linker may contain a hydroxylamine group, or a protected form thereof. The derivatives may be the same or different. For example, in a derivative, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 containing more than 20 different reactive groups , 13, 14, 15, 16, 17, 18, 19, 20 or more different sites.

[0278] A. Structure and synthesis of dolastatin linker derivatives: electrophilic and nucleophilic groups Dolastatin derivatives containing a hydroxylamine group (also called aminooxy) in the linker The conductors can be reacted with a variety of electrophilic groups, including but not limited to, with PEG and other water-soluble polymers. ) complexes such as hydrazine, hydrazide, and semicarbazide. As shown in Fig. 1, the enhanced nucleophilicity of the aminooxy group leads to the formation of aryl groups containing carbonyl or dicarbonyl groups. This allows the aminooxy group to react efficiently and selectively with a variety of molecules that react with the aminooxy group. The carbonyl or dicarbonyl group may be a ketone, aldehyde, or similar chemical reaction. Other functional groups that have a reactive property include, but are not limited to, those described in, for example, Shao, J. and Tam, J., J. Am. Chem. Soc. 117:3893-3899 (1995); H. Hang and C. Bertozzi, Acc. C See Hem. Res. 34(9): 727-736 (2001). Reaction with a hydrazine group results in the corresponding hydrazine. The oximes are generally obtained by cleaving the aminooxy group from a carbonyl or dicarbazole. These carbonyl or dicarbonyl groups are Examples of functional groups that may be used include ketones, aldehydes, or other functional groups with similar chemical reactivity. In some embodiments, the linker is not limited to an azide, an alkyne, or a silyl group. Chloroalkyne-containing dolastatin derivatives can be used in cycloaddition reactions (e.g., 1,3-dipolar addition). This allows the coupling of molecules via cyclization or azide-alkyne Huisgen cycloaddition. U.S. Pat. No. 7,807,619, which is incorporated by reference herein for the scope of the reaction. (described in ).

[0279] Thus, in certain embodiments, the linker may include a hydroxylamine, an aldehyde, a protected protected aldehydes, ketones, protected ketones, thioesters, esters, dicarbonyls, Hydrazines, amidines, imines, diamines, ketoamines, ketoalkynes, and enedioates Hydroxylamine group, (has the same reactivity as the hydroxylamine group, Hydroxylamine-like groups (structurally similar to amine groups) a masked hydroxylamine group (which can also be converted to a hydroxylamine group upon deprotection) or A dolastatin containing a protected hydroxylamine group (having a reactivity similar to that of a hydroxylamine group) Described herein are dolastatin linker derivatives having the formula: This includes compounds having the structures I), (III), (IV), (V), and (VI).

[0280] [ka] JPEG2024170587000059.jpg193169

[0281] (In the formula, Z has the structure

[0282] [ka]

[0283] R 5 H,COR 8 , alkyl having 1 to 6 carbon atoms, or thiazole; R 8 is OH or -NH-(alkylene-O) n -NH 2 and R 6 is OH or H, Ar is phenyl or pyridine; R 7 is an alkyl group having 1 to 6 carbon atoms or hydrogen; Y and V are hydroxylamine, methyl, aldehyde, and protected aryl groups, respectively. aldehydes, ketones, protected ketones, thioesters, esters, dicarbonyls, hydra azines, amidines, imines, diamines, azides, ketoamines, ketoalkynes, alkynes, cyclohexane selected from the group consisting of chloroalkynes, and enediones; L, L 1 , L 2 , L 3 , and L 4 are a bond, -alkylene-, and -alkylene, respectively. -C(O)-, -alkylene-J-, -(alkylene-O) n -Alkylene-, -(A alkylene-O) n -Alkylene-C(O)-, -(alkylene-O) n -J-, -(Alkyle N-O) n -J-alkylene, -(alkylene-O) n -(CH 2 ) n’ -NHC(O)- (CH 2 ) n’’ -C(Me) 2 -SS-(CH 2 ) n’’’ -NHC(O)-(Al Kiren-O) n’’’’ -Alkylene, -(alkylene-O) n -Alkylene-W-, - Alkylene-C(O)-W-, -(alkylene-O) n -Alkylene-J-, -Alkylene' -J-(alkylene-O) n -Alkylene-, -(alkylene-O) n -Alkylene-J-Alkyl '-, -J-(alkylene-O) n -Alkylene-, -(alkylene-O) n -Alkylene- J-(alkylene-O) n '-Alkylene-J'-, -W-, -Alkylene-W-, Alkylene'- J-(alkylene-NMe) n -Alkylene-W-, -J-(Alkylene-NMe) n -Alkylene-W- , -(alkylene-O) n -Alkylene-UC(O)-, -(alkylene-O) n -a -U-alkylene;-J-alkylene-NMe-alkylene'-NMe-alkylene''- W-, and -alkylene-J-alkylene'-NMe-alkylene''-NMe-alkylene'''- W-, W has the structure

[0284] [ka]

[0285] U has the structure

[0286] [ka]

[0287] Each J and J′ independently has the structure

[0288] [ka]

[0289] n, n', n'', n''', and n'''' are each independently an integer equal to or greater than 1. is a number, Or, L is absent, Y is methyl and R 5 is COR 8 and R 8 Ha-NH(A alkylene-O) n -NH 2 It is.) Such dolastatin linker derivatives may be in the form of a salt, and may be linked to an unnatural amino acid, It may be incorporated into a polymer, polysaccharide, or polynucleotide and optionally post-translationally modified. stomach.

[0290] In certain embodiments of compounds of Formula (I), (III), and (V), R 5 Is Thiazo Certain embodiments of the compounds of formula (I), (III), and (V) are In the embodiment, R 5 is hydrogen. In embodiments, R 5 is methyl, ethyl, propyl, isopropyl, butyl, isobutyl , sec-butyl, tert-butyl, pentyl, or hexyl. In certain embodiments of compounds (III) and (V), R 5 is -NH-(alkylene -O) n -NH 2 and the alkylene is -CH 2 -, -CH 2 CH 2 -, -CH 2 CH 2 CH 2 -, -CH 2 CH 2 CH 2 CH 2 -, -CH 2 CH 2 CH 2 CH 2 CH 2 - , -CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 -, -CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 -, -CH 2 CH 2 CH 2 CH 2 CH 2CH 2 CH 2 CH 2 -, -CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 -, -CH 2 CH 2 CH 2 CH 2 CH 2 C H 2 CH 2 CH 2 CH 2 CH 2 -, -CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 - or -CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 Certain embodiments of the compounds of formula (IV) and (VI) In this state, n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 1 4, 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, 5 4, 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, 9 4, 95, 96, 97, 98, 99, or 100.

[0291] In some embodiments, Y is azide. In other embodiments, Y is cycloalkyl. In certain embodiments, the cycloalkyne has the structure:

[0292] [ka]

[0293] (In the formula, Each R 19 are independently selected from alkyl having 1 to 6 carbon atoms, alkoxy having 1 to 6 carbon atoms, ester alkyl, ether, thioether, aminoalkyl, halogen, alkyl ester, aryl Esters, amides, arylamides, alkyl halides, alkylamines, alkyls Sulfonic acid, alkyl nitro, thioester, sulfonyl ester, halosulfonyl, nitro is selected from the group consisting of alkyl, alkylnitrile, and nitro; q is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11. In certain embodiments of compounds of Formula (I), (III), and (V), R 6 is hydrogen In some embodiments of the compounds of Formula (I), (III), and (V), R 6 teeth, It is hydroxy.

[0294] In certain embodiments of compounds of Formula (I), (III), and (V), Ar is phenyl. It is.

[0295] In certain embodiments of the compounds of formula (I), (III), (IV), (V), and (V), , R 7 is methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, isobutyric acid, butyl, tert-butyl, pentyl, or hexyl. In certain embodiments of compounds (IV), (V), and (V), R 7 is hydrogen.

[0296] In certain embodiments of compounds of Formula (I), (III), and (V), Y is hydroxy. Amines, aldehydes, protected aldehydes, ketones, protected ketones, thioesters amines, esters, dicarbonyls, hydrazines, amidines, imines, diamines, ketoamines, It is a ketoalkyne, an alkyne, or an enedione.

[0297] In certain embodiments of compounds of Formula (IV) and (VI), V is hydroxylamine, Methyl, aldehyde, protected aldehyde, ketone, protected ketone, thioester , esters, dicarbonyls, hydrazines, amidines, imines, diamines, ketoamines, ketoamines, Toarkyne, and enedione.

[0298] In certain embodiments of the compounds of formula (I), (III), (IV), (V), and (VI), , L, L 1 , L 2 , L 3 , and L 4 each independently represents a cleavable linker or Formula (I), (III), (IV), (V), and (VI) In certain embodiments of the compound of formula (I), L, L 1 , L 2, L 3 , and L 4 are each independently , an oligo(ethylene glycol)-derivatized linker.

[0299] In certain embodiments of the compounds of formula (I), (III), (IV), (V), and (VI), The alkylene, alkylene', alkylene'', and alkylene''' are Independently, -CH 2 -, -CH 2 CH 2 -, -CH 2 CH 2 CH 2 -, -CH 2 CH 2 CH 2 CH 2 -, -CH 2 CH 2 CH 2 CH 2 CH 2 -, -CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 -, -CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 -, -CH 2 CH 2 C H 2 CH 2 CH 2 CH 2 CH 2 CH 2 -, -CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 -, -CH2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 -, -CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 -,Also is -CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 - Formulae (XIV), (XV), (XVI), (XVII), and (XVIII) In certain embodiments of the compound, n, n', n'', n''', and n'''' are each , 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, 2 9, 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, 6 9, 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.

[0300] B. Structure and Synthesis of Dolastatin Linker Derivatives: Hydroxylamine Group Thus, in one embodiment, the linker may include a hydroxylamine group, (hydroxylamine It has the same reactivity as the hydroxylamine group and is structurally similar to the hydroxylamine group. Amine-like groups, masked hydroxyls (which can be converted to hydroxylamine groups at any time) Amine groups or protected groups (which have similar reactivity to hydroxylamine groups upon deprotection) Described herein are dolastatin derivatives that contain an incorporated hydroxylamine group. Such dolastatin linker derivatives include compounds having the structure of formula (I):

[0301] [ka]

[0302] (In the formula, Z has the structure

[0303] [ka]

[0304] R 5 H,COR 8 , alkyl having 1 to 6 carbon atoms, or thiazole; R 8 is OH or -NH-(alkylene-O) n -NH 2 and R 6 is OH or H, Ar is phenyl or pyridine; R 7is an alkyl group having 1 to 6 carbon atoms or hydrogen; Y is NH 2 -O- or methyl; L is -alkylene-, -alkylene-C(O)-, -(alkylene-O) n -Arki Rene-, -(alkylene-O) n -Alkylene-C(O)-, -(alkylene-O) n - (CH 2 ) n’ -NHC(O)-(CH 2 ) n’’ -C(Me) 2 -SS-(CH 2 ) n’’’ -NHC(O)-(alkylene-O) n’’’’ -Alkylene, -(alkylene -O) n -Alkylene-W-, -Alkylene-C(O)-W-, -(Alkylene-O) n -alkylene-U-alkylene-C(O)-, and -(alkylene-O) n -Arki is a linker selected from the group consisting of: aryl-U-alkylene; W has the structure

[0305] [ka]

[0306] U has the structure

[0307] [ka]

[0308] Or, L is absent, Y is methyl and R 5 is COR 8 and R 8 Ha-NH(A alkylene-O) n-NH 2 where n, n', n'', n''', and n'''' are , each independently an integer of 1 or greater.) Such dolastatin linker derivatives may be in the form of a salt, and may be linked to an unnatural amino acid, It may be incorporated into a polymer, polysaccharide, or polynucleotide and optionally post-translationally modified. stomach.

[0309] In certain embodiments of the compound of Formula (I), R 5 is thiazole. Compounds of formula (I) In some embodiments, R 5 is hydrogen. In certain embodiments of the compound of Formula (I), R 5 is methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl or hexyl. In this state, R 5 is -NH-(alkylene-O) n -NH 2 and the alkylene is - CH 2 -, -CH 2 CH 2 -, -CH 2 CH 2 CH 2 -, -CH 2 CH 2 CH 2 CH 2 - , -CH 2 CH 2 CH 2 CH 2 CH 2 -, -CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 -, -CH 2 CH 2 CH 2 CH 2 CH2 CH 2 CH 2 -, -CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 -, -CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 - , -CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 -, -CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 - or -CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 Formula (I) In certain embodiments of the compound, 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, 2 4, 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, 6 4, 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.

[0310] In certain embodiments of the compound of Formula (I), R 6 is hydrogen. Some of the compounds of formula (I) In some embodiments, R 6 is hydroxy.

[0311] In certain embodiments of a compound of Formula (I), Ar is phenyl.

[0312] In certain embodiments of the compound of Formula (I), R 7 is methyl, ethyl, propyl, isopropyl Pyrene, butyl, sec-butyl, isobutyl, tert-butyl, pentyl or heptyl In certain embodiments of the compound of Formula (I), R 7 is hydrogen.

[0313] In certain embodiments of the compound of Formula (I), Y is a hydroxylamine, an aldehyde, a protected Protected aldehydes, ketones, protected ketones, thioesters, esters, dicarbonyls , hydrazine, amidine, imine, diamine, ketoamine, ketoalkyne, or ene In certain embodiments of the compound of Formula (I), V is hydroxylamine, methyldione. aldehydes, aldehydes, protected aldehydes, ketones, protected ketones, thioesters, esters Stearyl, dicarbonyl, hydrazine, amidine, imine, diamine, ketoamine, ketoa Lukin, and Enzion.

[0314] In certain embodiments of a compound of Formula (I), each L is independently a cleavable linker or a non- In certain embodiments of a compound of Formula (I), each L is independently an oxidase. It is a oligo(ethylene glycol) derivatized linker.

[0315] In certain embodiments of a compound of Formula (I), each said alkylene is -CH 2 -, -CH 2 C H 2 -, -CH 2 CH 2 CH 2 -, -CH 2 CH 2 CH 2 CH 2 -, -CH 2 CH 2 CH 2 CH 2 CH 2 -, -CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 -, -CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 -, -CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 - , -CH 2 CH2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 -, -CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 -, -CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 - or -CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 Certain embodiments of the compounds of formula (I) In this state, n, n', n'', n''', and n'''' are 0, 1, 2, 3, and 4, respectively. , 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, 3 2, 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, 7 2, 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, It is either 99 or 100.

[0316] In certain embodiments, the dolastatin linker derivative is a compound having the structure of formula (II): Includes.

[0317] [ka]

[0318] In some embodiments of the compound of Formula (II), L is -(alkylene-O). n -Al In some embodiments, each alkylene is -CH 2 CH 2 -Yes n=3, R 7 is methyl. In some embodiments of the compound of formula (II), Each alkylene is -CH 2 CH 2 - and R 7 is methyl or hydrogen In some embodiments of the compound of Formula (II), L is -(alkylene-O). n -a In some embodiments of the compound of formula (II), each alkene is -C(O)-. N is -CH 2 CH 2 -, n=4, R 7 is methyl. In some embodiments of the compound, L is -(alkylene-O). n -(CH 2 ) n’ -NH C(O)-(CH 2 ) n’’ -C(Me) 2-SS-(CH 2 ) n’’’ -NHC(O )-(Alkylene-O) n’’’’ -alkylene. Some of the compounds of (II) In embodiments, each alkene is -CH 2 CH 2 -, n=1, n'=2 , n''=1, n''=2, n''=4, and R 7 is methyl Such dolastatin linker derivatives may be in the form of a salt and may include unnatural amino acids. by incorporation into acids, polymers, polysaccharides, or polynucleotides, optionally post-translationally modified. Good too.

[0319] In certain embodiments of a compound of Formula (II), each L is independently a cleavable linker or In certain embodiments of the compound of Formula (II), each L is independently , an oligo(ethylene glycol)-derivatized linker.

[0320] In certain embodiments of the compound of Formula (II), R 7 are methyl, ethyl, propyl, isopropyl propyl, butyl, sec-butyl, isobutyl, tert-butyl, pentyl, or In certain embodiments of the compound of Formula (II), R 7 is hydrogen.

[0321] In certain embodiments of a compound of Formula (II), each said alkylene is -CH 2 -, -CH 2 CH 2 -, -CH 2 CH 2 CH 2 -, -CH 2 CH 2 CH 2 CH 2 -, -CH 2CH 2 C H 2 CH 2 CH 2 -、-CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 -、-CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 -、-CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 -、-CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 -、-CH 2 CH 2 C H 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 -、-CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 -、または、-CH 2 CH 2 CH 2 CH 2 CH 2CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 In some embodiments, the compound of formula (II) In the embodiment, n, n', n'', n''', and n'''' are 0, 1, 2, , 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 1 8, 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, 5 8, 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, 9 8, 99, or 100.

[0322] Such dolastatin linker derivatives are represented by the formula (III), (IV), (V), or This includes compounds having the structure (VI).

