Composition containing a non-natural amino acid-bonded drastatin derivative, a method using the same, and its use

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 specification is based on U.S. Provisional Application No. 61 / 491,146 filed on 27 May 2011. A composition containing a non-natural amino acid-bonded drastatin derivative, a method using the same, and Priority is claimed from the "use of". The above application as a whole is referenced herein. It will be more integrated.

[0002] [Background of the Invention] Proteins contain amino acids that are not genetically coded (i.e., "non-natural amino acids"). The ability to incorporate naturally occurring functional groups (e.g., -NH2 at the ε position of lysine, cysta) We propose useful alternatives to sulfhydryl-SH and histidine imino groups. It enables the introduction of available chemical functional groups. Some chemical functional groups are genetically encoded. It is inactive to the functional groups found in the 20 normal amino acids, but is incorporated into non-natural amino acids. It reacts efficiently and accurately with functional groups that can be incorporated to form stable bonds. It is known that...

[0003] Here, we have the usual 20 amino acids that are not present in the protein but are genetically coded. It is chemically inert to all existing functional groups and efficient with reagents containing specific functional groups. Furthermore, select chemical functional groups that can react selectively and be used to form stable covalent bonds. We provide a method for implementing it effectively.

[0004] [Summary of the Invention] In this specification, a toxic moiety having one or more linkers and a non-natural amino acid bonded A toxic group and a method for producing such unnatural amino acids and polypeptides have been developed. It will be shown.

[0005] In some embodiments of the present invention, a compound represented by formula (I), or a salt thereof, is described. It is being done.

[0006] [ka]

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

[0008] [ka]

[0009] R5 is H, COR8, an alkyl group with 1 to 6 carbon atoms, or a thiazole. R8 is OH, or -NH- (alkylene-O) n -NH2, R6 is OH or H. Ar is phenyl or pyridine. R7 is an alkyl group with 1 to 6 carbon atoms, or hydrogen. Y is hydroxylamine, methyl, aldehyde, protected aldehyde, ketone, protected Protected ketones, thioesters, esters, dicarbonyls, hydrazines, amidines, imi N, diamines, azides, ketoamines, ketoalkynes, alkynes, cycloalkynes, and Selected from the group consisting of engines, L represents -alkylene-, -alkylene-C(O)-, and -(alkylene-O). n -Aruki Ren-, -(Alkiren-O) n -Alkylene-C(O)-, -(Alkylene-O) n - (CH2) n’ -NHC(O)-(CH2) 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-U-alkylene-C(O)-, and, -(alkylene-O) n -alk ylene-U-alkylene selected from the group consisting of, W has the following structure,

[0010]

Chemical formula

[0011] U has the following structure,

[0012]

Chemical formula

[0013] Or, L is absent, Y is methyl, R5 is COR8, R8 is -NH(alk ylene-O) n -NH2, and n, n’, n’’, n’’’, and, n’’’’ are , each independently, an integer of 1 or more.) In some embodiments, R5 is thiazole. In other embodiments, R6 is H is. In one embodiment, Ar is phenyl. Further, or additional embodiments forms, R7 is methyl. In some embodiments, n is an integer of 0 to 20, 0 to 10 , or 0 to 5.)

[0014] In some embodiments, the compound represented by formula (II) is described.

[0015]

Chemical formula

[0016] In one embodiment, L is -(alkylene-O) n -Alkilen-. Specific implementation form In this state, each alkylene is -CH2CH2-, n=3, and R7 is methyl In other embodiments, L is an alkylene. In specific embodiments, each Alkylenes are -CH2CH2-, and R7 is either methyl or hydrogen. In the application form, L is -(alkylene-O) n -Alkilen-C(O). A certain implementation form In this state, each alkylene is -CH2CH2-, n=4, and R7 is methyl. In further or additional embodiments, L is -(alkylene-O) n -(CH 2) n’ -NHC(O)-(CH2) n’’ -C(Me)2-SS-(CH2) n’’ ’ -NHC(O)-(alkylene-O) n’’’’ -It is alkylene. Several implementations In terms of form, each alkylene is -CH2CH2-, n is 1, and n' is 2. n'' is 1, n''' is 2, n'''' is 4, and R7 is methyl be.

[0017] In some embodiments, Y is azid. In other embodiments, Y is cyclooc It is cynothin. In a particular embodiment, the cyclooctin has the following structure.

[0018] [ka]

[0019] (In the formula, Each R 19 These are, independently, alkyl groups with 1 to 6 carbon atoms, alkoxy groups with 1 to 6 carbon atoms, and ester groups. ethers, thioethers, aminoalkyls, halogens, alkyl esters, aryls Esters, amides, arylamides, alkyl halides, alkylamines, alkyls Sulfonic acid, alkyl nitro, thioester, sulfonyl ester, halosulfonyl, nitrate Selected from the group consisting of lyl, 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, formulas (II), (III), (IV), (V), and (VI) describes the compound or salt thereof.