[0323] [ka] JPEG2024170587000071.jpg169169 JPEG2024170587000072.jpg108169

[0324] (In the formula, Z has the structure

[0325] [ka]

[0326] R 5 H,COR 8 , alkyl having 1 to 6 carbon atoms, or thiazole; R 8 is OH, R 6 is OH or H, Ar is phenyl or pyridine; R 7 is an alkyl group having 1 to 6 carbon atoms or hydrogen; Y is NH 2 -O-, V is O-NH 2 and L 1 , L 2 , L 3 , and L 4 each independently represents a bond, -alkylene-, -(a alkylene-O) n -Alkylene-J-, -Alkylene'-J-(Alkylene-O) n -Alkylene -, -J-(alkylene-O) n -Alkylene-, -(alkylene-O) n -Alkylene-J-( Alkylene-O) n '-Alkylene-J'-, -(Alkylene-O) n -Alkylene-J-Alkyl Alkylene'-, -W-, -Alkylene-W-, Alkylene'-J- (Alkylene-NMe) n -Alkire -W-, -J-(Alkylene-NMe) n -Alkylene-W-, -J-Alkylene-NMe-Alkylene -Alkylene'-NMe-alkylene''-W-, and -Alkylene-J-alkylene'-NMe-alkylene -NMe-alkylene-W-; W has the structure

[0327] [ka]

[0328] Each J and J′ independently has the structure

[0329] [ka]

[0330] Each n and n' is independently an integer greater than or equal to 1. The derivative may be in the form of a salt, and may be an unnatural amino acid, a polymer, a polysaccharide, or a polysaccharide. It may be incorporated into nucleotides and optionally post-translationally modified.

[0331] In certain embodiments of a compound of Formula (III), (IV), (V), or (VI), R 5 is thiazole. Any of the compounds of formula (III), (IV), (V) or (VI) In certain embodiments, R 5 is hydrogen. In certain embodiments of the compounds of formula (III), (IV), ( In certain embodiments of compounds of formula (V), (V), or (VI), R 7 is methyl. In certain embodiments of compounds of formula (IV), (V), or (VI), n and n′ are is an integer from 0 to 20. In some embodiments, n and n' are integers from 0 to 10. In certain embodiments of the compounds of formula (V), (VI), or (VI), n and n′ are integers from 0 to 5. do.

[0332] In certain embodiments of compounds of Formula (III) and (V), R 5 is thiazole or kaolin In certain embodiments of the compounds of formula (III) and (V), R 5 Is Meth ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert -butyl, pentyl or hexyl. In certain embodiments, R 5 is -NH-(alkylene-O) n -NH 2 wherein the alkylene N is -CH 2 -, -CH 2 CH 2 -, -CH 2 CH 2 CH 2 -, -CH 2 CH 2 CH 2 CH 2 -, -CH 2 CH 2 CH 2 CH 2 CH 2 -, -CH 2 CH 2 CH 2 CH 2 CH 2 C H 2 -, -CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 -, -CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 -, -CH 2 CH 2 CH 2 CH 2 CH2 CH 2 CH 2 CH 2 CH 2 -, -CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 -, -C H 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 - or -C H 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 -It is. In certain embodiments of the compound of Formula (I), 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, 3 6, 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, 7 6, 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 do.

[0333] In certain embodiments of compounds of Formula (III), (IV), (V), and (VI), R 6 is hydrogen. Some of the compounds of formula (III), (IV), (V), and (VI) In the embodiment, R 6 is hydroxy.

[0334] In certain embodiments of the compounds of Formula (III), (IV), (V), and (VI), Ar is phenyl.

[0335] In certain embodiments of compounds of Formula (III), (IV), (V), and (VI), R 7 is methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, isobutyl, tert-butyl, pentyl, or hexyl. ), and in certain embodiments of compounds of (VI), R 7 is hydrogen.

[0336] In certain embodiments of compounds of Formula (III) and (V), Y is hydroxylamine, Aldehydes, protected aldehydes, ketones, protected ketones, thioesters, esters dicarbonyl, hydrazine, amidine, imine, diamine, ketoamine, ketoalkyl In certain embodiments of the compounds of Formula (IV) and (VI), , V is hydroxylamine, methyl, aldehyde, protected aldehyde, ketone, protected Protected ketones, thioesters, esters, dicarbonyls, hydrazines, amidines, imines These include amines, diamines, ketoamines, ketoalkynes, and enediones.

[0337] A compound of formula (XIV), (XV), (XVI), (XVII), (XVII) In this embodiment, L 1 , L 2 , L 3 , and L 4 are each independently a cleavable linker or or a non-cleavable linker. In certain embodiments of compounds of formula (XVIII), L 1 , L 2 , L 3 , and L 4 teeth, Each is independently an oligo(ethylene glycol)-derivatized linker.

[0338] In certain embodiments of the compounds of formula (III), (IV), (V), and (VI), Each alkylene is -CH 2 -, -CH 2 CH 2 -, -CH 2 CH 2 CH 2 -, -CH 2 C H 2 CH 2 CH 2 -, -CH 2 CH 2 CH 2 CH 2 CH 2 -, -CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 -, -CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 -, -CH 2 CH 2 CH2 CH 2 CH 2 CH 2 CH 2 CH 2 -, -CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 C H 2 CH 2 CH 2 -, -CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 -, -CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 -,Ma Or, -CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 In certain embodiments of the compounds of formula (III), (IV), (V), and (VI), The alkylene is methylene, ethylene, propylene, butylene, pentylene, hexylene, It is silene or heptylene.

[0339] In certain embodiments of compounds of Formula (III), (IV), (V), and (VI), n, n' and n' are 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, 2 5, 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, 6 5, 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.

[0340] In certain embodiments, the dolastatin linker derivative is a compound having the structure of formula (II): Includes.

[0341] [ka]

[0342] In certain embodiments of the compound of Formula (VII), L 1 H-(alkylene-O) n -Alkylene- J- and L 2 is -alkylene'-J'-(alkylene-O) n '-Alkylene-, L 3 teeth,- J''-(alkylene-O) n ''-Alkylene-, alkylene is -CH 2 CH 2 -And, Al Kiren' is -(CH 2 ) 4 where n is 1, and n' and n'' are 3; J has the structure

[0343] [ka]

[0344] J′ and J″ have the following structure:

[0345] [ka]

[0346] R 7 In certain embodiments of a compound of Formula (VII), L 1 is -J-(A alkylene-O) n -alkylene-, and L 2 H-(alkylene-O) n' -Alkylene-J'- alkylene'-, L 3 H-(alkylene-O) n'' -alkylene-J''-, Ren is -CH 2 CH 2 -, and alkylene' is -(CH 2 ) 4 -, n is 1, n′ and n″ are 4, and J, J′, and J″ have the following structures:

[0347] [ka]

[0348] Such dolastatin linker derivatives may be in the form of a salt, and may be linked to a non-natural amino acid , polymers, polysaccharides, or polynucleotides, and optionally post-translationally modified. good.

[0349] In one embodiment, the compounds of Formulae (I)-(VII) can be prepared in aqueous solution under mildly acidic conditions. In one embodiment, the compounds of formula (I)-(VII) are In one embodiment, the compound of formula Compounds (I) to (VII) are stable in aqueous solution under weakly acidic conditions for at least 5 days. In one embodiment, the weakly acidic conditions are pH 2 to 8.

[0350] The methods and compositions provided and described herein comprise at least one carbonyl an alkyl or dicarbonyl group, an oxime group, a hydroxylamine group, or a protected or The present invention includes polypeptides that include dolastatin linker derivatives that contain masked forms. By introducing at least one reactive group into the dolastatin linker derivative, While not reacting with the amino acids that can be used, it is possible to react with one or more dolastatin linker derivatives in a specific This allows for applications of conjugation chemistry including, but not limited to, the ability to participate in chemical reactions. The dolastatin linker derivative side chains included herein are described or Utilizing chemical methodologies suited to specific functional groups or substituents present in the tatin linker derivatives It can be modified as follows.

[0351] The methods and compositions of dolastatin linker derivatives described herein are directed to a variety of A composite of a substance having a functional group, substituent, or moiety and another substance is provided. Other substances include polymers, water-soluble polymers, derivatives of polyethylene glycol, etc. 2. Protein or polypeptide or polypeptide analog, antibody or antibody fragment The present invention is not limited to the above.

[0352] In one embodiment, the compounds described herein include those of Formula (I)-(VII). The dolastatin linker derivatives, linkers, and reagents are synthesized in aqueous solution under mildly acidic conditions (pH 2 In another embodiment, the compound is stable under conditions including, but not limited to, conditions of Such compounds are stable in aqueous solution under mildly acidic conditions for at least one month. In one embodiment, such compounds have a molecular weight of at least 2.0, preferably 2.0, more ... In certain embodiments, such compounds are stable in aqueous solution under mildly acidic conditions. It is stable for at least 5 days.

[0353] In another aspect of the compositions, methods, techniques, and strategies described herein, the above-mentioned " Either modified or unmodified non-natural amino acid dolastatin linker derivatives were studied or This embodiment includes, by way of example only, methods for using "modified or unmodified" non-natural The present invention also benefits from the use of dolastatin linker derivatives that contain natural amino acid polypeptides or proteins. Therapeutic, diagnostic, assay-based, industrial and cosmetic uses , plant biology applications, environmental applications, energy production applications, consumer product applications, and and / or military uses.

[0354] Dolastatin linker derivatives include, but are not limited to, the following: do not have.

[0355] [ka] JPEG2024170587000081.jpg225169 JPEG2024170587000082.jpg212169 JPEG2024170587000083.jpg214169 JPEG2024170587000084.jpg216169 JPEG2024170587000085.jpg159169

[0356] IV. Unnatural Amino Acid Derivatives The unnatural amino acids used in the methods and compositions described herein include the following four types: That is, (1) the unnatural amino acid has at least one of the following properties in its side chain: At least one functional group in 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 Orthogonal to the chemical reactivity of valine, or a polypeptide containing the above unnatural amino acid A small number of amino acids that are at least orthogonal to the chemical reactivity of the naturally occurring amino acids present in peptides. (2) Having at least one property and / or activity and / or reactivity. The unnatural amino acids are chemically distinct from the 20 common genetically encoded amino acids. (3) The unnatural amino acid is preferably a naturally occurring amino acid. The amino acid has a similar stability to the amino acid described above, or is stable to polypeptides under typical physiological conditions. More preferably, such incorporation is via an in vivo system. (4) The unnatural amino acid can be produced by the addition of an oxime functional group. or preferably, the biological characteristics of the polypeptides containing the unnatural amino acids. under conditions that do not destroy the biological properties (provided that such destruction of biological properties is for the purpose of modification / transformation) (This is of course not the case if the desired pH is desired) or under aqueous conditions with a pH between about 4 and about 8. or under conditions that allow the transformation to occur in the presence of the unnatural amino acid. Transformation of the electrophilic moiety into an oxime group by reaction with a reagent under conditions in which the electrophilic moiety is an electrophilic moiety Any number of unnatural amino acids can be incorporated into the polypeptide. The unnatural amino acid can be introduced as a protected oxime or a masked oxime. oxime, or after deprotection or unmasking, can be transformed into an oxime group. The unnatural amino acid may further include a protecting or masking group that can be used to , which is transformed into a carbonyl or dicarbonyl group after deprotection or unmasking This allows the reaction with hydroxylamines or oximes to form oxime groups. a protected carbonyl group or a protected dicarbonyl group, which can be formed It may further comprise a masked carbonyl group or a masked dicarbonyl group. stomach.

[0357] Unnatural amino acids that can be used in the methods and compositions described herein include: Amino acids with novel functional groups, amino acids that interact covalently or non-covalently with other molecules modified by acids, glycosylated amino acids (e.g., sugar-substituted serine), and other carbohydrates. amino acids containing keto groups, aldehyde-containing amino acids, polyethylene glycols or Other polyether-containing amino acids, amino acids substituted with heavy elements, Chemically cleavable and / or photocleavable amino acids, which have extended chain length compared to natural amino acids Long side chains (polyethers or long chain hydrocarbons (greater than about 5 or about 10 carbons) These include, but are not limited to, amino acids that are soluble in water, amino acids that contain carbon-linked sugars, amino acids that are redox-active, amino acids that are These include thioacid-containing amino acids, as well as amino acids containing one or more toxic moieties. , but not limited to these.

[0358] In some embodiments, the unnatural amino acid comprises a glycan moiety. Examples of nonaic acids are N-acetylglucosaminyl-L-serine, N-acetylglucosaminyl-L-serine, -Galactosaminyl-L-serine, N-Acetyl-L-glucosaminyl-L-threonine , N-acetyl-L-glucosaminyl-L-asparagine and O-mannosaminyl-L Examples of such amino acids include, but are not limited to, -serine. The naturally occurring N- or O-type linkages between amino acids and glycans are not normally found in nature. Covalent bonds that cannot be formed (e.g., alkenes, oximes, thioethers, and amides) Examples of the substituted aryl groups include, but are not limited to, those described above. Examples of such amino acids include glycans not normally found in naturally occurring proteins (e.g., 2- deoxy-glucose, and 2-deoxygalactose).

[0359] Incorporating unnatural amino acids into a polypeptide Chemical moieties provide various advantages and manipulations of the polypeptide. For example, (keto functional groups or Specific reactions of carbonyl or dicarbonyl functional groups (such as aldehyde or aldehyde functional groups) The activity was evaluated using a number of either hydrazine- or hydroxylamine-containing reagents. Allows for selective modification of proteins in vivo and in vitro. Natural amino acids can be useful, for example, for phasing X-ray structure data. Site-specific introduction of heavy atoms using amino acids provides selectivity and flexibility in choosing the positions for the heavy atoms. Photoreactive unnatural amino acids (benzophenones and aryl azides ( and amino acids having side chains such as, but not limited to, phenylazide. (including, but not limited to, in vivo and in vitro expression of polypeptides) An example of a photoreactive amino acid is p-azido- Examples of phenylalanine include, but are not limited to, p-benzoyl-phenylalanine and p-benzoyl-phenylalanine. Polypeptides having photoreactive amino acids do not, as a result, provide photoreactive groups. In one non-limiting example, the photocrosslinking can be performed at will by excitation of the temporal control that is provided. Methyl groups of unnatural amino acids can be characterized using, but not limited to, nuclear magnetic resonance and vibrational microscopy. These include, but are not limited to, radiolabeled probes of local structure and dynamics. The alkyl group may be substituted with an alkyl group, including but not limited to, a methyl group.

[0360] A. Structure and synthesis of unnatural amino acid derivatives: carbonyl groups, carbonyl-like groups, mass (protected carbonyl group and protected carbonyl group) Amino acids with electrophilic reactive groups are capable of various reactions and can undergo various chemical reactions (nucleophilic The electrophilic cations can be bonded to the molecules through electrochemical reactions, including but not limited to addition reactions. The reactive groups are carbonyl or dicarbonyl groups (keto or aldehyde groups). groups), carbonyl-like groups or dicarbonyl-like groups (carbonyl or di It has the same reactivity as a carbonyl group and has the same structure as a carbonyl group or dicarbonyl group. similar to the aryl group), masked carbonyl group or masked dicarbonyl group ( can be readily converted to a carbonyl or dicarbonyl group), or A protected dicarbonyl group or a protected dicarbonyl group (which, when deprotected, becomes a carbonyl group or a dicarbonyl group) Such amino acids include those of the formula ( XXXVII)

[0361] [ka]

[0362] (In the formula, A is optional and, when present, is lower alkylene, substituted lower alkylene, lower cycloalkylene, cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, Alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene aryl, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroaryl aryl, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or is a substituted aralkylene; B is optional and, when present, is lower alkylene, substituted lower alkylene, lower alkoxy, aryl, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene , or substituted alkylene)-, -S(O) k- (where k is 1, 2, or 3) -S(O) k (Alkylene or substituted alkylene)-, -C(O)-, -N S(O) 2 -, -OS(O) 2 -, -C(O)-(alkylene or substituted alkylene )-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N( R')-, -NR'-(alkylene or substituted alkylene)-, -C(O)N(R' )-, -CON(R')-(alkylene or substituted alkylene)-, -CSN(R' )-, -CSN(R')-(alkylene or substituted alkylene)-, -N(R')C O-(alkylene or substituted alkylene)-, -N(R')C(O)O-, -S(O ) k N(R')-, -N(R')C(O)N(R')-, -N(R')C(S)N(R' )-, -N(R')S(O) k N(R')-, -N(R')-N=, -C(R')=N- , -C(R')=NN(R')-, -C(R')=NN=, -C(R') 2 -N=N - and -C(R') 2 -N(R')-N(R')- where each R' is independently H, alkyl, or substituted alkyl. , K is

[0363] [ka]

[0364] and R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl. can be, Each R" is independently H, alkyl, substituted alkyl, or a protecting group, or two or more When the above R″ group is present, two R″ groups optionally form a heterocycloalkyl; R 1 is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide. It is R 2 is an OH, ester protecting group, resin, amino acid, polypeptide, or polynucleotide. It is a punch line, R 3 and R 4 are each independently H, halogen, lower alkyl, or substituted lower alkyl. alkyl or R 3 and R 4 , or two R 3 The group is a cycloalkyl group. alkyl or heterocycloalkyl, or -ABKR groups each have at least one carbonyl group (including dicarbonyl groups). ), a protected carbonyl group (including a protected dicarbonyl group), or a masked Bicyclic or tricyclic rings containing a masked carbonyl group (including a masked dicarbonyl group) or forming a cycloalkyl or heterocycloalkyl of -KR groups each have at least one carbonyl group (including a dicarbonyl group), Protected carbonyl groups (including protected dicarbonyl groups) or masked carbonyl groups Monocyclic or bicyclic cycloalkyl groups containing a cycloalkyl group (including a masked dicarbonyl group) forming an alkyl or heterocycloalkyl; where A is phenylene and each R 3 is H, B is present and A is -(CH 2 ) 4 - and each R 3is H, B is -NHC(O)(CH 2 CH 2 )--not , A and B do not exist, and each R 3 When is H, R is not methyl. The non-natural amino acid may be in the form of a salt or may be a polypeptide, polymer, It may be incorporated into a polysaccharide or polynucleotide and optionally further modified post-translationally. may be used.) In one embodiment, the compound of formula (XXXVII) is at least In one embodiment, the compound represented by formula (XXXVII) is stable in aqueous solution for one month. The compound is stable under mildly acidic conditions for at least two weeks. The compound shown in formula XVII) is stable under weakly acidic conditions for at least 5 days. In an embodiment, the acidic conditions are a pH of 2-8.

[0365] In certain embodiments of the compound 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 is a substituted alkylene)-, -S(alkylene or substituted alkylene)-, -S(O) (alkylene or substituted alkylene)- or -S(O) 2 (Alkylene, also In certain embodiments of the compound of formula (XXXVII), B is -O(CH 2 )-, -CH=N-, -CH=N-NH-, -NHCH 2 -, -N HCO-, -C(O)-, -C(O)-(CH 2 )-, -CONH-(CH 2 )-, -S CH 2 -, -S(=O)CH 2 - or -S(O) 2 CH 2 -. Formula (XXXV In some embodiments of the compound of formula II), R is alkyl or cycloalkyl having 1 to 6 carbon atoms. In certain embodiments of the compound of formula (XXXVII), R is -CH 3 , -CH(CH 3 ) 2 or cyclopropyl. In certain embodiments of the compound, R 1 H, tert-butyloxycarbonyl (Boc) , 9-fluorenylmethoxycarbonyl (Fmoc), N-acetyl, tetrafluoroethylene cetyl (TFA) or benzyloxycarbonyl (Cbz). In some embodiments of the compound shown in II), R 1 Resins, amino acids, polypeptides, and antibodies In one embodiment of the compound of formula (XXXVII), In terms of form, R 2 is OH, O-methyl, O-ethyl, or Ot-butyl. In some embodiments of the compound shown in formula XXXVII, R 2 Resins, amino acids, polypeptides The compound represented by formula (XXXVII) is a peptide, an antibody, or a polynucleotide. In one embodiment, R 2 is a polynucleotide. In one embodiment of the invention, R 2 is ribonucleic acid (RNA).

[0366] In certain embodiments of the compound of Formula (XXXVII),

[0367] [ka]

[0368] is selected from the group consisting of: (i) A is a substituted lower alkylene, an arylene having 4 carbon atoms, a substituted arylene, or a heteroaryl. aryl, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or is a substituted aralkylene; B is optional and, when present, is lower alkylene, substituted lower alkylene, lower alkoxy, substituted lower alkenylene, -O-, -O-(alkylene, or substituted alkylene N)-, -S-, -S(O)-, -S(O) 2 -, -NS(O) 2 -, -OS(O) 2 - , -C(O)-, -C(O)-(alkylene or substituted alkylene)-, -C(S) -, -N(R')-, -C(O)N(R')-, -CON(R')-(alkylene, is a substituted alkylene)-, -CSN(R')-, -N(R')CO-(alkylene, or is a substituted alkylene)-, -N(R')C(O)O-, -N(R')C(S)-, -S( O)N(R'), -S(O) 2 N(R'), -N(R')C(O)N(R')-, -N( R')C(S)N(R')-, -N(R')S(O)N(R')-, -N(R')S(O ) 2 N(R')-, -N(R')-N=, -C(R')=NN(R')-, -C(R' )=NN=, -C(R') 2 -N=N- and -C(R') 2 -N(R')-N( R')-, (ii) A is an optional group, and when present, it is a substituted lower alkylene, an arylene having 4 carbon atoms, a substituted Substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkali aralkylene, aralkylene, or substituted aralkylene; B is a lower alkylene, a substituted lower alkylene, a lower alkenylene, or a substituted lower alkenylene. -O-, -O-(alkylene or substituted alkylene)-, -S-, -S(O)- , -S(O) 2 -, -NS(O) 2 -, -OS(O) 2 -, -C(O)-, -C(O)- (Alkylene or substituted alkylene)-, -C(S)-, -N(R')-, -C(O )N(R')-, -CON(R')-(alkylene or substituted alkylene)-, -C SN(R')-, -N(R')CO-(alkylene or substituted alkylene)-, -N (R')C(O)O-, -N(R')C(S)-, -S(O)N(R'), -S(O) 2 N(R'), -N(R')C(O)N(R')-, -N(R')C(S)N(R')-, -N(R')S(O)N(R')-, -N(R')S(O) 2 N(R')-, -N(R' )-N=, -C(R')=NN(R')-, -C(R')=NN=, -C(R') 2 -N=N- and -C(R') 2 -N(R')-N(R')- is a bivalent linker (iii) A is a lower alkylene; B is optional and, when present, is lower alkylene, substituted lower alkylene, lower alkoxy, substituted lower alkenylene, -O-, -O-(alkylene, or substituted alkylene N)-, -S-, -S(O)-, -S(O) 2 -, -NS(O) 2 -, -OS(O) 2 - , -C(O)-, -C(O)-(alkylene or substituted alkylene)-, -C(S) -, -N(R')-, -C(O)N(R')-, -CSN(R')-, -CON(R') -(alkylene or substituted alkylene)-, -N(R')C(O)O-, -N(R' )C(S)-, -S(O)N(R'), -S(O) 2 N(R'), -N(R')C(O) N(R')-, -N(R')C(S)N(R')-, -N(R')S(O)N(R')- , -N(R')S(O) 2 N(R')-, -N(R')-N=, -C(R')=NN( R')-, -C(R')=NN=, -C(R') 2 -N=N- and -C(R') 2 -N(R')-N(R')-, (iv) A is phenylene; B is a lower alkylene, a substituted lower alkylene, a lower alkenylene, or a substituted lower alkenylene. -O-, -O-(alkylene or substituted alkylene)-, -S-, -S(O)- , -S(O) 2 -, -NS(O) 2 -, -OS(O) 2 -, -C(O)-, -C(O)- (Alkylene or substituted alkylene)-, -C(S)-, -N(R')-, -C(O )N(R')-, -CON(R')-(alkylene or substituted alkylene)-, -C SN(R')-, -N(R')CO-(alkylene or substituted alkylene)-, -N (R')C(O)O-, -N(R')C(S)-, -S(O)N(R'), -S(O) 2 N(R'), -N(R')C(O)N(R')-, -N(R')C(S)N(R')-, -N(R')S(O)N(R')-, -N(R')S(O) 2 N(R')-, -N(R' )-N=, -C(R')=NN(R')-, -C(R')=NN=, -C(R') 2 -N=N- and -C(R') 2 -N(R')-N(R')- is a bivalent linker K is

[0369] [ka]

[0370] and each R' is independently H, alkyl, or substituted alkyl; R 1 is optional and, if present, represents H, an amino protecting group, a resin, an amino acid, a polypeptide or a polynucleotide, R 2 is optional and, if present, represents an OH, ester protecting group, resin, amino acid, polypeptide a peptide or polynucleotide, Each R 3 and R 4 are independently H, halogen, lower alkyl, or substituted lower alkoxy. It is a kill. R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl. be.

[0371] Also included are amino acids having the structure of formula (XXXVIII):

[0372] [ka]

[0373] (In the formula, A is optional and, when present, is lower alkylene, substituted lower alkylene, lower cycloalkylene, cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, Alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene aryl, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroaryl aryl, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or is a substituted aralkylene; B is optional and, when present, is lower alkylene, substituted lower alkylene, lower alkoxy, aryl, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene , or substituted alkylene)-, -S(O) k - (where k is 1, 2, or 3) -S(O) k (Alkylene or substituted alkylene)-, -C(O)-, -N S(O) 2 -, -OS(O) 2 -, -C(O)-(alkylene or substituted alkylene )-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N( R')-, -NR'-(alkylene or substituted alkylene)-, -C(O)N(R' )-, -CON(R')-(alkylene or substituted alkylene)-, -CSN(R' )-, -CSN(R')-(alkylene or substituted alkylene)-, -N(R')C O-(alkylene or substituted alkylene)-, -N(R')C(O)O-, -S(O ) k N(R')-, -N(R')C(O)N(R')-, -N(R')C(S)N(R' )-, -N(R')S(O) k N(R')-, -N(R')-N=, -C(R')=N- , -C(R')=NN(R')-, -C(R')=NN=, -C(R') 2 -N=N - and -C(R') 2 -N(R')-N(R')- where each R' is independently H, alkyl, or substituted alkyl. , R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl. can be, R 1 is H, an amino protecting group, a resin, at least one amino acid, a polypeptide, or , a polynucleotide, R 2 can be any combination of OH, ester protecting groups, resin, at least one amino acid, polypeptide, or or a polynucleotide, provided that when A is phenylene, B is present and A is -(CH 2 ) 4 -, B is -N HC(O)(CH 2 CH 2 )-, and if A and B are absent, R is not methyl. The unnatural amino acids may be in the form of a salt or may be polysaccharides of unnatural amino acids. It may be incorporated into a peptide, polymer, polysaccharide, or polynucleotide, and It may be optionally modified after translation.) Also included are amino acids having the structure of formula (XXXIX):

[0374] [ka]

[0375] (In the formula, B is a lower alkylene, a substituted lower alkylene, a lower alkenylene, or a substituted lower alkenylene. lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene, or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene) -, -S(O) k - (where k is 1, 2, or 3), -S(O) k (Archi olefin or substituted alkylene)-, -C(O)-, -NS(O) 2 -, -OS(O) 2 -, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S )-(alkylene or substituted alkylene)-, -N(R')-, -NR'-(alkyl aryl, or substituted alkylene)-, -C(O)N(R')-, -CON(R')-(aryl, or substituted alkylene)-, -C(O)N(R')-, -CON(R')-(aryl alkylene, or substituted alkylene)-, -CSN(R')-, -CSN(R')-(a -N(R')CO-(alkylene or substituted alkylene)-, -N(R')CO-(alkylene or substituted -N(R')C(O)O-, -S(O) k N(R')-, -N(R' )C(O)N(R')-, -N(R')C(S)N(R')-, -N(R')S(O) k N(R')-, -N(R')-N=, -C(R')=N-, -C(R')=NN(R' )-, -C(R')=NN=, -C(R') 2 -N=N- and -C(R') 2 - N(R')-N(R')-, wherein each R' is independently H, alkyl, or substituted alkyl; R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl. can be, R 1 is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide. It is R 2 is an OH, ester protecting group, resin, amino acid, polypeptide, or polynucleotide. It is a punch line, Each R a is H, halogen, alkyl, substituted alkyl, -N(R') 2 , -C(O) k R ' (where k is 1, 2, or 3), -C(O)N(R') 2 ,-OR',o And -S(O) k R', wherein each R' is independently selected from the group consisting of , H, alkyl, or substituted alkyl). The unnatural amino acid may be in the form of a salt. Alternatively, the unnatural amino acid may be a polypeptide, polymer, polysaccharide, or polynucleotide. It may be incorporated into a nucleic acid fragment and optionally modified post-translationally. The following amino acids are also included:

[0376] [ka]

[0377] The unnatural amino acid is optionally a protected group, a carboxyl protecting group, and / or Alternatively, it may be in the form of a salt, or may be a polypeptide, polymer, or mixture of non-natural amino acids. It may be incorporated into a polysaccharide or polynucleotide and optionally post-translationally modified. Good too.

[0378] Also included are the following amino acids having the structure of formula (XXXX):

[0379] [ka]

[0380] (In the formula, B is -NS(O) 2 -, -OS(O) 2 -, optional, if present, lower alk alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroaromatic alkylene alkylene, substituted lower heteroalkylene, -O-, -O-(alkylene, or substituted aryl alkylene)-, -S-, -S-(alkylene or substituted alkylene)-, -S(O) k - (where k is 1, 2, or 3), -S(O) k (Alkylene or -substituted alkylene)-, -C(O)-, -NS(O) 2 -, -OS(O) 2 -, -C(O)- (Alkylene or substituted alkylene)-, -C(S)-, -C(S)-(alkylene , or substituted alkylene)-, -N(R')-, -NR'-(alkylene or substituted -C(O)N(R')-, -CON(R')-(alkylene, or , substituted alkylene)-, -CSN(R')-, -CSN(R')-(alkylene, or , substituted alkylene)-, -N(R')CO-(alkylene or substituted alkylene)- , -N(R')C(O)O-, -S(O) k N(R')-, -N(R')C(O)N(R ')-, -N(R')C(S)N(R')-, -N(R')S(O) k N(R')-, - N(R')-N=, -C(R')=N-, -C(R')=NN(R')-, -C(R' )=NN=, -C(R') 2 -N=N- and -C(R') 2 -N(R')-N( R')-, where each R' is independently selected from the group consisting of H, a alkyl, or substituted alkyl; R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl. can be, R 1 is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide. It is R 2 is an OH, ester protecting group, resin, amino acid, polypeptide, or polynucleotide. It is a punch line, Each R a is H, halogen, alkyl, substituted alkyl, -N(R') 2 , -C(O) k R ' (where k is 1, 2, or 3), -C(O)N(R') 2 ,-OR',o And -S(O) k R′, wherein each R′ is independently selected from the group consisting of: H, alkyl, or substituted alkyl), n is 0 to 8; However, A is -(CH 2 ) 4 -, B is -NHC(O)(CH 2 CH 2 )-in The unnatural amino acids may be in the form of a salt or may be polyamines such as cyclic amines. It may be incorporated into a polypeptide, polymer, polysaccharide, or polynucleotide; may be optionally post-translationally modified.) The following amino acids are also included:

[0381] [ka]

[0382] wherein the compound is optionally an amino protecting group and optionally a carboxyl protecting group; Optionally, an amino protecting group and a carboxyl protecting group, or a salt thereof; and The invention relates to the incorporation of unnatural amino acids into polypeptides, polymers, polysaccharides, or polynucleotides. It may also be optionally modified after translation.) Also included are the following amino acids having the structure of formula (XXXXI):

[0383] [ka]

[0384] (In the formula, A is optional and, when present, is lower alkylene, substituted lower alkylene, lower cycloalkylene, cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, Alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene aryl, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroaryl aryl, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or is a substituted aralkylene; B is optional and, when present, is lower alkylene, substituted lower alkylene, lower alkoxy, aryl, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene , or substituted alkylene)-, -S(O) k - (where k is 1, 2, or 3) -S(O)k (Alkylene or substituted alkylene)-, -C(O)-, -N S(O) 2 -, -OS(O) 2 -, -C(O)-(alkylene or substituted alkylene )-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N( R')-, -NR'-(alkylene or substituted alkylene)-, -C(O)N(R' )-, -CON(R')-(alkylene or substituted alkylene)-, -CSN(R' )-, -CSN(R')-(alkylene or substituted alkylene)-, -N(R')C O-(alkylene or substituted alkylene)-, -N(R')C(O)O-, -S(O ) k N(R')-, -N(R')C(O)N(R')-, -N(R')C(S)N(R' )-, -N(R')S(O) k N(R')-, -N(R')-N=, -C(R')=N- , -C(R')=NN(R')-, -C(R')=NN=, -C(R') 2 -N=N - and -C(R') 2 -N(R')-N(R')- where each R' is independently H, alkyl, or substituted alkyl. , R 1 is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide. It is R 2 is an OH, ester protecting group, resin, amino acid, polypeptide, or polynucleotide. It is a punch line, The unnatural amino acid may be in the form of a salt or may be a polypeptide of the unnatural amino acid. The polypeptide may be incorporated into a peptide, polymer, polysaccharide, or polynucleotide, and may be further translated. It may be optionally modified later.) Also included are the following amino acids having the structure of formula (XXXXII):

[0385] [ka]

[0386] (In the formula, B is optional and, when present, is lower alkylene, substituted lower alkylene, lower alkoxy, aryl, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene , or substituted alkylene)-, -S(O) k - (where k is 1, 2, or 3) -S(O) k (Alkylene or substituted alkylene)-, -C(O)-, -N S(O) 2 -, -OS(O) 2 -, -C(O)-(alkylene or substituted alkylene )-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N( R')-, -NR'-(alkylene or substituted alkylene)-, -C(O)N(R' )-, -CON(R')-(alkylene or substituted alkylene)-, -CSN(R' )-, -CSN(R')-(alkylene or substituted alkylene)-, -N(R')C O-(alkylene or substituted alkylene)-, -N(R')C(O)O-, -S(O ) k N(R')-, -N(R')C(O)N(R')-, -N(R')C(S)N(R' )-, -N(R')S(O) k N(R')-, -N(R')-N=, -C(R')=N- , -C(R')=NN(R')-, -C(R')=NN=, -C(R') 2 -N=N - and -C(R') 2 -N(R')-N(R')- where each R' is independently H, alkyl, or substituted alkyl. , R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl. can be, R 1 is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide. It is R 2 is an OH, ester protecting group, resin, amino acid, polypeptide, or polynucleotide. It is a punch line, Each R a is H, halogen, alkyl, substituted alkyl, -N(R') 2 , -C(O) k R ' (where k is 1, 2, or 3), -C(O)N(R') 2 ,-OR',o And -S(O) k R', wherein each R' is independently selected from the group consisting of , H, alkyl, or substituted alkyl).