[0020] [ka] JPEG0007911040000008.jpg115169

[0021] Here, Z has the following structure:

[0022] [ka]

[0023] R5 is H, COR8, an alkyl group with 1 to 6 carbon atoms, or a thiazole. R8 is OH, Ar is phenyl or pyridine. R7 is an alkyl group with 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 N, amidine, imine, diamine, azido, ketoamine, ketoalkyne, alkyne, cyk Selected from the group consisting of roalkynes and engiones, L1, L2, L3, and L4 are each independently linked, -alkylene-, -(A Lukilen-O) n -alkylene-J-, -alkylene'-J-(alkylene-O) n -Alkilen -, -J-(alkylene-O) n -Alkylene-, -(Alkylene-O) n -Alkilen-J-( Alkylene-O) n '-Alkilen-J'-, -(Alkilen-O) n -Alkilen-J-Alki Len'-, -W-, -Alkilen-W-, Alkilen'-J-(Alkilen-NMe) n -Archile N-W-, -J-(alkylene-NMe) n -Alkilen-W-, -J-Alkilen-NMe-Alki Len'-NMe-alkylene''-W-, and -alkylene-J-alkylene'-NMe-alkylene A linker selected from the group consisting of n''-NMe-alkylene'''-W-, W has the following structure:

[0024] [ka]

[0025] Each J and J' independently has the following structure:

[0026] [ka]

[0027] Each n and n' is an independent integer greater than or equal to 1.

[0028] In one embodiment, a compound comprising formula (VII) is described.

[0029] [ka]

[0030] In one embodiment, L1 is -(alkylene-O) n -Alkilen-J-, and L2 is -A Lukilen'-J'-(Alkilen-O) n '-Alkylene-, L3 is -J''-(Alkylene-O) n ' '-alkylene-, alkylene is -CH2CH2-, and alkylene' is -(CH 2) 4-, n is 1, n' and n'' are 3, J has the following structure:

[0031] [ka]

[0032] J' and J'' have the following structure:

[0033] [ka]

[0034] R7 is methyl. In another embodiment, L1 is -J-(alkylene-O) n -Aruki It is ylene-, and L2 is -(alkylene-O) n' -alkylene-J'-alkylene'-, L 3 is -(alkylene-O) n'' -Alkylene-J''-, and alkylene is -CH2CH2- And alkylene' is -(CH2)4-, n is 1, and n' and n'' are 4. Yes, J, J', and J'' have the following structure:

[0035] [ka]

[0036] In one embodiment, Y is azide. In another embodiment, Y is cyclooctane. In certain embodiments, cyclooctin has the following structure.

[0037] [ka]

[0038] (In the ceremony Each R 19 These are, independently, alkyl groups with 1 to 6 carbon atoms, alkoxy groups with 1 to 6 carbon atoms, and ester groups. ethers, thioethers, aminoalkyls, halogens, alkyl esters, aryls Esters, amides, arylamides, alkyl halides, alkylamines, alkyls Sulfonic acid, alkyl nitro, thioester, sulfonyl ester, halosulfonyl, nitrate Selected from the group consisting of lyl, 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 represented by formula (VIII) or (IX), and The active metabolite, pharmaceutically acceptable prodrug, or solvate is described. .

[0039] [ka]

[0040] (In the formula, A is optional, and if present, it may be a lower alkylene, substituted lower alkylene, or lower alkylene. Roalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, Alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene Len, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene , substituted heteroarylene, alkalilene, substituted alkalilene, aralkylene, or substituted heteroarylene, alkalilene, alkalilene, or substituted It is a converted aralkylene, B is optional and, if present, may be a lower alkylene, a substituted lower alkylene, or a lower alkylene. Kenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene, or substituted alkylene)-, -S-, -S-(alkylene (N, or substitution alkylene)-, -S(O) k -(where k is 1, 2, or 3 (There is), -S(O) k (Alkylene, or substituted alkylene)-, -C(O)-, -C (O)-(alkylene, or substituted alkylene)-, -C(S)-, -C(S)-(A Alkilen, or substituted alkylene)-, -N(R')-, -NR'-(alkylene, For example, substitution alkylenes: -C(O)N(R')-, -CON(R')-(alkylenes) , or substituted alkylene)-, -CSN(R')-, -CSN(R')-(alkylene) , or substituted alkylene)-, -N(R')CO-(alkylene, or substituted alkyl 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' A linker selected from the group consisting of )-N(R')- (where each R' is independently (H, alkyl, or substituted alkyl), R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl. can be, R1 is H, amino protecting group, resin, at least one amino acid, polypeptide, or , it is a polynucleotide, R2 is OH, ester protecting group, resin, at least one amino acid, polypeptide, or is a polynucleotide, R3 and R4 are independently H, halogen, lower alkyl, or substituted lower alkyl. It is either a cycloalkyl group, or R3 and R4, or two R3 groups are cycloalkyl groups. Kill or, optionally, heterocycloalkyl groups may be formed. Z has the following structure:

[0041] [ka]

[0042] R5 is H, COR8, an alkyl group with 1 to 6 carbon atoms, or a thiazole. R8 is OH, R6 is OH or H. Ar is phenyl or pyridine. R7 is an alkyl group with 1 to 6 carbon atoms, or hydrogen. L represents -alkylene-, -alkylene-C(O)-, and -(alkylene-O). n -Aruki Ren-, -(Alkiren-O) n -Alkylene-C(O)-, -(Alkylene-O) n - (CH2) n’-NHC(O)-(CH2) 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 -Alkilen-U-A A linker selected from the group consisting of lukilen. W has the following structure:

[0043] [ka]

[0044] U has the following structure:

[0045] [ka]

[0046] n, n', n'', n''', and n'''' are each independently one or more integers. It is a number. In some embodiments, R1 is a polypeptide. In certain embodiments, the p The lipeptide is an antibody. In certain embodiments, the antibody is Herceptin. In other embodiments, R2 is a polypeptide. In specific embodiments, the polypeptide Tide is an antibody. In certain embodiments, the antibody is Herceptin.