[0387] The unnatural amino acid may be in the form of a salt or may be a polypeptide of the unnatural amino acid. The polypeptide may be incorporated into a peptide, polymer, polysaccharide, or polynucleotide, and may be further translated. It may be optionally modified later.) The following amino acids are also included:

[0388] [ka]

[0389] wherein the compound is optionally an amino protecting group and optionally a carboxyl protecting group; Optionally, an amino protecting group and a carboxyl protecting group, or a salt thereof; and The invention relates to the incorporation of unnatural amino acids into polypeptides, polymers, polysaccharides, or polynucleotides. It may also be optionally modified after translation.) Also included are the following amino acids having the structure of formula (XXXXIV):

[0390] [ka]

[0391] (In the formula, B is optional and, when present, is lower alkylene, substituted lower alkylene, lower alkoxy, aryl, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene , or substituted alkylene)-, -S(O) k - (where k is 1, 2, or 3) -S(O) k (Alkylene or substituted alkylene)-, -C(O)-, -N S(O) 2 -, -OS(O) 2 -, -C(O)-(alkylene or substituted alkylene )-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N( R')-, -NR'-(alkylene or substituted alkylene)-, -C(O)N(R' )-, -CON(R')-(alkylene or substituted alkylene)-, -CSN(R' )-, -CSN(R')-(alkylene or substituted alkylene)-, -N(R')C O-(alkylene or substituted alkylene)-, -N(R')C(O)O-, -S(O ) k N(R')-, -N(R')C(O)N(R')-, -N(R')C(S)N(R' )-, -N(R')S(O) k N(R')-, -N(R')-N=, -C(R')=N- , -C(R')=NN(R')-, -C(R')=NN=, -C(R') 2 -N=N - and -C(R') 2 -N(R')-N(R')- where each R' is independently H, alkyl, or substituted alkyl. , R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl. can be, R 1 is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide. It is R 2 is an OH, ester protecting group, resin, amino acid, polypeptide, or polynucleotide. It is a punch line, Each R a is H, halogen, alkyl, substituted alkyl, -N(R') 2 , -C(O) k R ' (where k is 1, 2, or 3), -C(O)N(R') 2 ,-OR',o And -S(O) k R′, wherein each R′ is independently selected from the group consisting of: H, alkyl, or substituted alkyl), and n is 0 to 8.

[0392] The unnatural amino acid may be in the form of a salt or may be a polypeptide of the unnatural amino acid. The polypeptide may be incorporated into a peptide, polymer, polysaccharide, or polynucleotide, and may be further translated. It may be optionally modified later.) The following amino acids are also included:

[0393] [ka]

[0394] wherein the compound is optionally an amino protecting group and optionally a carboxyl protecting group; Optionally, an amino protecting group and a carboxyl protecting group, or a salt thereof; and The invention relates to the incorporation of unnatural amino acids into polypeptides, polymers, polysaccharides, or polynucleotides. It may also be optionally modified after translation.) The unnatural amino acids described herein can contain, in addition to monocarbonyl structures, dicarbonyl groups, Dicarbonyl-like groups, masked dicarbonyl groups, and protected dicarbonyl groups It may contain any group.

[0395] For example, the following amino acids having the structure of formula (XXXXV) are included:

[0396] [ka]

[0397] (In the formula, A is optional and, when present, is lower alkylene, substituted lower alkylene, lower cycloalkylene, cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, Alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene aryl, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroaryl aryl, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or is a substituted aralkylene; B is optional and, when present, is lower alkylene, substituted lower alkylene, lower alkoxy, aryl, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene , or substituted alkylene)-, -S(O) k - (where k is 1, 2, or 3) -S(O) k (Alkylene or substituted alkylene)-, -C(O)-, -N S(O) 2 -, -OS(O) 2 -, -C(O)-(alkylene or substituted alkylene )-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N( R')-, -NR'-(alkylene or substituted alkylene)-, -C(O)N(R' )-, -CON(R')-(alkylene or substituted alkylene)-, -CSN(R' )-, -CSN(R')-(alkylene or substituted alkylene)-, -N(R')C O-(alkylene or substituted alkylene)-, -N(R')C(O)O-, -S(O ) k N(R')-, -N(R')C(O)N(R')-, -N(R')C(S)N(R' )-, -N(R')S(O) k N(R')-, -N(R')-N=, -C(R')=N- , -C(R')=NN(R')-, -C(R')=NN=, -C(R') 2 -N=N - and -C(R') 2 -N(R')-N(R')- where each R' is independently H, alkyl, or substituted alkyl. , R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl. can be, R 1 is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide. It is R 2 is an OH, ester protecting group, resin, amino acid, polypeptide, or polynucleotide. It is a punch line, The unnatural amino acid may be in the form of a salt or may be a polypeptide of the unnatural amino acid. The polypeptide may be incorporated into a peptide, polymer, polysaccharide, or polynucleotide, and may be further translated. It may be optionally modified later.) Also included are the following amino acids having the structure of formula (XXXXVI):

[0398] [ka]

[0399] (In the formula, B is optional and, when present, is lower alkylene, substituted lower alkylene, lower alkoxy, aryl, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene , or substituted alkylene)-, -S(O) k - (where k is 1, 2, or 3) -S(O) k (Alkylene or substituted alkylene)-, -C(O)-, -N S(O) 2 -, -OS(O) 2 -, -C(O)-(alkylene or substituted alkylene )-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N( R')-, -NR'-(alkylene or substituted alkylene)-, -C(O)N(R' )-, -CON(R')-(alkylene or substituted alkylene)-, -CSN(R' )-, -CSN(R')-(alkylene or substituted alkylene)-, -N(R')C O-(alkylene or substituted alkylene)-, -N(R')C(O)O-, -S(O ) k N(R')-, -N(R')C(O)N(R')-, -N(R')C(S)N(R' )-, -N(R')S(O) k N(R')-, -N(R')-N=, -C(R')=N- , -C(R')=NN(R')-, -C(R')=NN=, -C(R') 2 -N=N - and -C(R') 2 -N(R')-N(R')- where each R' is independently H, alkyl, or substituted alkyl. , R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl. can be, R 1 is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide. It is R 2 is an OH, ester protecting group, resin, amino acid, polypeptide, or polynucleotide. It is a punch line, Each R a is H, halogen, alkyl, substituted alkyl, -N(R') 2 , -C(O) k R ' (where k is 1, 2, or 3), -C(O)N(R') 2 ,-OR',o And -S(O) k R', wherein each R' is independently selected from the group consisting of , H, alkyl, or substituted alkyl).

[0400] The unnatural amino acid may be in the form of a salt or may be a polypeptide of the unnatural amino acid. The polypeptide may be incorporated into a peptide, polymer, polysaccharide, or polynucleotide, and may be further translated. It may be optionally modified later.) The following amino acids are also included:

[0401] [ka]

[0402] wherein the compound optionally has an amino protecting group and a carboxyl protecting group, or The unnatural amino acid may be in the form of a salt or may be a salt of the unnatural amino acid. The acid may be incorporated into a polypeptide, polymer, polysaccharide, or polynucleotide. , and may be optionally modified after translation.) Also included are the following amino acids having the structure of formula (XXXXVII):

[0403] [ka]

[0404] (In the formula, B is optional and, when present, is lower alkylene, substituted lower alkylene, lower alkoxy, aryl, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene , or substituted alkylene)-, -S(O) k - (where k is 1, 2, or 3) -S(O) k (Alkylene or substituted alkylene)-, -C(O)-, -N S(O) 2 -, -OS(O) 2-, -C(O)-(alkylene or substituted alkylene )-, -C(S)-, -C(S)-(alkylene or substituted alkylene)-, -N( R')-, -NR'-(alkylene or substituted alkylene)-, -C(O)N(R' )-, -CON(R')-(alkylene or substituted alkylene)-, -CSN(R' )-, -CSN(R')-(alkylene or substituted alkylene)-, -N(R')C O-(alkylene or substituted alkylene)-, -N(R')C(O)O-, -S(O ) k N(R')-, -N(R')C(O)N(R')-, -N(R')C(S)N(R' )-, -N(R')S(O) k N(R')-, -N(R')-N=, -C(R')=N- , -C(R')=NN(R')-, -C(R')=NN=, -C(R') 2 -N=N - and -C(R') 2 -N(R')-N(R')- where each R' is independently H, alkyl, or substituted alkyl. , R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl. can be, R 1 is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide. It is R 2 is an OH, ester protecting group, resin, amino acid, polypeptide, or polynucleotide. It is a punch line, Each R a is H, halogen, alkyl, substituted alkyl, -N(R') 2 , -C(O) k R ' (where k is 1, 2, or 3), -C(O)N(R') 2,-OR',o And -S(O) k R′, wherein each R′ is independently selected from the group consisting of: H, alkyl, or substituted alkyl), and n is 0 to 8.

[0405] The unnatural amino acid may be in the form of a salt or may be a polypeptide of the unnatural amino acid. The polypeptide may be incorporated into a peptide, polymer, polysaccharide, or polynucleotide, and may be further translated. It may be optionally modified later.) The following amino acids are also included:

[0406] [ka]

[0407] wherein the compound optionally has an amino protecting group and a carboxyl protecting group, or or a polypeptide, polymer, polysaccharide, or polysaccharide of a non-natural amino acid. It may be incorporated into a nucleotide and may be optionally modified post-translationally. Also included are the following amino acids having the structure of formula (XXXXVIII):

[0408] [ka]

[0409] (In the formula, A is optional and, when present, is lower alkylene, substituted lower alkylene, lower cycloalkylene, cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, Alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene aryl, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroaryl aryl, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or is a substituted aralkylene; R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl. can be, R 1 is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide. It is R 2 is an OH, ester protecting group, resin, amino acid, polypeptide, or polynucleotide. It is a punch line, X 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. ), The unnatural amino acid may be in the form of a salt or may be a polypeptide of the unnatural amino acid. The polypeptide may be incorporated into a peptide, polymer, polysaccharide, or polynucleotide, and may be further translated. It may be optionally modified later.) Also included are the following amino acids having the structure of formula (XXXXIX):

[0410] [ka]

[0411] (In the formula, A is optional and, when present, is lower alkylene, substituted lower alkylene, lower cycloalkylene, cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, Alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene aryl, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroaryl aryl, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or is a substituted aralkylene; R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl. can be, R 1 is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide. It is R 2 is an OH, ester protecting group, resin, amino acid, polypeptide, or polynucleotide. It is a punch line, L is alkylene, substituted alkylene, N(R')(alkylene), or N(R') (substituted alkylene) (wherein R' is H, alkyl, substituted alkyl, cycloalkyl, aryl, or substituted cycloalkyl; The unnatural amino acid may be in the form of a salt or may be a polypeptide of the unnatural amino acid. The polypeptide may be incorporated into a peptide, polymer, polysaccharide, or polynucleotide, and may be further translated. It may be optionally modified later.) Also included are the following amino acids having the structure of formula (XXXXX):

[0412] [ka]

[0413] (In the formula, A is optional and, when present, is lower alkylene, substituted lower alkylene, lower cycloalkylene, cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, Alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene aryl, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroaryl aryl, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or is a substituted aralkylene; R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl. can be, R 1 is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide. It is R 2 is an OH, ester protecting group, resin, amino acid, polypeptide, or polynucleotide. It is a punch line, L is alkylene, substituted alkylene, N(R')(alkylene), or N(R') (substituted alkylene) (wherein R' is H, alkyl, substituted alkyl, cycloalkyl, cycloalkyl, or substituted cycloalkyl.

[0414] The unnatural amino acid may be in the form of a salt or may be a polypeptide of the unnatural amino acid. The polypeptide may be incorporated into a peptide, polymer, polysaccharide, or polynucleotide, and may be further translated. It may be optionally modified later.) Also included are the following amino acids having the structure of formula (XXXXXI):

[0415] [ka]

[0416] (In the formula, A is optional and, when present, is lower alkylene, substituted lower alkylene, lower cycloalkylene, cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, Alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene aryl, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroaryl aryl, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or is a substituted aralkylene; R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl. can be, R 1 is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide. It is R 2 is an OH, ester protecting group, resin, amino acid, polypeptide, or polynucleotide. It is a punch line, X 1 is C, S, or S(O), and n is 0, 1, 2, 3, 4, or 5. the law of nature, Each CR 8 R 9 Each R in the group 8 and R 9 is H, alkoxy, alkylamine, halo R is independently selected from the group consisting of alkyl, alkyl, and aryl, or any R 8 oh YobiR 9 can be taken together to form =O or cycloalkyl, or R 8 Any groups adjacent to the group can be taken together to form a cycloalkyl.

[0417] The unnatural amino acid may be in the form of a salt or may be a polypeptide of the unnatural amino acid. The polypeptide may be incorporated into a peptide, polymer, polysaccharide, or polynucleotide, and may be further translated. It may be optionally modified later.) Also included are the following amino acids having the structure of formula (XXXXXII):

[0418] [ka]

[0419] (In the formula, A is optional and, when present, is lower alkylene, substituted lower alkylene, lower cycloalkylene, cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, Alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene aryl, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroaryl aryl, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or is a substituted aralkylene; R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl. can be, R 1 is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide. It is R 2 is an OH, ester protecting group, resin, amino acid, polypeptide, or polynucleotide. It is a punch line, n is 0, 1, 2, 3, 4, or 5, and each CR 8 R 9 Each R in the group 8 and R 9 consists of H, alkoxy, alkylamine, halogen, alkyl, and aryl. or any R 8 and R 9 Both are =O or cycloalkenyl A kill can be formed or R 8 Any groups adjacent to the group are both cycloalkyl A kill can be formed.

[0420] The unnatural amino acid may be in the form of a salt or may be a polypeptide of the unnatural amino acid. The polypeptide may be incorporated into a peptide, polymer, polysaccharide, or polynucleotide, and may be further translated. It may be optionally modified later.) Also included are the following amino acids having the structure of formula (XXXXXIII):

[0421] [ka]

[0422] (In the formula, A is optional and, when present, is lower alkylene, substituted lower alkylene, lower cycloalkylene, cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, Alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene aryl, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroaryl aryl, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or is a substituted aralkylene; R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl. can be, R 1 is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide. It is R 2 is an OH, ester protecting group, resin, amino acid, polypeptide, or polynucleotide. It is a punch line, n is 0, 1, 2, 3, 4, or 5, and each CR 8 R 9 Each R in the group 8 and R 9 consists of H, alkoxy, alkylamine, halogen, alkyl, and aryl. or any R 8 and R 9Both are =O or cycloalkenyl A kill can be formed or R 8 Any groups adjacent to the group are both cycloalkyl A kill can be formed.

[0423] The unnatural amino acid may be in the form of a salt or may be a polypeptide of the unnatural amino acid. The polypeptide may be incorporated into a peptide, polymer, polysaccharide, or polynucleotide, and may be further translated. It may be optionally modified later.) Also included are the following amino acids having the structure of formula (XXXXXIV):

[0424] [ka]

[0425] (In the formula, A is optional and, when present, is lower alkylene, substituted lower alkylene, lower cycloalkylene, cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, Alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene aryl, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroaryl aryl, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or is a substituted aralkylene; R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl. can be, R 1 is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide. It is R 2 is an OH, ester protecting group, resin, amino acid, polypeptide, or polynucleotide. It is a punch line, X 1is 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. ).