[0047] In one embodiment of the present invention, formulas (X), (XI), (XII), or (XIII) The compound or its salt is shown and described.

[0048] [ka] JPEG0007911040000022.jpg169169

[0049] (In the formula, A is optional, and if present, it may be a lower alkylene, substituted lower alkylene, or lower alkylene. Roalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, Alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene Len, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene , substituted heteroarylene, alkalilene, substituted alkalilene, aralkylene, or substituted heteroarylene, alkalilene, alkalilene, or substituted It is a converted aralkylene, B is optional and, if present, may be a lower alkylene, a substituted lower alkylene, or a lower alkylene. Kenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene, or substituted alkylene)-, -S-, -S-(alkylene (N, or substitution alkylene)-, -S(O) k -(where k is 1, 2, or 3 (There is), -S(O) k (Alkylene, or substituted alkylene)-, -C(O)-, -C (O)-(alkylene, or substituted alkylene)-, -C(S)-, -C(S)-(A Alkilen, or substituted alkylene)-, -N(R')-, -NR'-(alkylene, For example, substitution alkylenes: -C(O)N(R')-, -CON(R')-(alkylenes) , or substituted alkylene)-, -CSN(R')-, -CSN(R')-(alkylene) , or substituted alkylene)-, -N(R')CO-(alkylene, or substituted alkyl 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' A linker selected from the group consisting of )-N(R')- (where each R' is independently (H, alkyl, or substituted alkyl), R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl. can be, R1 is H, amino protecting group, resin, at least one amino acid, polypeptide, or , it is a polynucleotide, R2 is OH, ester protecting group, resin, at least one amino acid, polypeptide, or is a polynucleotide, R3 and R4 are independently H, halogen, lower alkyl, or substituted lower alkyl. It is either a cycloalkyl group, or R3 and R4, or two R3 groups are cycloalkyl groups. Kill or, optionally, heterocycloalkyl groups may be formed. Z has the following structure:

[0050] [ka]

[0051] R5 is H, COR8, an alkyl group with 1 to 6 carbon atoms, or a thiazole. R8 is OH, R6 is OH or H. Ar is phenyl or pyridine. R7 is an alkyl having 1 to 6 carbon atoms or hydrogen, L1, L2, L3, and L4 are each independently a bond, alkylene, -(alki ylene-O) n -alkylene-J-, -alkylene'-J-(alkylene-O) n -alkylene-, -J-(alkylene-O) n -alkylene-, -(alkylene-O) n -alkylene-J-(alk ylene-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- and is a linker selected from the group consisting of W has the following structure

[0052]

Chemical formula

[0053] Each J and J' independently has the following structure

[0054]

Chemical formula

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

[0056] In some embodiments, R1 is a polypeptide. In certain embodiments, the said po The lipeptide is an antibody. In certain embodiments, the antibody is Herceptin. In other embodiments, R2 is a polypeptide. In specific embodiments, the polypeptide Tide is an antibody. In certain embodiments, the antibody is Herceptin.

[0057] In one embodiment, the formulas shown are (I), (III), (IV), (V), or (VI) A method for derivatizing a drastatin analog, The method involves contacting the drastatin analog with the reagent of formula (XXXVII). Including, Formulas (I), (III), (IV), (V), or (VI) correspond to the following:

[0058] [ka] JPEG0007911040000027.jpg185169

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

[0060] [ka]

[0061] R5 is H, COR8, an alkyl group with 1 to 6 carbon atoms, or a thiazole. R8 is OH, or -NH- (alkylene-O) n -NH2, R6 is OH or H. Ar is phenyl or pyridine. R7 is an alkyl group with 1 to 6 carbon atoms, or hydrogen. Y is NH2-O-, or methyl. L, L1, L2, L3, and L4 are each independently a bond, -alkylene-, - alkylene-C(O)-, -(alkylene-O) n -alkylene-, -(alkylene-O ) n -alkylene-C(O)-, -(alkylene-O) n -(CH2) n’ -NHC(O )-(CH2) n’’ -C(Me)2-S-S-(CH2) n’’’ -NHC(O)-( alkylene-O) n’’’’ -alkylene, -(alkylene-O) n -alkylene-W- , -alkylene-C(O)-W-, -(alkylene-O) n -alkylene-J-, -alky len'-J-(alkylene-O) n -alkylene,-(alkylene-O) n -alkylene-J-alk ylene'-,-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 -alk ylene-W-,-(alkylene-O) n -alkylene-U-alkylene; -J-alkylene- NMe-alkylene'-NMe-alkylene''-W-, and -alkylene-J-alkylene'-NM e-alkylene''-NMe-alkylene'''-W- selected from the group consisting of W has the following structure

[0062]

Chemical Structure

[0063] U has the following structure:

[0064] [ka]

[0065] Each J and J' independently has the following structure:

[0066] [ka]

[0067] Alternatively, L is absent, Y is methyl, R5 is COR8, and R8 is -NH(A Lukilen-O) n -NH2, where n, n', n'', n''', and n'''' are (Each of these is an independent integer greater than or equal to 1.) Formula (XXXVII) corresponds to the following method, which is described herein.

[0068] [ka]

[0069] (In the formula, A is optional, and if present, it may be a lower alkylene, a substituted lower alkylene, or a lower alkylene. Kenylene, substituted lower alkenylene, arylene, substituted arylene, heteroarylene, Substitute heteroarylene, alkalilene, substituted alkalilene, aralkylene, or substituted a It is Larkiren, B is optional and, if present, may be a lower alkylene, a substituted lower alkylene, or a lower alkylene. Kenylene, substituted lower alkene, -O-, -O-(alkylene, or substituted alkylene -, -S-, -S-(alkylene, or substituted alkylene)-, -S(O) k -( Here, k is 1, 2, or 3), -S(O) k (Alkylene, or substitute 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-( Lukilen, 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-, approximately The linker is selected from the group consisting of -C(R')2-N(R')-N(R')- (Note that 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, R1 is H, an amino protecting group, a resin, at least one amino acid, or a polynucleotide. It is Chido, R2 is an OH group, an ester protecting group, a resin, at least one amino acid, or a polynucleotide. It is a leotide, R3 and R4 can each be independently H, halogen, lower alkyl, or substituted. It is either a lower alkyl group, or R3 and R4, or two R3 groups are cycloa (A lukyl or heterocycloalkyl group may be optionally formed.) In some embodiments, the derivatized drastatin analog is derived from formula (VIII Having the structure of ), (IX), (X), (XI), (XII), or (XIII): It is an amino acid that contains at least one oxime.

[0072] [ka] JPEG0007911040000035.jpg176169 JPEG0007911040000036.jpg169169

[0073] In certain embodiments, the drastatin analog is expressed in the form shown in formula (XXXVII) The reagent is brought into contact with the other reagents in an aqueous solution under weakly acidic conditions.

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

[0075] [ka] JPEG0007911040000038.jpg224169 JPEG0007911040000039.jpg103169

[0076] (wherein, Z has the following structure,

[0077]

Chemical formula

[0078] R5 is H, CO2H, alkyl having 1 to 6 carbon atoms, or thiazole, R6 is OH or H, Ar is phenyl or pyridine, R1 is H, an amino protecting group, a resin, at least one amino acid, a polypeptide, or a polynucleotide, R2 is OH, an ester protecting group, a resin, at least one amino acid, a polypeptide, or a polynucleotide, R4 is H, halogen, lower alkyl, or substituted lower alkyl, R7 is alkyl having 1 to 6 carbon atoms or hydrogen, L, L1, L2, L3, and L4 are each independently 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 -(CH2) n’ -NHC (O)-(CH2) n’’ -C(Me)2-S-S-(CH2) n’’’ -NHC(O) -(alkylene-O) n’’’’ -alkylene, -(alkylene-O) n-alkylene- W-, -alkylene-C(O)-W-, -(alkylene-O) n -alkylene-J-, -al kylene'-J-(alkylene-O) n -alkylene-, -(alkylene-O) n -alkylene-J -alkylene'-, -J-(alkylene-O) n -alkylene-, -(alkylene-O) n -al kylene-J-(alkylene-O) n '-alkylene-J'-, -W-, -alkylene-W-, al kylene'-J-(alkylene-NMe) n -alkylene-W-, -J-(alkylene-NMe) n -alk ylene-W-, -(alkylene-O) n -alkylene-U-C(O)-, -(alkylene- O) n -alkylene-U-alkylene; -J-alkylene-NMe-alkylene'-NMe-al kylene''-W-, and -alkylene-J-alkylene'-NMe-alkylene''-NMe-al kylene'''-W- selected from the group consisting of, W has the following structure,

[0079]

Chemical formula

[0080] U has the following structure,

[0081]

Chemical formula

[0082] Each J and J' independently has the following structure,

[0083] [ka]

[0084] n and n' are independent integers greater than or equal to 1. Each R 16 These are independently hydrogen, halogen, alkyl, NO2, CN, and substituted alkyl (Selected from a group consisting of kills.) In some embodiments, R1 is a polypeptide. In certain embodiments, the p The lipeptide is an antibody. In certain embodiments, the antibody is Herceptin. In other embodiments, R2 is a polypeptide. In specific embodiments, the polypeptide Tide is an antibody. In certain embodiments, the antibody is Herceptin.

[0085] In some embodiments of the present invention, formulas (XXXI), (XXXII), (XXXIII The compounds listed are those shown in (XXXIV), (XXXV), or (XXXVI). It is being done.

[0086] [ka] JPEG0007911040000045.jpg195169 JPEG0007911040000046.jpg93169

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

[0088] [ka]