[0426] The unnatural amino acid may be in the form of a salt or may be a polypeptide of the unnatural amino acid. The polypeptide may be incorporated into a peptide, polymer, polysaccharide, or polynucleotide, and may be further translated. It may be optionally modified later.) Also included are the following amino acids having the structure of formula (XXXXXV):

[0427] [ka]

[0428] (In the formula, A is optional and, when present, is lower alkylene, substituted lower alkylene, lower cycloalkylene, cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, Alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene aryl, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroaryl aryl, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or is a substituted aralkylene; R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl. can be, R 1 is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide. It is R 2is an OH, ester protecting group, resin, amino acid, polypeptide, or polynucleotide. It is a punch line, L is alkylene, substituted alkylene, N(R')(alkylene), or N(R') (substituted alkylene) (wherein R' is H, alkyl, substituted alkyl, cycloalkyl, cycloalkyl, or substituted cycloalkyl.

[0429] The unnatural amino acid may be in the form of a salt or may be a polypeptide of the unnatural amino acid. The polypeptide may be incorporated into a peptide, polymer, polysaccharide, or polynucleotide, and may be further translated. It may be optionally modified later.) Also included are the following amino acids having the structure of formula (XXXXXVI):

[0430] [ka]

[0431] (In the formula, A is optional and, when present, is lower alkylene, substituted lower alkylene, lower cycloalkylene, cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, Alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene aryl, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroaryl aryl, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or is a substituted aralkylene; R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl. can be, R 1 is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide. It is R2 is an OH, ester protecting group, resin, amino acid, polypeptide, or polynucleotide. It is a punch line, L is alkylene, substituted alkylene, N(R')(alkylene), or N(R') (substituted alkylene) (wherein R' is H, alkyl, substituted alkyl, cycloalkyl, cycloalkyl, or substituted cycloalkyl.

[0432] The unnatural amino acid may be in the form of a salt or may be a polypeptide of the unnatural amino acid. The polypeptide may be incorporated into a peptide, polymer, polysaccharide, or polynucleotide, and may be further translated. It may be optionally modified later.) Also included are amino acids having the structure of formula (XXXXXVII):

[0433] [ka]

[0434] (In the formula, A is optional and, when present, is lower alkylene, substituted lower alkylene, lower cycloalkylene, cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, Alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene aryl, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroaryl aryl, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or is a substituted aralkylene; M is

[0435] [ka]

[0436] and (a) shows the bond to the A group, while (b) shows the bond to the respective carbonyl group. R 3 and R 4 is H, halogen, alkyl, substituted alkyl, cycloalkyl, if or substituted cycloalkyl, or R 3 and R 4 , two R 3 Group or two R 4 The group may be any of cycloalkyl and heterocycloalkyl. Formed into R is H, halogen, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl. is alkyl, T 3 is a bond, C(R)(R), O, or S, where R is H, halogen, alkyl, or aryl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; R 1 is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide. It is R 2 is an OH, ester protecting group, resin, amino acid, polypeptide, or polynucleotide. It is a punch line, The unnatural amino acid may be in the form of a salt or may be a polypeptide of the unnatural amino acid. The polypeptide may be incorporated into a peptide, polymer, polysaccharide, or polynucleotide, and may be further translated. It may be optionally modified later.) Also included are amino acids having the structure of formula (XXXXXVIII):

[0437] [ka]

[0438] (In the formula, M is

[0439] [ka]

[0440] and (a) shows the bond to the A group, while (b) shows the bond to the respective carbonyl group. R 3 and R 4 is H, halogen, alkyl, substituted alkyl, cycloalkyl, if or substituted cycloalkyl, or R 3 and R 4 , two R 3 Group or two R 4 The group may be any of cycloalkyl and heterocycloalkyl. Formed into R is H, halogen, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl. is alkyl, T 3 is a bond, C(R)(R), O, or S, where R is H, halogen, alkyl, or aryl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; R 1 is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide. It is R 2 is an OH, ester protecting group, resin, amino acid, polypeptide, or polynucleotide. It is a punch line, Each R a is H, halogen, alkyl, substituted alkyl, -N(R') 2 , -C(O) k R ' (where k is 1, 2, or 3), -C(O)N(R') 2 ,-OR',o And -S(O) k R', wherein each R' is independently selected from the group consisting of , H, alkyl, or substituted alkyl).

[0441] The unnatural amino acid may be in the form of a salt or may be a polypeptide of the unnatural amino acid. The polypeptide may be incorporated into a peptide, polymer, polysaccharide, or polynucleotide, and may be further translated. It may be optionally modified later.) Also included are amino acids having the structure of formula (XXXXXIX):

[0442] [ka]

[0443] (In the formula, R is H, halogen, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl. is alkyl, T 3 is O or S.

[0444] The unnatural amino acid may be in the form of a salt or may be a polypeptide of the unnatural amino acid. The polypeptide may be incorporated into a peptide, polymer, polysaccharide, or polynucleotide, and may be further translated. It may be optionally modified later.) Also included are amino acids having the structure of formula (XXXXXX).

[0445] [ka]

[0446] (In the formula, R is H, halogen, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl. It is an alkyl group. Also included are the following amino acids having the structure of formula (XXXXXX):

[0447] [ka]

[0448] The unnatural amino acid may be in the form of a salt or may be a polypeptide of the unnatural amino acid. The polypeptide may be incorporated into a peptide, polymer, polysaccharide, or polynucleotide, and may be further translated. It may be optionally modified later.

[0449] Carbonyl or dicarbonyl functional groups are easily oxidized in aqueous solutions under mild conditions. can be selectively reacted with hydroxylamine-containing reagents to form the corresponding oxime bonds. This oxime bond is stable under physiological conditions. For example, Jencks, W. P., J. Am. Chem. Soc. 81, 475-481 (1959); Shao, J. and Tam, JP, J. Am. Chem. Soc. 117(14):3893-3899 (1995). In addition, the carbonyl group or dicarboxyl group may be The unique reactivity of the carboxyl group allows for selective modification in the presence of other amino acid side chains. See, e.g., Cornish, VW, et al., J. Am. Chem. Soc. 118:8150-8151 (1996); hegan, KF & Stroh, JG, Bioconjug. Chem. 3:138-146 (1992); Mahal, LK, et al., Science 276:1125-1128 (1997).

[0450] p-Acetyl-(+ / -)-phenylalanine and m-Acetyl-(+ / -)-phenylalanine The synthesis of niylalanine is described in Zhang, Z. et al, Biochemistry 42: 673, which is incorporated by reference. 5-6746 (2003). Other carbonyl-containing or dicarbonyl-containing amino acids are described in Amino acids can be prepared similarly.

[0451] In some embodiments, the polypeptides comprising unnatural amino acids are chemically modified. The reaction is carried out in a manner that produces reactive carbonyl or dicarbonyl functional groups. Aldehyde functional groups useful in the reaction have vicinal amino and hydroxyl groups. When the biologically active molecule is a polypeptide, it can be generated from a functional group that is An N-terminal serine or threonine (which may be present normally or may be chemically or enzymatically The cleavage of the α-terminal end of the β ... These can be used to generate aldehyde functional groups. 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 include cleaving an amino acid sequence at the N-terminus of a peptide or protein. Limited to acids.

[0452] Additionally, by way of example, non-natural amino acids having adjacent hydroxyl and amino groups can be used. The amino acid may be incorporated into a polypeptide as a "masked" aldehyde functional group. For example, 5-hydroxylysine has a hydroxyl group adjacent to the amine at the epsilon position. The reaction conditions for generating aldehydes are chosen to avoid oxidation at other sites in the polypeptide. This involves the addition of a molar excess of sodium metaperiodate in a mild environment. The pH of the oxidation reaction is typically about 7.0. Adding approximately 1.5 molar excess of sodium metaperiodate to the buffer solution and then incubating in the dark For example, U.S. Patent No. 6,339,663, which is incorporated herein by reference, includes incubating the solution at room temperature for about 10 minutes. See US Pat. No. 423,685.

[0453] B. Structure and Synthesis of Unnatural Amino Acids: Dicarbonyl Groups, Dicarbonyl-Like Groups, and Masks protected dicarbonyl group and protected dicarbonyl group Amino acids with electrophilic reactive groups are capable of various reactions, particularly via nucleophilic addition reactions. The electrophilic reactive group can be a dicarbonyl group (a diketone group, a ketone group, These include aldehyde groups, keto acid groups, keto ester groups, and ketothioester groups. ), carbonyl-like or dicarbonyl-like groups (having the same reactivity as dicarbonyl groups and is structurally similar to a dicarbonyl group), masked dicarbonyl group (dicarbonyl a protected dicarbonyl group, which, when deprotected, (having a reactivity similar to that of a dicarbonyl group). An example is an amino acid having the structure of formula (XXXVII).

[0454] [ka]

[0455] (In the formula, A is optional and, when present, is lower alkylene, substituted lower alkylene, lower cycloalkylene, cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, Alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene aryl, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroaryl aryl, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or is a substituted aralkylene; B is optional and, if present, is a phosphorus bonded at one end to a diamine-containing moiety. The linker is a lower alkylene, a substituted lower alkylene, a lower alkenylene, , substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-( Alkylene or substituted alkylene)-, -S-(alkylene or substituted alkylene) N)-, -C(O)R”-, -S(O) k (Alkylene or substituted alkylene)-( where k is 1, 2, or 3), -C(O)-(alkylene or substituted alkylene; 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 and -N(R")-CO-(alkylene or substituted alkylene)- wherein each R″ is independently H, alkyl, or substituted alkyl; K is

[0456] [ka]

[0457] and Where: T 1is a bond, an optionally substituted alkylene having 1 to 4 carbon atoms, 1-4 alkenylene or optionally substituted heteroalkyl; Each optional substituent is a lower alkylene, a substituted lower alkylene, a lower cycloalkylene, a substituted Substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower Heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroa Arylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene More independently selected, T 2 is a lower alkylene, a substituted lower alkylene, a lower alkenylene, a substituted lower alkenylene, Heteroalkylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene , or substituted alkylene)-, -S-, -S-(alkylene or substituted alkylene )-, -S(O) k - (where k is 1, 2, or 3), -S(O) k (Al alkylene or substituted alkylene)-, -C(O)-, -NS(O) 2 -, -OS(O) 2 -, -C(O)-(alkylene or substituted alkylene)-, -C(S)-, -C( S)-(alkylene or substituted alkylene)-, -N(R')-, -NR'-(alkyl alkylene or substituted alkylene)-, -C(O)N(R')-, -CON(R')-( Alkylene or substituted alkylene)-, -CSN(R')-, -CSN(R')-( alkylene or substituted alkylene)-, -N(R')CO-(alkylene or Substituted alkylene)-, -N(R')C(O)O-, -S(O) k N(R')-, -N(R ')C(O)N(R')-, -N(R')C(S)N(R')-, -N(R')S(O) k N(R')-, -N(R')-N=, -C(R')=N-, -C(R')=NN(R ')-, -C(R')=NN=, -C(R') 2 -N=N- and -C(R') 2 -N(R')-N(R')-, wherein each R' is independently selected from the group consisting of H, alkyl or substituted alkyl); T 3 teeth,

[0458] [ka]

[0459] and Where: each X 1 is -O-, -S-, -N(H)-, -N(R)-, -N(Ac)-, and -N(OMe)-, 2 -OR, -OAc, -SR , -N(R) 2 , -N(R)(Ac), -N(R)(OMe), or N 3 And and each R' is independently H, alkyl, or substituted alkyl; R is H, halogen, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl. is alkyl, R 1 is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide. It is R 2 is an OH, ester protecting group, resin, amino acid, polypeptide, or polynucleotide. It is an Ochido, or -ABKR groups each have at least one carbonyl group (including dicarbonyl groups). ), a protected carbonyl group (including a protected dicarbonyl group), or a masked Bicyclic or tricyclic rings containing a masked carbonyl group (including a masked dicarbonyl group) or forming a cycloalkyl or heterocycloalkyl of -KR groups each have at least one carbonyl group (including a dicarbonyl group), Protected carbonyl groups (including protected dicarbonyl groups) or masked carbonyl groups Monocyclic or bicyclic cycloalkyl groups containing a cycloalkyl group (including a masked dicarbonyl group) alkyl or heterocycloalkyl. Non-limiting examples of dicarbonyl amino acids having the structure of formula (XXXVII) include the following: The following are some examples.

[0460] [ka] JPEG2024170587000125.jpg63169

[0461] Further included are the following amino acids having the structure of formula (XXXVII):

[0462] [ka]

[0463] The unnatural amino acid may be in the form of a salt or may be a polypeptide of the unnatural amino acid. The polypeptide may be incorporated into a peptide, polymer, polysaccharide, or polynucleotide, and may be further translated. It may be optionally modified later.

[0464] C. Structure and synthesis of unnatural amino acids: ketoalkyne groups, ketoalkyne-like groups, mercaptoalkyne-like groups, and ketoalkyne-like groups. Protected ketoalkyne group, protected ketoalkyne group (Groupk), alkyne group, and , cycloalkyne group) Amino acids containing reactive groups with dicarbonyl-like reactivity can be decomposed via nucleophilic addition reactions. Such electrophilic reactive groups include ketoalkyne groups and ketoalkyne-like groups. (It has the same reactivity as the ketoalkyne group and is structurally similar to the ketoalkyne group. ), a masked ketoalkyne group (which can be readily converted to a ketoalkyne group), or a protected The ketoalkyne group, which, when deprotected, has the same reactivity as the ketoalkyne group, is listed. In some embodiments, a terminal alkyne, an internal alkyne, or a cycloalkyne is Amino acids containing quinone-bearing reactive groups are suitable for cycloaddition reactions (e.g., 1,3-dipolar cycloaddition). The molecules may be attached via an azide-alkyne Huisgen cycloaddition, etc. Such amino acids include those represented by the chemical formula (XXXXXXI-A) or (XXXXXXI- Examples of the amino acid include those having the structure B).

[0465] [ka]

[0466] (In the formula, A is optional and, when present, is lower alkylene, substituted lower alkylene, lower cycloalkylene, cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, Alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene aryl, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroaryl aryl, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or is a substituted aralkylene; B is optional and, if present, is a linker attached at one end to the diamine-containing moiety. and the linker is a lower alkylene, a substituted lower alkylene, a lower alkenylene, a substituted Lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-(alkyl -S-(alkylene or substituted alkylene)- , -C(O)R”-, -S(O) k (alkylene or substituted alkylene)-(wherein , k is 1, 2, or 3), -C(O)-(alkylene or substituted alkylene -C(S)-(alkylene or substituted alkylene)-, -NR"-(alkylene -CON(R")-(alkylene or substituted alkylene)-, -CON(R")-(alkylene or substituted alkylene) -CSN(R")-(alkylene or substituted alkylene)-, -CSN(R")-(alkylene or substituted alkylene)-, and , -N(R")CO-(alkylene or substituted alkylene)- wherein each R″ is independently H, alkyl, or substituted alkyl; G is optional and if present,

[0467] [ka]

[0468] and T 4 is a carbonyl protecting group, and examples of the carbonyl protecting group include the following: These include, but are not limited to:

[0469] [ka]

[0470] (In the formula, each 1 are independently -O-, -S-, -N(H)-, -N(R)-, -N(A c) - and -N(OMe)-, 2 -OR, -OAc , -SR, -N(R) 2 , -N(R)(Ac), -N(R)(OMe), or N 3 in and each R' is independently H, alkyl, or substituted alkyl; R is H, halogen, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl. is alkyl, R 1 is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide. It is R 2 is an OH, ester protecting group, resin, amino acid, polypeptide, or polynucleotide. It is a punch line, R 3 and R 4 each independently represents H, halogen, lower alkyl, or substituted lower alkyl or R 3 and R 4 , or two R 3 The group is a cycloa cycloalkyl or heterocycloalkyl may be optionally formed, R 19 are independently an alkyl group having 1 to 6 carbon atoms, an alkoxy group having 1 to 6 carbon atoms, or an ester group. , ether, thioether, amino alkyl, halogen, alkyl ester, aryl ester Stearate, amide, arylamide, alkyl halide, alkylamine, alkylsulfate Phosponic acid, alkyl nitro, thioester, sulfonyl ester, halosulfonyl, nitrile is selected from the group consisting of alkyl, alkylnitrile, and nitro; q is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11. D. Structure and Synthesis of Unnatural Amino Acids: Ketoamine Groups, Ketoamine-like Groups, Masks protected ketoamine group and protected ketoamine group Amino acids containing reactive groups with dicarbonyl-like reactivity can be decomposed via nucleophilic addition reactions. Such reactive groups include ketoamine groups, ketoamine-like groups (ketoamine-like groups, etc.). It has the same reactivity as the amine group and is structurally similar to the ketoamine group, and is masked a protected ketoamine group (which can be readily converted to a ketoamine group) or groups, which, when deprotected, have similar reactivity to a ketoamine group. The amino acid includes an amino acid having the structure of the chemical formula (XXXXXXII).