[0089] R5 is H, CO2H, an alkyl group with 1 to 6 carbon atoms, or a thiazole. R6 is OH or H. Ar is phenyl or pyridine. R1 is H, amino protecting group, resin, at least one amino acid, polypeptide, or , it is a polynucleotide, R2 is OH, ester protecting group, resin, at least one amino acid, polypeptide, or is a polynucleotide, R4 is H, halogen, lower alkyl, or substituted lower alkyl. R7 is an alkyl group with 1 to 6 carbon atoms, or hydrogen. L, L1, L2, L3, and L4 are independently linked, -alkylene-, - Alkylene-C(O)-, -Alkylene-J-, -(Alkylene-O) n -Alkilen- ,-(alkylene-O) n -Alkylene-C(O)-, -(Alkylene-O) n -J-,-( Alkylene-O) n -J-alkylene, -(alkylene-O) n -(CH2) n’ -NHC (O)-(CH2) n’’ -C(Me)2-SS-(CH2) n’’’ -NHC(O) -(Alkylene-O) n’’’’ -Alkylene, -(Alkylene-O) n -Alkilen- W-,-alkylene-C(O)-W-,-(alkylene-O) n -Alkilen-J-, -Al Kiren'-J-(Alkiren-O) n -Alkilen-, -(Alkilen-O) n -Alkilen-J -Alkilen'-, -J-(Alkilen-O) n -Alkilen-, -(Alkilen-O) n - Al Kiren-J-(Alkilen-O) n'-Alkilen-J'-, -W-, -Alkilen-W-, Al KIRENE-J-(alkylene-NMe) n -Alkilen-W-, -J-(Alkilen-NMe) n -Aruki Ren-W-, -(Alkiren-O) n -Alkylene-UC(O)-, -(Alkylene- O) n -Alkilen-U-Alkilen;-J-Alkilen-NMe-Alkilen'-NMe-Al Chilen''-W-, and -alkylene-J-alkylene'-NMe-alkylene''-NMe-alkylene A linker selected from the group consisting of kylene'''-W-, W has the following structure:

[0090] [ka]

[0091] U has the following structure:

[0092] [ka]

[0093] Each J and J' independently has the following structure:

[0094] [ka]

[0095] n and n' are independent integers greater than or equal to 1. D has the following structure:

[0096] [ka]

[0097] Each R17 These are independently H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, Alkynyl, Substituted Alkynyl, Alkoxy, Substituted Alkoxy, Alkylalkoxy, Substituted Alkylalkoxy, polyalkylene oxide, substituted polyalkylene oxide, ali aryl, substituted aryl, heteroaryl, substituted heteroaryl, alkali, substituted alkali Lu, aralkyl, substituted aralkyl, -(alkylene, or substituted alkylene)-ON (R")2, -(alkylene or substituted alkylene)-C(O)SR'', -(al (Chilene, or substituted alkylene)-SS-(aryl, or substituted aryl) From the group consisting of -C(O)R'', -C(O)2R'', or -C(O)N(R''2 Selected (Note that each R'' can independently be hydrogen, alkyl, substituted alkyl, alkenyl, or substituted alkyl.) Substituting alkenyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl (alkalinus, substituted alkalil, aralkyl, or substituted aralkyl), Each Z1 is a bond, CR 17 R 17 , O, S, NR', CR 17 R 17 -CR 17 R 17 CR 17 R 17 -O, O-CR 17 R 17 CR 17 R 17 -S, S-CR 17 R 17 CR 17 R 17 -NR', or NR'-CR 17 R 17 And, Each R' is H, alkyl, or substituted alkyl. Each Z2 is a bond, -C(O)-, -C(S)-, or an optionally substituted atom with 1 to 3 carbon atoms. Chelen, optionally substituted C1-C3 alkenylenes, and optionally substituted hetene Selected from the group consisting of rhalkyl groups, Each Z3 consists of a bond, an optionally substituted alkylene with 1 to 4 carbon atoms, and an optionally substituted carbon atom. Alkenylenes with 1 to 4 terms, arbitrarily substituted heteroalkyls, -O-, -S-, -C(O Selected from the group consisting of -, -C(S)-, and -N(R')-, Each T3 is a bond, C(R")(R"), O, or S (wherein T3 is O, Alternatively, when it is S, R'' is not a halogen. Each R is H, halogen, alkyl, substituted alkyl, cycloalkyl, or substituted alkyl It is a chloroalkyl, m and p are 0, 1, 2, or 3 (where at least one of m or p is 3). (One is not 0), M2 is

[0098] [ka]

[0099] (Note that (a) indicates bonding to the B group, and (b) indicates bonding in the heterocyclic group, respectively) (Indicates connection to the position), The M3 is

[0100] [ka]

[0101] (Note that (a) indicates bonding to the B group, and (b) indicates bonding in the heterocyclic group, respectively) (Indicates connection to the position), The M4 is

[0102] [ka]

[0103] (where (a) indicates the bond to the B group, and (b) indicates the bond to the heterocyclic group, respectively) (This indicates a connection to this position), Each R 19 These are, independently, alkyl groups with 1 to 6 carbon atoms, alkoxy groups with 1 to 6 carbon atoms, and ester groups. ethers, thioethers, aminoalkyls, halogens, alkyl esters, aryls Esters, amides, arylamides, alkyl halides, alkylamines, alkyls Sulfonic acid, alkyl nitro, thioester, sulfonyl ester, halosulfonyl, nitrate Selected from the group consisting of lyl, alkylnitrile, and nitro, q is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11. Each R 16 These are independently hydrogen, halogen, alkyl, NO2, CN, and substituted alkyl (Selected from a group consisting of kills.) In some embodiments, R1 is a polypeptide. In certain embodiments, the p The lipeptide is an antibody. In certain embodiments, the antibody is Herceptin. In other embodiments, R2 is a polypeptide. In specific embodiments, the polypeptide Tide is an antibody. In certain embodiments, the antibody is Herceptin.

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

[0105] [ka]

[0106] In one embodiment, any of the above compounds are used, along with a pharmaceutically acceptable carrier, excipient, and The following describes a pharmaceutical composition comprising a binder and [another substance].

[0107] In further or alternative embodiments, the presence of polypeptides in the patient is detected. A method for doing so, comprising a polypeptide containing at least one heterocyclic nonnatural amino acid The administration of a heterocyclic nonnatural amino acid polypeptide is said to be the same as the polypeptide This method alters the immunogenicity of tide compared to similar naturally occurring amino acids.