[0471] [ka]

[0472] (In the formula, A is optional and, when present, is lower alkylene, substituted lower alkylene, lower cycloalkylene, cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, Alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene aryl, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroaryl aryl, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or is a substituted aralkylene; B is optional and, if present, is a linker attached at one end to the diamine-containing moiety. and the linker is a lower alkylene, a substituted lower alkylene, a lower alkenylene, a substituted Lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-(alkyl -S-(alkylene or substituted alkylene)- , -C(O)R”-, -S(O) k (alkylene or substituted alkylene)-(wherein , k is 1, 2, or 3), -C(O)-(alkylene or substituted alkylene -C(S)-(alkylene or substituted alkylene)-, -NR"-(alkylene -CON(R")-(alkylene or substituted alkylene)-, -CON(R")-(alkylene or substituted alkylene) -CSN(R")-(alkylene or substituted alkylene)-, -CSN(R")-(alkylene or substituted alkylene)-, and , -N(R")CO-(alkylene or substituted alkylene)- wherein each R″ is independently H, alkyl, or substituted alkyl; G is

[0473] [ka]

[0474] and T 1 is an optionally substituted alkylene having 1 to 4 carbon atoms, or an optionally substituted heteroalkyl; T 4 is a carbonyl protecting group, and examples of the carbonyl protecting group include the following: These include, but are not limited to:

[0475] [ka]

[0476] (In the formula, each 1 are independently -O-, -S-, -N(H)-, -N(R')-, -N( X is selected from the group consisting of -N(OMe)- and -Ac)-; 2 -OR, -OA c, -SR', -N(R') 2 , -N(R')(Ac), -N(R')(OMe), and is N 3 and each R' is independently H, alkyl, or substituted alkyl. ), R is H, halogen, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl. is alkyl, R 1 is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide. It is R 2 is an OH, ester protecting group, resin, amino acid, polypeptide, or polynucleotide. It is a punch line, R 3 and R 4 each independently represents H, halogen, lower alkyl, or substituted lower alkyl or R 3 and R 4 , or two R 3 The group is a cycloa The aryl group may optionally form an alkyl, alkyl, or heterocycloalkyl. The amino acid having the structure of the chemical formula (XXXXXXII) is III) and amino acids having the structure of formula (XXXXXXIV):

[0477] [ka]

[0478] (In the formula, each R aare independently H, halogen, alkyl, substituted alkyl, -N(R') 2 , -C(O) k R' (where k is 1, 2, or 3), -C(O)N(R') 2 , -OR', and -S(O) k R', wherein each R' is selected from the group consisting of: are independently H, alkyl, or substituted alkyl. E. Structure and Synthesis of Unnatural Amino Acids: Diamines, Diamine-like, Masked Diamines Amines, protected amines and azides Amino acids that have nucleophilic reactive groups can form bonds with other molecules through electrophilic addition reactions. The nucleophilic reactive group is a diamine group (hydrazine group, imine group, 1,1-diamine group, 1,2-diamine group, 1,3-diamine group, and 1,4-diamine groups), diamine-like groups (reactivity similar to that of diamine groups). and structurally similar to a diamine group), a masked diamine group ( a protected diamine group (which, when deprotected, becomes a diamine In some embodiments, the azide-containing Amino acids containing reactive groups that can be reacted with cycloaddition reactions (e.g., 1,3-dipolar cycloaddition, azido- The molecules may be attached via alkyne Huisgen cycloaddition, etc.

[0479] In another embodiment, a hydroxyl group is used for derivatization of a carbonyl-substituted dolastatin derivative. Methods for the chemical synthesis of hydrazine substituted molecules are described. In one embodiment, The hydrazine substituted molecule can be a dolastatin linked derivative. In the paper, a method for preparing hydrazine-substituted molecules is described. For example, it is suitable for derivatizing carbonyl-containing unnatural amino acid polypeptides, By way of example only, unnatural amino acid containing polypeptides include ketone-containing unnatural amino acids. Polypeptides or aldehyde-containing non-natural amino acid polypeptides. In additional or additional embodiments, the unnatural amino acid is an in vivo In further or additional embodiments, the nucleic acid is site-specifically incorporated during translation in The hydrazine-substituted dolastatin derivatives are then site-specifically linked to nitrogen-containing heterocycles. to form a heterocyclic derivatized polypeptide, such as a heterocyclic derivatized polypeptide. Site-specific derivatization of carbonyl-containing unnatural amino acids via nucleophilic attack of the carbonyl group In further or additional embodiments, the hydrazine-substituted drass may be The method for producing tatin derivatives allows for a wide variety of site-specifically derivatized polypeptides. In further or additional embodiments, the hydrazine moiety can be obtained. Method for synthesizing functionalized polyethylene glycol (PEG)-linked dolastatin derivatives is described.

[0480] Such amino acids include those represented by the formula (XXXVII-A) or (XXXVII-B) ) structure.

[0481] [ka]

[0482] (In the formula, A is optional and, when present, is lower alkylene, substituted lower alkylene, lower cycloalkylene, cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, Alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene aryl, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroaryl aryl, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or is a substituted aralkylene; B is optional and, if present, is a linker attached at one end to the diamine-containing moiety. and the linker is a lower alkylene, a substituted lower alkylene, a lower alkenylene, a substituted Lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-(alkyl -S-(alkylene or substituted alkylene)- , -C(O)R"-, -C(O)R"-, -S(O) k (Alkylene or substituted alkene -C(O)-(alkylene, or or substituted alkylene)-, -C(S)-(alkylene or substituted alkylene)-, -NR"-(alkylene or substituted alkylene)-, -CON(R")-(alkylene -CSN(R")-(alkylene or substituted alkylene)-, -CSN(R")-(alkylene or substituted alkylene) -N(R")CO-(alkylene or substituted alkylene)- and -N(R")CO-(alkylene or substituted alkylene)- wherein each R″ is independently selected from the group consisting of H, alkyl, or substituted alkyl. (It is a rule), K is

[0483] [ka]

[0484] (In the formula, R 8 and R 9 are independently H, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl, a chloroalkyl or an amine protecting group; T 1 is a bond, an optionally substituted alkylene having 1 to 4 carbon atoms, 1-4 alkenylene or optionally substituted heteroalkyl; T 2 is an optionally substituted alkylene having 1 to 4 carbon atoms, an optionally substituted heteroalkyl, an optionally substituted aryl, or , optionally substituted heteroaryl; wherein each optional substituent is independently lower alkyl, substituted lower alkyl, lower cycloalkyl, Alkyl, substituted lower cycloalkyl, lower alkenyl, substituted lower alkenyl, alkynyl , lower heteroalkyl, substituted heteroalkyl, lower heterocycloalkyl, substituted lower heterocycloalkyl cycloalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, a aryl, substituted alkaryl, aralkyl, or substituted aralkyl; R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl. can be, R 1 is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide. It is R 2 is an OH, ester protecting group, resin, amino acid, polypeptide, or polynucleotide. It is a punch line, R 3 and R 4 each independently represents H, halogen, lower alkyl, or substituted lower alkyl or R 3 and R4 , or two R 3 The group is a cycloa or optionally forming an alkyl, alkyl or heterocycloalkyl; -ABKR groups are both at least one diamine group, a protected diamine group, or a bicyclic or tricyclic cycloalkyl or heterocyclic group having a masked diamine group. Form a heterocycloalkyl (wherein at least one amine group on -ABKR is is an optionally protected amine, or Each of the -BKR groups is selected from the group consisting of at least one diamine group, a protected diamine group, and is a bicyclic or tricyclic cycloalkyl or cycloalkyl group having a masked diamine group; forming an aryl or heterocycloalkyl; or Both -KR groups are at least one diamine group, protected diamine group, or Monocyclic or bicyclic cycloalkyl or heterocyclic cycloalkyl groups having a masked diamine group It forms a aryl alkyl group. It is.

[0485] In some embodiments, there is a compound having the following Structure 1 or Structure 2:

[0486] [ka]

[0487] (In the formula, A is optional and, when present, is lower alkylene, substituted lower alkylene, lower cycloalkylene, cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, Alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene aryl, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroaryl aryl, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or is a substituted aralkylene; B is optional and, if present, is a linker attached at one end to the diamine-containing moiety. and the linker is a lower alkylene, a substituted lower alkylene, a lower alkenylene, a substituted Lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-(alkyl -S-(alkylene or substituted alkylene)- , -C(O)R”-, -S(O) k (alkylene or substituted alkylene)-(wherein , k is 1, 2, or 3), -C(O)-(alkylene or substituted alkylene -C(S)-(alkylene or substituted alkylene)-, -NR"-(alkylene -CON(R")-(alkylene or substituted alkylene)-, -CON(R")-(alkylene or substituted alkylene) -CSN(R")-(alkylene or substituted alkylene)-, -CSN(R")-(alkylene or substituted alkylene)-, and , -N(R")CO-(alkylene or substituted alkylene)- wherein each R″ is independently H, alkyl, or substituted alkyl; T 1 is a bond or CH 2 and T 2 is CH, wherein each optional substituent is independently lower alkyl, substituted lower alkyl, lower cycloalkyl, Alkyl, substituted lower cycloalkyl, lower alkenyl, substituted lower alkenyl, alkynyl , lower heteroalkyl, substituted heteroalkyl, lower heterocycloalkyl, substituted lower heterocycloalkyl cycloalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, a is selected from alkyl, aryl, substituted alkaryl, aralkyl, or substituted aralkyl; R 1 is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide. It is R 2 is an OH, ester protecting group, resin, amino acid, polypeptide, or polynucleotide. It is a punch line, R 3 and R 4 each independently represents H, halogen, lower alkyl, or substituted lower alkyl or R 3 and R 4 , or two R 3 The group is a cycloa or optionally forming an alkyl, alkyl or heterocycloalkyl; Both -AB-diamine-containing moieties contain at least one diamine group, a protected diamine Bicyclic cycloalkyl or heterocyclic rings having a diamine group or a masked diamine group Form a cycloalkyl (wherein at least one amine of the -AB-diamine-containing moiety is The amine group may be an optionally protected amine or may be an active metabolite, salt, or derivative thereof. or a pharma- ceutically acceptable prodrug or solvate thereof, -B-a group consisting of a diamine-containing moiety, or a bicyclic or tricyclic cycloalkyl group having a masked diamine group. cycloaryl or heterocycloalkyl. Non-limiting examples of amino acids having the structure of formula (XXXVII) include the following: Some examples include:

[0488] [ka]

[0489] The unnatural amino acids may be in the form of a salt or may be used as unnatural amino acid polypeptides. and / or optionally incorporated into a tide, polymer, polysaccharide, or polynucleotide. It may be post-translationally modified.

[0490] In one embodiment, the compound of formula (XXXVII) can be reacted with at least In one embodiment, the compound represented by the formula (XXXVI) is stable in aqueous solution for at least one month. The compound of formula I) is stable under weakly acidic conditions for at least two weeks. The compound of formula (XXXVII) is stable for at least 5 days under weakly acidic conditions. In one embodiment, the acidic conditions have a pH of about 2 to 8.

[0491] In certain embodiments of the compound 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)-(Al (alkylene or substituted alkylene)-, CON(R')(alkylene or substituted alkylene)- -S(alkylene or substituted alkylene)-, -S(O)(alkylene , or substituted alkylene)- or -S(O) 2 (Alkylene or substituted alkene In certain embodiments of the compound of formula (XXXVII), B is - O(CH 2 )-, -CH=N-, CH=NNH-, -NHCH 2 -, -NHCO-, C( O)-, C(O)(CH 2 )-, CONH(CH 2 )-, -SCH 2-, -S(=O)C H 2 - or -S(O) 2 CH 2 -. The compound of formula (XXXVII) In an embodiment, R is an alkyl or cycloalkyl having 1 to 6 carbon atoms. In certain embodiments of the compound of (XXXVII), R is -CH 3 , -CH(CH3) 2 or cyclopropyl. In certain embodiments of the compound of formula (XXXVII), Hey, R 1 H, tert-butyloxycarbonyl (t-Boc), 9-fluorene N-acetylglucosamine (Fmoc), N-acetylglucosamine (TFA ), or benzyloxycarbonyl (Cbz). In certain embodiments of the compound, R 1 Resins, amino acids, polypeptides, or polynucleotides In certain embodiments of the compound of formula (XXXVII), R 1 teeth, An antibody, an antibody fragment, or a monoclonal antibody. A compound of formula (XXXVII) In some embodiments, R 2 is OH, O-methyl, O-ethyl, or Ot-bromo. In certain embodiments of the compound of formula (XXXVII), R 2 Resin, It is at least one amino acid, polypeptide, or polynucleotide. In some embodiments of compounds of formula XXXVII, R 2 The antibody, antibody fragment, or monoclonal antibody It is a monoclonal antibody.

[0492] Non-limiting examples of amino acids having the structure of formula (XXXVII) include the following: Some examples include:

[0493] [ka]

[0494] Non-limiting examples of protected amino acids having the structure of formula (XXXVII) include the following: The following are some of them.

[0495] [ka]

[0496] F. Structure and Synthesis of Unnatural Amino Acids: Aromatic Amines Unnatural amino acids with nucleophilic reactive groups can be used to link molecules together via a variety of reactions. Various reactions include the synthesis of aldehyde-containing dolastatin linker derivatives. Such nucleophiles include, but are not limited to, reductive alkylation reactions using Non-natural amino acids with reactive groups include, by way of example only, aromatic amine groups (secondary and and tertiary amine groups), masked aromatic amine groups (directly attached to aromatic amine groups), or a protected aromatic amine group (which, when deprotected, becomes an aromatic amine). Such unnatural amino acid-containing aromatic amines have reactivity similar to that of the aryl amine groups. An example of such an amino acid is an amino acid having the structure of the chemical formula (XXXXXXV).

[0497] [ka]

[0498] (In the formula,

[0499] [ka]

[0500] is a monocyclic aryl ring, a bicyclic aryl ring, a polycyclic aryl ring, a monocyclic heteroaryl ring , a bicyclic heteroaryl ring, and a polycyclic heteroaryl ring; A is independently CR a , or N, B is independently a , N, O, or S; Each R a are independently H, halogen, alkyl, -NO 2 , -CN, substituted alkyl, - N(R') 2 , -C(O) k R', -C(O)N(R') 2 ,-OR', and,-S( O) k R′, where k is 1, 2, or 3; and n is selected from the group consisting of: 0, 1, 2, 3, 4, 5, or 6; R 1 is H, an amino protecting group, a resin, at least one amino acid, a polypeptide, or , a polynucleotide, R 2 can be any combination of OH, ester protecting groups, resin, at least one amino acid, polypeptide, or or a polynucleotide, R 3 and R 4 each independently represents H, halogen, lower alkyl, or substituted lower alkyl or R 3 and R 4 , or two R 3 The group is a cycloa cycloalkyl or heterocycloalkyl; M is H or -CH 2 R 5 or MNC(R 5 ) part is 4 or may form a seven-membered ring structure, R 5 is an alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkenyl, Alkynyl, alkoxy, substituted alkoxy, alkylalkoxy, substituted alkylalkoxy , polyalkylene oxide, substituted polyalkylene oxide, cycloalkyl, substituted cyclo Alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocycle, Substituted heterocycles, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl, -C(O) R”, ​​-C(O)OR”, -C(O)N(R”) 2 , -C(O)NHCH(R") 2 , - (alkylene or substituted alkylene)-N(R") 2 , -(alkenylene, or Substituted alkenylene)-N(R”) 2 , -(alkylene or substituted alkylene)-(a aryl or substituted aryl), -(alkenylene or substituted alkenylene)-( Aryl or substituted aryl), -(alkylene or substituted alkylene)-ON (R”) 2 , -(alkylene or substituted alkylene)-C(O)SR", -(alkyl aryl or substituted aryl)-SS-(aryl or substituted aryl); (wherein each R" is independently hydrogen, alkyl, substituted alkyl, alkenyl, substituted aryl, aryl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted Heteroaryl, heterocycle, substituted heterocycle, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl or -C(O)OR', or The Two R's 5 The group optionally forms a cycloalkyl or heterocycloalkyl. Or, R 5 and any R a and optionally cycloalkyl or heterocycloalkyl. You can do it, Each R' is independently H, alkyl, or substituted alkyl. The natural amino acids may be in the form of a salt or may be used in combination with non-natural amino acid polypeptides, poly and optionally reductively alkylated. It's fine. The following structure (as shown in all examples herein):

[0501] [ka]

[0502] are "A", "B", "NH-M" and "R a Although the relative orientation of These four configurations of the structure are as shown in the examples of this specification. The ions may be oriented (along with other structures) in any suitable chemical manner.

[0503] Unnatural amino acids containing an aromatic amine moiety having the structure of formula (A) include the following: Non-natural amino acids having the structures below are included.

[0504] [ka]

[0505] (wherein each A' is independently CR a , N, or

[0506] [ka]

[0507] and up to two A' are selected from

[0508] [ka]

[0509] And the remaining A' is CR a , or N. The unnatural amino acids may be in the form of a salt or may be unnatural amino acid polysaccharides. Incorporated into a peptide, polymer, polysaccharide, or polynucleotide, optionally reductively acylated It may be alkylated.