[0108] The methods and compositions described herein, the methods, protocols, and cell systems described herein. It is understood that the composition, composition, and reagents are not limited to, but may be changed. The terms used herein are for the sole purpose of describing specific embodiments and are not included in the attached claims. The scope of the methods and compositions described herein is limited only by the foregoing, and is not limited to the methods and compositions described herein. It is understood that it does not exist.

[0109] Terms written in the singular form, as used in this specification and the attached claims, are This includes cases where the plural form is used, regardless of whether it is the first or previous appearance, but the singular form is used depending on the context. This does not apply if it is clear that it refers only to the scope described in the document.

[0110] Unless otherwise specified, the technical and scientific terms used herein refer to the terminology to which the present invention belongs. This term shall have the same meaning as that commonly understood by those skilled in the art. Regarding the methods, apparatus, and materials used when carrying out or testing the inventions described in the details: Therefore, any method, apparatus, and materials similar to or equivalent to the methods, apparatus, and materials described herein, While various methods, apparatuses, and materials can be used, preferred methods, apparatuses, and materials are described below. .

[0111] The publications and patent documents referenced herein may, for example, be used in connection with the present invention. This document describes the constituent concepts and methodologies. The aim is to explain these constituent concepts and methodologies. Specifically, by referring to the above publications and patent documents, their entire contents are incorporated into this application. The above publications discussed in this specification only refer to disclosures made before the filing date of this application. This specification does not imply that any description herein is within the scope of the inventors described herein. If the person who made the invention prior to the present invention is not recognized by prior art or for other reasons This does not endorse such a thing.

[0112] The term "aldol-based bonding" or "mixed aldol-based bonding" is a term used in some cultures. Bonyl compounds as enolates of other carbonyl compounds, which may be identical or different thereto. / Enols are subjected to acid-catalyzed or base-catalyzed condensation to form β-hydroxycarbonyl compounds ( This refers to the process of creating Aldor.

[0113] As used herein, the term “affinity label” means a label that is reversible to other molecules. Alternatively, a label that irreversibly binds to modify or destroy it, or forms a compound with it. It refers to identification. For example, affinity labels are used for enzymes and their substrates, or antibodies and It contains the antigen of biology.

[0114] The terms "alkoxy," "alkylamino," and "alkylthio" (or thioal Coxy is used in its traditional sense, and is used in the sense of a molecule via an oxygen atom, amino group, or sulfur atom. These refer to each of the linked alkyl groups.

[0115] Unless otherwise specified, the term "alkyl" refers to fully saturated molecules, either alone or as part of another molecule. It may be in a monounsaturated or polyunsaturated state, and may have a specified number of carbon atoms (i.e., C1 ~C 10 It can contain divalent and polyvalent groups having 1 to 10 carbon atoms. This refers to linear, segmented, or cyclic hydrocarbon groups, or combinations thereof. Examples of hydrogen radicals include methyl, ethyl, n-propyl, isopropyl, n-butyl, and t -Butyl, isobutyl, sec-butyl, cyclohexyl, (cyclohexyl)methyl, and cyclopropylmethyl, as well as, for example, n-pentyl, n-hexyl, n-heptyl This includes, but is not limited to, groups such as, and congeners and isomers of 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, clotyl, 2-isopentenyl, 2-( Butadienyl, 2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl Includes 1-propynyl, 3-propynyl, 3-butynyl, higher congeners, and higher isomers. However, it is not limited to these. The term "alkyl" also applies here unless otherwise specified. Alkyl derivatives ("heteroalkyl", "haloalkyl", etc.) will be explained in more detail below. It is intended to include (and "homoalkyl," etc.)

[0116] The term "alkylene" refers to (-CH2-) when used alone or as part of another molecule. n (n is 1~ Divalent radicans derived from alkanes, such as those exemplified by (which may be numbers up to approximately 24) It means -CH2CH2- and -CH2CH2C. This includes, but is not limited to, groups with 10 or fewer carbon atoms, such as H2CH2-. No. "Lower alkyl" or "lower alkylene" usually has 8 or fewer carbon atoms. It is a short-chain alkyl or alkylene group. The term "alkylene" is also used in specific cases. Unless otherwise specified, the group is intended to contain the group described as “heteroalkylene”. ru.

[0117] The term "amino acid" refers to natural and non-natural amino acids, as well as those that function similarly to natural amino acids. This refers to amino acid analogs and amino acid mimetics. Naturally encoded amino acids No acids are the 20 common amino acids (alanine, arginine, asparagine, asparagus). Ginic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine leucine, lysine, methionine, phenylalanine, proline, serine, threonine, (Tryptophan, tyrosine, and valine), pyrrolidine, and selenocysteine Amino acid analogs refer to compounds whose basic chemical structure is identical to that of natural amino acids. (Example) Examples include hydrogen, carboxyl groups, amino groups, and α-carbons bonded to R groups. These amino acid analogs have the same basic chemical structure as natural amino acids. Therefore, those with modified R groups (norleucine as an example) or those with modified peptide skeletons It may also be an amino acid analog. Non-limiting examples of amino acid analogs include homoserine and norleucine. It contains methionine sulfoxide and methionine methylsulfonium.

[0118] In this specification, amino acids may be indicated by their names, or by IUPAC classifications. -IUB Biochemical Nomenclature C It may also be indicated by a three-letter or one-letter abbreviation, as recommended by the commission. Yes. Also, nucleotides are generally represented by a single-letter code. There is.

[0119] An "amino-terminal modification group" refers to any molecule that can be bonded to a terminal amine group. As such, these terminal amine groups are found in polymers (polypeptides, polynucleotides, and Terminal modification group (including, but not limited to, polysaccharides) This includes, but is not limited to, a variety of water-soluble polymers, peptides, or proteins. For example, terminal modifying groups include polyethylene glycol or serum albumin. End-modifying groups may be used to modify the therapeutic properties of polymers. Such modifications can be performed on peptides. This includes, but is not limited to, increasing the blood half-life of [the substance].

[0120] "Antibody fragment" means any form of antibody other than the full-length form. The antibody fragments used herein are This includes the antibody, which is a smaller element present within the full-length antibody, and the modified antibody. The somatic fragments are Fv, Fc, Fab, (Fab')2, single-chain Fv(scFv), diabody, Triabody, tetrabody, bifunctional hybrid antibody, CDR1, CDR2, CDR 3. CDR combination, variable region, framework region, steady region, heavy chain, light chain, variable This includes, but is not limited to, regions, selective skeletal 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 basic structures are linked by peptide linkers. The single chain Fv( is composed of variable regions of covalently bound immunoglobulin heavy and light chains.) (scFv) (Sz Hu et al., 1996, Cancer Research, 56, 3055-3061). Usually, These small molecules (Mr 25,000) proteins are antigens in a single polypeptide. Because it maintains specificity and affinity to the antigen, it is possible to construct larger antigen-specific molecules. It can provide useful building blocks. Unless otherwise specified, A description or claim using the term "antigen" (singular or plural) should not be referred to as "antigen fragment" (singular or plural). This includes, in particular, the number and plural forms.

[0121] As used herein, “antibody and drug conjugate” or “ADC (anti A "body-drug conjugate" is a molecule covalently bonded to one or more biologically active molecules. This refers to antibody molecules or fragments thereof. Bioactive molecules include linkers, polymers, or The antibody may also be bound via other covalent bonds.

[0122] As used herein, the terms “aromatic” or “aryl” refer to a bonded π-electron system. This refers to a ring-closed structure having at least one ring, comprising a carbocyclic aryl group and a heterocyclic aryl group. It contains both a reel group (or a "heteroaryl group" or a "heteroaromatic group"). Aromatic groups of carbocyclic or heterocyclic rings contain approximately 5 to 20 ring atoms. The above terms refer to covalently bonded monocyclic or polycyclic fused rings (i.e., adjacent rings). It includes a ring group that shares a pair of carbon atoms. The aromatic group can be unsubstituted. Aromatic groups can also be substituted. Non-limiting examples of "aromatic" or "aryl" groups include phenyl, 1-naphthyl, 2-naphthyl, 4-biphenyl, anthracenyl, and phenyl Contains xenanthracenyl. Substituents of the aryl ring system and heteroaryl ring system described above. The substituents are selected from the group consisting of acceptable substituents as described herein.

[0123] Simply put, the term "aryl" is related to other terms (aryloxy, arylthiox). When used in combination with (including, but not limited to, shi and aralkil) It includes both aryl rings and heteroaryl rings as defined earlier. Therefore, The term "alalkyl" or "alkalil" refers to alkyl groups (benzyl, phenethyl, An aryl group is bonded to (including, but not limited to, pyridylmethyl) It is intended to contain radicals. Note that the alkyl group above is a carbon atom (me A group containing, but not limited to, a ethylene group, is formed by a heteroatom (for example, an oxygen atom). It contains substituted alkyl groups. Examples of such aryl groups include phenoxymethyl, 2-propyl aryl group. It contains lysyloxymethyl and 3-(1-naphthyloxy)propyl, but Not limited.

[0124] As used herein, the term "arylene" refers to a divalent aryl radical. Non-limiting examples of "arirene" include phenylene, pyridinylene, pyrimidinylene, and thiof. It contains ernylene. The substituents of the arylene group consist of the acceptable substituents described herein. Selected from the group.

[0125] A "bifunctional polymer," also called a "bifunctional linker," reacts specifically with other parts. This refers to a polymer containing two functional groups capable of forming covalent or non-covalent bonds. The eel portion contains side chain groups of natural or unnatural amino acids, or natural or unnatural amino acids. Includes, but is not limited to, the side chain groups of the peptide. Difunctional linker or difunctional The other parts that can bond with the polymer may be the same or different parts. For example, The bifunctional linker has a functional group that is reactive to the base of the first peptide, and the second peptide The base of the first peptide has other functional groups that are reactive, and thereby the two functional groups A complex is formed containing a potential linker and the second peptide. Various compounds are peptide Numerous methods and binding molecules for binding to tides are known. For example, European patent applications. U.S. Patent No. 188,256, U.S. Patent No. 4,671,958, U.S. Patent No. 4,659,839 U.S. Patent No. 4,414,148, U.S. Patent No. 4,699,784, U.S. Patent No. 4 See U.S. Patent No. 680,338 and U.S. Patent No. 4,569,789. The above patent applications and By referring to the patent, the entire content thereof is incorporated into this application. "Multifunctional polymer" is Also known as a "polyfunctional linker," it contains two or more functional groups that can react with other parts. This refers to polymers that form covalent or non-covalent bonds. It contains side chain groups of unnatural amino acids or side chain groups of peptides containing natural or unnatural amino acids. However, it is not limited to this. (It includes, but is not limited to, side chain groups of amino acids.) Difunctional The functional polymer or polyfunctional polymer may have a desired length or molecular weight. A particular relationship exists between one or more molecules that bind to a compound and the molecule to which this molecule binds or the compound itself. It may be selected to provide a specific desired gap or three-dimensional structure.