[0510] An unnatural amino acid containing an aromatic amine moiety having the structure of formula (XXXXXXV): Non-limiting examples of the formula (XXXXXXVI) and the formula (XXXXXXV Examples of unnatural amino acids include those having the structure II).

[0511] [ka]

[0512] (wherein G is an amine protecting group, including but not limited to the following: do not have.)

[0513] [ka]

[0514] The unnatural amino acids may be in the form of a salt or may be incorporated into unnatural amino acid polypeptides. and optionally reductively alkylated. It may be embodied.

[0515] The unnatural amino acid containing an aromatic amine moiety has the structure:

[0516] [ka]

[0517] (In the formula, each R a are independently H, halogen, alkyl, -NO 2 , -CN, substituted alkyl Ru, -N(R') 2 , -C(O) k R', -C(O)N(R') 2 , -OR', and - S(O) k R′, where k is 1, 2, or 3; M is H or -CH 2 R 5 or MNC(R 5 ) part is 4 or may form a seven-membered ring structure, R 1 is H, an amino protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide. It is R 2 is an OH, ester protecting group, resin, amino acid, polypeptide, or polynucleotide. It is a punch line, R 5 is an alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkenyl, Alkynyl, alkoxy, substituted alkoxy, alkylalkoxy, substituted alkylalkoxy , polyalkylene oxide, substituted polyalkylene oxide, cycloalkyl, substituted cyclo Alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocycle, Substituted heterocycles, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl, -C(O) R”, ​​-C(O)OR”, -C(O)N(R”) 2, -C(O)NHCH(R") 2 , - (alkylene or substituted alkylene)-N(R") 2 , -(alkenylene, or Substituted alkenylene)-N(R”) 2 , -(alkylene or substituted alkylene)-(a aryl or substituted aryl), -(alkenylene or substituted alkenylene)-( Aryl or substituted aryl), -(alkylene or substituted alkylene)-ON (R”) 2 , -(alkylene or substituted alkylene)-C(O)SR", -(alkyl aryl or substituted aryl)-SS-(aryl or substituted aryl); (wherein each R" is independently hydrogen, alkyl, substituted alkyl, alkenyl, substituted aryl, aryl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted Heteroaryl, heterocycle, substituted heterocycle, alkaryl, substituted alkaryl, aralkyl, substituted aralkyl or -C(O)OR', or R 5 and any R a and optionally cycloalkyl or heterocycloalkyl. You can do it, Each R' is independently H, alkyl, or substituted alkyl. The unnatural amino acids may be in the form of a salt or may be unnatural amino acid polysaccharides. It may be incorporated into a peptide, polymer, polysaccharide, or polynucleotide.

[0518] The above unnatural amino acids of formula (XXXXXXV) are those in which the aromatic moiety of the unnatural amino acid They may also be formed by reduction of a protected or masked amine moiety. Such protected or masked amine moieties include imines, hydrazines, nitriles, and amines. The protected or azide substituents include, but are not limited to, The reducing agents used to reduce the masked amine moiety include TCEP, Na 2 S, Na 2 S 2 O 4 , LiAlH 4 , NaBH 4 , or NaBCNH 3 Examples include But not limited to these.

[0519] (V. Non-natural amino acid-linked dolastatin derivatives) Other embodiments described herein include at least one of the above dolastatin linker derivatives. The present invention relates to methods, strategies, and techniques for incorporating unnatural amino acids into the body. The above dolastatin linker derivatives containing the above unnatural amino acids are produced, purified, and characterized. Additionally, the present embodiment includes methods of characterizing and using at least one unnatural amine. The compound is used, at least in part, to prepare a dolastatin linker derivative containing a cyclic acid. compositions of oligonucleotides (including DNA and RNA) that can be used in Methods for producing, purifying, characterizing, and using such oligonucleotides are also included. The embodiment includes a dolastatin linker derivative that contains at least one unnatural amino acid. The oligonucleotides described above can be used to at least partially produce Compositions of cells capable of expressing the inflammatory cytokines and methods for producing, purifying, characterizing, and using said cells - Patents.com The method includes:

[0520] Therefore, carbonyls, dicarbonyls, alkynes, cycloalkynes, azides, oxy or at least one unnatural amino acid having a hydroxylamine group, or Dolastatin linker derivatives having modified unnatural amino acids are referred to herein as: In one embodiment, a carbonyl, a dicarbonyl, an alkyne, At least one cycloalkyne, azide, oxime, or hydroxylamine group Dolastatin linkers containing one unnatural amino acid or a modified unnatural amino acid A derivative contains at least one post-translational modification at a location on the polypeptide. In some embodiments, the co-translational or post-translational modifications are mediated by cellular mechanisms (e.g., Glycosylation, acetylation, acylation, lipid modification, palmitoylation, palmitic acid addition, In most cases, the above cellular mechanisms are involved. Co-translational or post-translational modifications are modifications that occur at naturally occurring amino acid sites on a polypeptide. In some embodiments, the cellular mechanisms underlying the co-translational modification or translation The post-modification occurs at the unnatural amino acid site on the polypeptide.

[0521] In another embodiment, the post-translational modifications do not utilize cellular machinery, but instead , the first reactive group (ketone, aldehyde, acetal, hemiacetal, alkyne, cyclo Unnatural amines containing alkyne, azide, oxime, or hydroxylamine functional groups at least one unnatural amino acid having a cyclic or cyclic amine group, A molecule having a second reactive group (polymer, water-soluble polymer, polyethylene glycol) is a derivative of the polypeptide, a second protein or polypeptide or a polypeptide analogue, an antibody or antibody fragments, and any combination thereof) by the chemical procedures described herein. or alternatively, by coupling using appropriate reactive groups as specified above: In one embodiment, the co-translational or post-translational modification is In some embodiments, the method is performed in vivo in a eukaryotic or non-eukaryotic cell. The post-translational modification is carried out in vivo without the use of cellular machinery. at least one translationally or post-translationally modified unnatural amino acid as described above. The present invention also includes methods for making, purifying, characterizing, and using the above dolastatin linker derivatives. Can be enjoyed.

[0522] Further, the scope of the methods, compositions, strategies, and techniques described herein includes the above To effect any of the post-translational modifications of the polypeptide, the polypeptide may be modified in any of the following ways: nyl or dicarbonyl group, oxime group, alkyne, cycloalkyne, azide, hydrochloride containing xylamine groups or masked or protected forms thereof Also included are reagents that can react with dolastatin linker derivatives. The resulting post-translationally modified dolastatin linker derivatives have at least one o- The resulting modified oxime-containing dolastatin phosphorus The car derivative may then be modified. The above reagents capable of any of the above post-translational modifications of the tin linker derivatives are prepared, purified and characterized. This section also includes how to use the

[0523] In certain embodiments, the polypeptide or non-natural amino acid linked dolastatin derivative The body comprises at least one co-translational modification made in vivo by a host cell, and includes post-translational modifications that are not normally performed by a foreign host cell. In one embodiment, the polypeptide is a small polypeptide produced in vivo by a eukaryotic cell. At least one co-translational or post-translational modification, Post-translational modifications are not usually carried out by non-eukaryotic cells. Examples include glycosylation, acetylation, acylation, lipid modification, palmitoylation, palmitoylation, and palmitoylation. These include, but are not limited to, tin acid addition, phosphorylation, glycolipid linkage modification, and the like. In one embodiment, the co-translational or post-translational modification is GlcNAc-aspartate. Laginate bond (oligosaccharide is (GlcNAc-Man) 2 -Man-GlcNAc-Glc (including, but not limited to, cases with NAc, etc.) In another embodiment, the co-translational or post-translational modification comprises a linkage of an oligosaccharide that is linked to the nucleotide sequence of ... The decoration may be GalNAc-serine, GalNAc-threonine, GlcNAc-serine or Oligosaccharides (Ga) attached to serine or threonine by GlcNAc-threonine linkage These include, but are not limited to, l-GalNAc, Gal-GlcNAc, etc. In one embodiment, the protein or polypeptide is a secreted polypeptide. sequence or localization sequence, epitope tag, FLAG tag, polyhistidine tag, and / or Alternatively, it may comprise a GST fusion, etc. Furthermore, in this embodiment, at least one of the above Producing, purifying and characterizing said polypeptides containing co-translational or post-translational modifications; In another embodiment, a method of using a glycosylated non-natural amino acid polypeptide is provided. The glycosylated polypeptide is produced in a non-glycosylated form. The non-glycosylated forms of natural amino acids can be prepared by the following steps, or any combination of the following steps: The isolated or substantially purified or an oligonucleotide from a non-purified glycosylated non-natural amino acid polypeptide. Chemical or enzymatic removal of sugar groups; deglycosylated said non-natural amino acid polypeptides. The host (such a host may be modified so as not to glycosylate the polypeptide) may be unnatural amino acids in prokaryotes or eukaryotes that have been modified or mutated or the non-natural amino acid polypeptide is typically produced by glycosylating the polypeptide. Glycosylation inhibitors of polypeptides produced by eukaryotic organisms into cell culture media. Further, as used herein, the introduction of a non-natural amino acid polypeptide that is normally glycosylated is The above non-glycosylated forms of tides are described (usually glycosylated, by When a naturally occurring polypeptide is produced in a glycosylated state, the glycosylation (This refers to a polypeptide that may be glycosylated.) Of course, this does not necessarily mean a non-glycosylated polypeptide. of a natural amino acid polypeptide (or, indeed, any polypeptide described herein). The non-glycosylated form may be in a non-purified form, a substantially purified form, or a purified form. It may be in a separated form.

[0524] In some embodiments, the non-natural amino acid polypeptide is synthesized in the presence of a reaction enhancer. The post-translational modification may be stoichiometric, stoichiometric-like, or In another embodiment, the polypeptide is reacted with the polypeptide in the presence of a reaction enhancer. In another embodiment, the reaction accelerating agent is contacted with a reagent of formula (XIX) under , selected from the group consisting of:

[0525] [ka]

[0526] A. Chemical synthesis of non-natural amino acid-linked dolastatin derivatives: Oxime-containing linked dolastatin derivatives Rastatin derivatives) The non-natural amino acid dolastatin-linked derivatives containing an oxime group have a certain reactive carbomer. aryl or dicarbonyl groups (ketones, aldehydes, or similarly reactive Reactions with a variety of reagents containing aryl groups, including but not limited to aryl groups, This allows the synthesis of new unnatural amino acids with new oxime groups. The oxime exchange reaction allows further functionalization of the dolastatin-linked derivatives. The original dolastatin-linked derivatives containing oxime groups are the products of the oxime bond that links the amino acid to the polypeptide. As long as the nucleotides are stable under conditions required for incorporation into the tide (e.g., in vivo as described herein), , in vitro, and chemical synthesis methods) would be useful in that capacity.

[0527] Thus, in certain embodiments described herein, oxime groups, oxime-like groups (It has the same reactivity as the oxime group and is structurally similar to the oxime group), protected oxime group (which can be readily converted to an oxime group) or A non-naturally occurring compound having a side chain bearing a group (which, when deprotected, has a reactivity similar to that of an oxime group). There are amino acid dolastatin conjugates.

[0528] Such non-natural amino acid dolastatin-linked derivatives include those represented by formula (VIII) or Examples of the dolastatin-linked derivative include those having the structure (IX).

[0529] [ka]

[0530] (In the formula, A is optional and, when present, is lower alkylene, substituted lower alkylene, lower cycloalkylene, cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, Alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene aryl, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroaryl aryl, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or is a substituted aralkylene; B is optional and, when present, is lower alkylene, substituted lower alkylene, lower alkoxy, aryl, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene , or substituted alkylene)-, -S(O) k - (where k is 1, 2, or 3) -S(O) k (Alkylene or substituted alkylene)-, -C(O)-, -C (O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(a -N(R')-, -NR'-(alkylene, or substituted alkylene)-, -N(R')-, -NR'-(alkylene, or 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)-, -N(R')C(O)O-, -S(O) k N(R')-, -N(R')C(O) N(R')-, -N(R')C(S)N(R')-, -N(R')S(O) k N(R') -, -N(R')-N=, -C(R')=N-, -C(R')=NN(R')-, -C (R') = NN =, -C(R') 2 -N=N-, and -C(R') 2 N(R')-N (R')-, wherein each R' is independently H, alkyl or substituted alkyl); R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl. can be, R 1 is H, an amino protecting group, a resin, at least one amino acid, a polypeptide, or , a polynucleotide, R 2 can be any combination of OH, ester protecting groups, resin, at least one amino acid, polypeptide, or or a polynucleotide, R 3 and R 4 are each independently H, halogen, lower alkyl, or substituted lower alkyl. alkyl or R 3 and R 4 , or two R 3 The group is a cycloalkyl group. alkyl or heterocycloalkyl, Z has the structure

[0531] [ka]

[0532] R 5 H,COR 8 , alkyl having 1 to 6 carbon atoms, or thiazole; R 8 is OH, R 6 is OH or H, Ar is phenyl or pyridine; R 7 is an alkyl group having 1 to 6 carbon atoms or hydrogen; L is alkylene-, -alkylene-C(O)-, -(alkylene-O) n -Alkire -, -(alkylene-O) n -Alkylene-C(O)-, -(alkylene-O) n -( CH 2 ) n’ -NHC(O)-(CH 2 ) n’’ -C(Me) 2 -SS-(CH 2 ) n ’’’ -NHC(O)-(alkylene-O) n’’’’ -Alkylene, -(alkylene- O) n -Alkylene-W-, -Alkylene-C(O)-W-, -(Alkylene-O) n - Alkylene-U-alkylene-C(O)-, and -(alkylene-O) n -Alkire is a linker selected from the group consisting of alkylene-U-alkylene; W has the structure

[0533] [ka]

[0534] U has the structure

[0535] [ka]

[0536] n, n', n'', n''', and n'''' are each independently an integer equal to or greater than 1. is a number, or An active metabolite or an active pharma- ceutically acceptable prodrug, or a solvate thereof. be.

[0537] In certain embodiments of the compounds of Formula (VIII) and (IX), R 5 is thiazo In certain embodiments of the compounds of formula (VIII) and (IX), R 6 is H. In certain embodiments of the compounds of formula (VIII) and (IX), A In certain embodiments of the compounds of formula (VIII) and (IX), And R 7 is methyl. In this embodiment, n is an integer from 0 to 20. Compounds of formula (VIII) and (IX) In some embodiments, n is an integer from 0 to 10. In some embodiments of the compound of X), n is an integer from 0-5.

[0538] In certain embodiments of the compounds of Formula (VIII) and (IX), R 5 is thiazo In certain embodiments of the compounds of formula (VIII) and (IX), R 5 is hydrogen. In certain embodiments of the compounds of formula (VIII) and (IX), R 5is methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl butyl, pentyl, or hexyl.

[0539] In certain embodiments of the compounds of Formula (VIII) and (IX), R 5 is -NH -(alkylene-O) n -NH 2 and the alkylene is -CH 2 -, -CH 2 CH 2 -, -CH 2 CH 2 CH 2 -, -CH 2 CH 2 CH 2 CH 2 -, -CH 2 CH 2 CH 2 CH 2 CH 2 -, -CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 -, -CH 2 CH 2 CH 2 C H 2 CH 2 CH 2 CH 2 -, -CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 -, -CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 -, -CH2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 -, -CH 2 CH 2 CH 2 CH 2 CH 2 C H 2 CH 2 CH 2 CH 2 CH 2 CH 2 - or -CH 2 CH 2 CH 2 CH 2 CH 2 C H 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 -. Chemical formulas (VIII) and (IX In some embodiments of the compound of formula (I), the alkylene is methylene, ethylene, propylene. butylene, pentylene, hexylene, or heptylene.

[0540] In certain embodiments of the compounds of Formula (VIII) and (IX), R 8 is -NH-( Alkylene-O) n -NH 2 where 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, 2 3, 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, 6 3, 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 .

[0541] In certain embodiments of the compounds of Formula (VIII) and (IX), R 6 is hydrogen be.

[0542] In certain embodiments of the compounds of Formula (VIII) and (IX), Ar is phenyl. It is.

[0543] In certain embodiments of the compounds of Formula (VIII) and (IX), R 7 is methyl , ethyl, propyl, isopropyl, butyl, sec-butyl, isobutyl, tert- butyl, pentyl, or hexyl. Compounds of formula (VIII) and (IX) In some embodiments of the product, R 7 is hydrogen.

[0544] In certain embodiments of compounds of Formula (VIII) and (IX), each L is independently The linker is a cleavable or non-cleavable linker. In certain embodiments of the compound of formula (I), each L is independently an oligo(ethylene glycol) derivative. It is a modified linker.

[0545] In certain embodiments of the compounds of formula (VIII) and (IX), alkylene, a Alkylene', alkylene'', and alkylene''' each independently represent -CH2 -、-CH 2 CH 2 -、-CH 2 CH 2 CH 2 -、-CH 2 CH 2 CH 2 CH 2 -、-C H 2 CH 2 CH 2 CH 2 CH 2 -、-CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 -、-CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 -、-CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 -、-CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 -、-C H 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 -、-CH 2 CH 2 CH 2 CH 2 CH2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 - or -CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 CH 2 - is represented by the chemical formula (VII In certain embodiments of compounds of I) and (IX), alkylene is methylene, ethyl ethylene, propylene, butylene, pentylene, hexylene, or heptylene.

[0546] In certain embodiments of the compounds of formula (VIII) and (IX), n, n', n' ', n', and n' are each independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 2 1, 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, 6 1, 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 The number is 100.

[0547] In certain embodiments of the compound of Formula (VIII) or (IX), R 1 Polype In certain embodiments of the compound of formula (VIII) or (IX), R 2 is a polypeptide. Certain embodiments of the compound of formula (VIII) or (IX) wherein the polypeptide is an antibody. In some embodiments of the invention, the antibody is Herceptin.

[0548] Such non-natural amino acid dolastatin-linked derivatives include those represented by formula (X), (XI) (XII), or (XIII) .

[0549] [ka] JPEG2024170587000155.jpg94169

[0550] (In the formula, A is optional and, when present, is lower alkylene, substituted lower alkylene, lower cycloalkylene, cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, Alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene aryl, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroaryl aryl, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or is a substituted aralkylene; B is optional and, when present, is lower alkylene, substituted lower alkylene, lower alkoxy, aryl, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene or substituted alkylene)-, -S-, -S-(alkylene , or substituted alkylene)-, -S(O) k - (where k is 1, 2, or 3) -S(O) k (Alkylene or substituted alkylene)-, -C(O)-, -C (O)-(alkylene or substituted alkylene)-, -C(S)-, -C(S)-(a -N(R')-, -NR'-(alkylene, or substituted alkylene)-, -N(R')-, -NR'-(alkylene, or 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)-, -N(R')C(O)O-, -S(O) k N(R')-, -N(R')C(O) N(R')-, -N(R')C(S)N(R')-, -N(R')S(O) k N(R') -, -N(R')-N=, -C(R')=N-, -C(R')=NN(R')-, -C (R') = NN =, -C(R') 2 -N=N-, and -C(R') 2 N(R')-N (R')-, wherein each R' is independently H, alkyl or substituted alkyl); R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl. can be, R 1 is H, an amino protecting group, a resin, at least one amino acid, a polypeptide, or , a polynucleotide, R 2can be any combination of OH, ester protecting groups, resin, at least one amino acid, polypeptide, or or a polynucleotide, R 3 and R 4 are each independently H, halogen, lower alkyl, or substituted lower alkyl. alkyl or R 3 and R 4 , or two R 3 The group is a cycloalkyl group. alkyl or heterocycloalkyl, Z has the structure

[0551] [ka]

[0552] R 5 H,COR 8 , alkyl having 1 to 6 carbon atoms, or thiazole; R 8 is OH, R 6 is OH or H, Ar is phenyl or pyridine; R 7 is an alkyl group having 1 to 6 carbon atoms or hydrogen; L 1 , L 2 , L 3 , and L 4 each independently represents a bond, -alkylene-, -(a alkylene-O) n -Alkylene-J-, -Alkylene'-J-(Alkylene-O) n -Alkylene -, -J-(alkylene-O) n -Alkylene-, -(alkylene-O) n -Alkylene-J-( Alkylene-O) n '-Alkylene-J'-, -(Alkylene-O) n -Alkylene-J-Alkyl Alkylene'-, -W-, -Alkylene-W-, Alkylene'-J- (Alkylene-NMe) n -Alkire -W-, -J-(Alkylene-NMe) n -Alkylene-W-, -J-Alkylene-NMe-Alkylene -Alkylene'-NMe-alkylene''-W-, and -Alkylene-J-alkylene'-NMe-alkylene a linker selected from the group consisting of NMe-N-alkylene-NMe-, W has the structure

[0553] [ka]

[0554] Each J and J′ independently has the structure

[0555] [ka]

[0556] n and n are each independently an integer of 1 or greater.

[0557] In certain embodiments of a compound of Formula (X), (XI), (XII), or (XIII), R 5 is thiazole. Compounds of formula (X), (XI), (XII) or (XIII) In certain embodiments of the compound, R 5 is hydrogen. In some embodiments of the compound of formula (XIII), Ar is phenyl. In certain embodiments of I), (XII), or (XIII), R 7 is methyl. In certain embodiments of compounds of (X), (XI), (XII), or (XIII), n and and n' is an integer of 0 to 20. In certain embodiments of the compound of formula (I), n and n' are integers from 0 to 10. In certain embodiments of compounds of (XI), (XII), or (XIII), n and n' is an integer from 0 to 5.

[0558] In certain embodiments of a compound of Formula (X), (XI), (XII), or (XIII), R 5 is thiazole. Compounds of formula (X), (XI), (XII) or (XIII) In certain embodiments of the compound, R 5 is hydrogen. In certain embodiments of...

Claims

1. A compound represented by formula (VIII) or (IX), or an active metabolite, pharmaceutically acceptable prodrug, or solvate thereof: 【Chemical 1】 During the ceremony, A is optionally, if present, lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene; B is optional and, when present, is lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, —O—, —O-(alkylene or substituted alkylene)-, —S—, —S-(alkylene or substituted alkylene)-, —S(O) k — (where k is 1, 2, or 3), —S(O) k (alkylene or substituted alkylene)-, —C(O)—, —C(O)-(alkylene or substituted alkylene)-, —C(S)—, —C(S)-(alkylene or substituted alkylene)-, —N(R′)—, —NR′-(alkylene or substituted alkylene)-, —C(O)N(R′)—, —CON(R′)-(alkylene or substituted alkylene)-, —CSN(R′)—, —CSN(R′)-(alkylene or substituted alkylene)-, —N(R′)CO-(alkylene or substituted alkylene)-, —N(R′)C(O)O—, —S(O) k a linker selected from the group consisting of N(R')-, -N(R')C(O)N(R')-, -N(R')C(S)N(R')-, -N(R')S(O) k N(R')-, -N(R')-N=, -C(R')=N-, -C(R')=N-N(R')-, -C(R')=N-N=, -C(R') 2 -N=N-, and -C(R') 2 -N(R')-N(R')-, wherein each R' is independently H, alkyl, or substituted alkyl; R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; R 1 is H, an amino protecting group, a resin, at least one amino acid, a polypeptide, or a polynucleotide; R 2 is OH, an ester protecting group, a resin, at least one amino acid, polypeptide, or polynucleotide; R 3 and R 4 are independently H, halogen, lower alkyl, or substituted lower alkyl, or R 3 and R 4 or two R 3 groups may optionally form a cycloalkyl or heterocycloalkyl; Z has the following structure: 【Chemistry 2】 R 5 is H, COR 8 , alkyl having 1 to 6 carbon atoms, or thiazole; R 8 is OH; R 6 is OH or H; Ar is phenyl or pyridine; R 7 is alkyl having 1 to 6 carbon atoms or hydrogen; L is a linker selected from the group consisting of -alkylene-, -alkylene-C(O)-, -(alkylene-O) n -alkylene-, -(alkylene-O) n -alkylene-C(O)-, -(alkylene-O) n -(CH 2 ) n' -NHC(O)-(CH 2 ) n'' -C(Me) 2 -S-S-(CH 2 ) n''' -NHC(O)-(alkylene-O) n'''' -alkylene, -(alkylene-O) n -alkylene-W-, -alkylene-C(O)-W-, -(alkylene-O) n -alkylene-U-alkylene-C(O)-, and -(alkylene-O) n -alkylene-U-alkylene-; W has the following structure: 【Chemistry 3】 U has the following structure: 【Chemistry 4】 n, n', n'', n''', and n'''' are independently integers of 1 or greater.

2. A compound represented by formula (VIII): The compound of claim 1.

3. R 1 and R 2 are antibodies.

3. The compound of claim 1 or 2.

4. The antibody is trastuzumab. The compound of claim 3.

5. A is optional, and when present, is alkylene having 1 to 8 carbon atoms, heteroalkylene having 1 to 8 carbon atoms, or arylene having 5 to 20 ring atoms; The compound according to any one of claims 1 to 4.

6. A is an arylene having 5 to 20 ring atoms. The compound of claim 5.

7. A is phenylene. The compound of claim 6.

8. B is optional, and when present, is selected from alkylene having 1 to 8 carbon atoms, substituted alkylene having 1 to 8 carbon atoms, -O-, -O-(alkylene having 1 to 10 carbon atoms)-, -S-, -S-(alkylene having 1 to 10 carbon atoms)-, -C(O)-, -C(O)-(alkylene having 1 to 10 carbon atoms or substituted alkylene having 1 to 10 carbon atoms)-, -N(R')-, -NR'-(alkyl ... a linker selected from the group consisting of -(C1-C10 alkylene)-, -C(O)N(R')-, -CON(R')-(C1-C10 alkylene)-, -N(R')CO-(C1-C10 alkylene)-, -C(R')=N-, and -C(R')=N-N(R')- (wherein each R' is independently H or alkyl having 1 to 10 carbon atoms); The compound according to any one of claims 1 to 7.

9. B is absent or a linker selected from the group consisting of alkylene having 1 to 8 carbon atoms, -O-(alkylene having 1 to 10 carbon atoms)-, -S-(alkylene having 1 to 10 carbon atoms)-, -C(O)-(alkylene having 1 to 10 carbon atoms)-, -NR'-(alkylene having 1 to 10 carbon atoms)-, -CON(R')-(alkylene having 1 to 10 carbon atoms)-, and -N(R')CO-(alkylene having 1 to 10 carbon atoms)-, wherein each R' is independently H or alkyl having 1 to 10 carbon atoms; The compound of claim 8.

10. B is absent or a linker selected from the group consisting of alkylene having 1 to 8 carbon atoms, -C(O)-(alkylene having 1 to 10 carbon atoms)-, -O(CH 2 )-, -CH═N-, -CH═N-NH-, -NHCH 2 -, -NHCO-, -C(O)-, -C(O)-(CH 2 )-, -CONH-(CH 2 )-, -SCH 2 -, -S(═O)CH 2 -, and -S(O) 2 CH 2 -; The compound of claim 9.

11. B does not exist. The compound of claim 10.

12. R is H, alkyl having 1 to 6 carbon atoms, substituted alkyl having 1 to 6 carbon atoms, cycloalkyl having 3 to 6 carbon atoms, or substituted cycloalkyl having 3 to 6 carbon atoms. The compound according to any one of claims 1 to 11.

13. R is H, alkyl having 1 to 6 carbon atoms, or cycloalkyl having 3 to 6 carbon atoms.

13. The compound of claim 12.

14. R is —CH 3 , —CH(CH 3 ) 2 , or cyclopropyl. The compound of claim 13. Claim 15: R 3 is H. The compound according to any one of claims 1 to 14.

16. R 4 is H. The compound according to any one of claims 1 to 15.

17. The compound according to claim 1, wherein R 5 is COR 8 . The compound according to any one of claims 1 to 16.

18. The compound according to claim 1, wherein R 6 is H. The compound according to any one of claims 1 to 17.

19. Ar is phenyl. The compound according to any one of claims 1 to 18.

20. The compound according to claim 1, wherein R 7 is methyl. The compound according to any one of claims 1 to 19.

21. L is -(C1-C3 alkylene-O)n-C1-C3 alkylene-; The compound according to any one of claims 1 to 20.

22. L is —(CH 2 CH 2 —O) n —CH 2 CH 2 —; 22. The compound of claim 21.

23. n is 3. The compound according to any one of claims 1 to 22.

24. A is phenylene, B does not exist, R is methyl; L is —(CH 2 CH 2 —O) n —CH 2 CH 2 —; R 1 and R 2 are an antibody, and the antibody is trastuzumab; The compound according to any one of claims 1 to 23.

25. A method for derivatizing a dolastatin analog of formula (I), formula (III), formula (IV), formula (V), or formula (VI), comprising: The method comprises contacting the dolastatin analog with a reagent of formula (XXXVII): Formula (I), Formula (III), Formula (IV), Formula (V), or Formula (VI) corresponds to the following: 【Chemistry 5】 【change】 During the ceremony, Z has the following structure: 【Chemistry 6】 R 5 is H, COR 8 , alkyl having 1 to 6 carbon atoms, or thiazole; R 8 is OH or —NH-(alkylene-O) n —NH 2 ; R 6 is OH or H; Ar is phenyl or pyridine; R 7 is alkyl having 1 to 6 carbon atoms or hydrogen; Y is NH 2 —O— or methyl; L, L 1 , L 2 , L 3 and L 4 independently represent a bond, -alkylene-, -alkylene-C(O)-, -(alkylene-O) n -alkylene-, -(alkylene-O) n -alkylene-C(O)-, -(alkylene-O) n -(CH 2 ) n' -NHC(O)-(CH 2 ) n'' -C(Me) 2 -S-S-(CH 2 ) n''' -NHC(O)-(alkylene-O) n'''' -alkylene-, -(alkylene-O) n -alkylene-W-, -alkylene-C(O)-W-, -(alkylene-O) n -alkylene-J-, -alkylene'-J-(alkylene-O) n -Alkylene-, -(alkylene-O) n -Alkylene-J-alkylene', -J-(alkylene-O) n -Alkylene-, -(alkylene-O) n -Alkylene-J-(alkylene-O) n' -Alkylene-J'-, -W-, -Alkylene-W-, alkylene'-J-(alkylene-NMe) n -Alkylene-W-, J-(alkylene-NMe) n -Alkylene-W-, -(alkylene-O) n -Alkylene-U-alkylene-C(O)-, -(alkylene-O) n a linker selected from the group consisting of -alkylene-U-alkylene-, -J-alkylene-NMe-alkylene'-NMe-alkylene''-W-, and -alkylene-J-alkylene'-NMe-alkylene''-NMe-alkylene'''-W-; W has the following structure: 【Chemistry 7】 U has the following structure: 【Chemistry 8】 Each J and J′ independently has the structure: 【Chemistry 9】 or L is absent, Y is methyl, R 5 is COR 8 , and R 8 is —NH(alkylene-O) n —NH 2 ; each n, n', n'', n''', and n'''' is independently an integer greater than or equal to 1; Formula (XXXVII) corresponds to the following process: 【Chemistry 10】 During the ceremony, A is optionally lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkarylene, substituted alkarylene, aralkylene, or substituted aralkylene, if present; B is optional and, when present, is lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, —O—, —O-(alkylene or substituted alkylene)-, —S—, —S-(alkylene or substituted alkylene)-, —S(O) k — (wherein k is 1, 2, or 3), —S(O) k (alkylene or substituted alkylene)-, —C(O)—, —C(O)-(alkylene or substituted alkylene)-, —C(S)—, —C(S)-(alkylene or substituted alkylene)-, —N(R′)—, —NR′-(alkylene or substituted alkylene)-, —C(O)N(R′)—, —CON(R′)-(alkylene or substituted alkylene)-, —CSN(R′)—, —CSN(R′)-(alkylene or substituted alkylene)-, —N(R′)CO-(alkylene or substituted alkylene)-, —N(R′)C(O)O—, —S(O) k a linker selected from the group consisting of N(R')-, -N(R')C(O)N(R')-, -N(R')C(S)N(R')-, -N(R')S(O) k N(R')-, -N(R')-N=, -C(R')=N-, -C(R')=N-N(R')-, -C(R')=N-N=, -C(R') 2 -N=N-, and -C(R') 2 -N(R')-N(R')-, wherein each R' is independently H, alkyl, or substituted alkyl; K is one of the following: 【Chemistry 11】 R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; R 1 is H, an amino protecting group, a resin, at least one amino acid, or a polynucleotide; R 2 is OH, an ester protecting group, a resin, at least one amino acid, or a polynucleotide; R 3 and R 4 are independently H, halogen, lower alkyl, or substituted lower alkyl, or R 3 and R 4 or two R 3 groups may optionally form a cycloalkyl or heterocycloalkyl.

26. The derivatized dolastatin analog is an amino acid containing at least one oxime having the structure of formula (VIII), formula (IX), formula (X), formula (XI), formula (XII), or formula (XIII): The method of claim 25: 【Chemistry 12】 【change】 27. The dolastatin analog is a compound shown in formula (I):

26. The method of claim 25.

28. The derivatized dolastatin analog has the structure shown in formula (VIII):

28. The method of claim 26 or 27.

29. K is one of the following: The method according to any one of claims 25 to 28, 【Chemistry 13】 30. The method of claim 30, wherein the dolastatin analog is contacted with a reagent of formula (XXXVII) in an aqueous solution under weakly acidic conditions.

30. The method according to any one of claims 25 to 29.

31. R 1 , R 2 , A, B, R, R 3 , R 4 , R 5 , R 6 , Ar, R 7 , L, and n are as defined in any one of claims 3 to 24. The method according to any one of claims 25 to 30.

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