[0126] As used herein, the term "bioavailability" refers to the activity of a substance or its active ingredients. The rate at which the active portion of the drug is delivered from the dosage form and becomes available at the site of action or in systemic circulation. It refers to the degree of bioavailability. Increased bioavailability means that the active portion of a substance or drug is Increased rate and extent of delivery from the dosage form to the site of action or systemic circulation This demonstrates that, for example, increased bioavailability is due to other substances or other active parts. This can be indicated by an increased blood concentration of the substance or its active portion compared to the previous state. For a method to evaluate the increase in bioavailability, see Example 2 for a non-limiting example. The bioavailability of any polypeptide can be determined using this method. You may perform an evaluation.

[0127] As used herein, the terms “bioactive molecule,” “bioactive moiety,” or “biological” may be used interchangeably. "Activators" are organisms (viruses, bacteria, bacteriophages, transposons, prions). Biology of insects, fungi, plants, animals, and humans (including, but not limited to, insects, fungi, plants, animals, and humans) It is possible to influence the physical or biochemical properties of a particular system, pathway, molecule, or interaction. This means any substance that is capable of doing so. In particular, as used herein, a bioactive molecule is a human or for the purpose of diagnosing, curing, alleviating, treating, or preventing diseases of other animals, or for the purpose of treating, It contains any substance that improves the physical or mental health of a person or animal, but this It is not limited to these. Examples of bioactive molecules include peptides, proteins, enzymes, small molecule drugs, and herbs. Drugs, soft drugs, prodrugs, carbohydrates, inorganic atoms or molecules, dyes, fats Substance, nucleoside, radionuclide, oligonucleotide, toxin, cell, virus, liposome This includes, but is not limited to, particles, fine particles, and micelles. Suitable types of bioactive molecules for use in combination with the composition include drugs, prodrugs, and radioactive molecules. radionuclides, contrast agents, polymers, antibiotics, disinfectants, antivirals, anti-inflammatory agents, antitumor agents, cardiovascular blood Tuberculosis agents, anxiolytics, hormones, growth factors, steroids, and bacterial toxins, etc. This includes, but is not limited to, these items.

[0128] "Modifying biological activity" means increasing or decreasing the reactivity of polypeptides. , modifying the selectivity of polypeptides, or improving the substrate selectivity of polypeptides This means reducing the bioactivity. Analysis of modified bioactivity of non-natural polypeptides This can be done by comparing the sex with the biological activity of natural polypeptides.

[0129] As used herein, the term “biomaterial” refers to a material that is biologically derived. The above-mentioned biologically derived materials are used in bioreactors and / or recombinant methods and technologies. This includes, but is not limited to, materials obtained from.

[0130] As used herein, the term “biophysical probe” refers to a change in the structure of a molecule. It includes a probe capable of detecting or observing such molecules, including proteins. However, it is not limited to this. Using "biophysical probes," proteins and other macros... Interactions with molecules may be detected or observed. An example of a biophysical probe is a spin marker. This includes, but is 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). Any method utilizing the system, which involves polynucleotides, codons, tRNA, and ribonucleotides. This refers to a method that involves using at least one element such as a some. The method and technique described in the specification and non-limiting example 20 ("Polypropylene containing non-natural amino acids") Using a method for in vivo production of lipeptides, non-natural amino acid polypeptides are produced. The "biosynthetic incorporation" of amino acids may also be performed. Furthermore, non-natural amino acid polypeptides may be used. A method for selecting useful non-natural amino acids that can be "biosynthetically incorporated" into is non-limiting. This is described in Example 20.

[0132] As used herein, the term “biotin analog” (or “biotin mimetics”) (also known as "crack") binds with high affinity to avidin and / or streptavidin. It is any molecule other than biotin.

[0133] As used herein, the term "carbonyl" means -C(O)-, -S(O)-, This refers to a group containing a moiety selected from the group consisting of -S(O)2- and -C(S)-. And, although not limited to these, one ketone group and at least one aldehyde group , at least one ester group, at least one carboxylic acid group, and / or less Both groups encapsulate a group containing one thioester group. Such carbonyl groups are ketones. It encapsulates aldehydes, carboxylic acids, esters, and thioesters. Furthermore, this The na group may be part of a linear, segmented, or cyclic molecule.

[0134] The term "carboxy-terminal modification group" refers to any group that can be attached to a terminal carboxyl group. It refers to a child. For example, such terminal carboxyl groups are found in polymers (polypeptides, polynuclear polymers). Located at the ends of creotides and polysaccharides (including, but not limited to, these) It may be present. Terminal modification groups include a variety of water-soluble polymers, peptides, or proteins. However, it is not limited to these. For example, terminal modification groups include polyethylene glycol and It contains serum albumin. Terminal modification groups may be used to modify the therapeutic properties of the polymer. The above modifications include, but are not limited to, extending the half-life of the peptide in the blood.

[0135] As used herein, the term “chemically cleavable group” is used in the sense of “chemically unstable.” It is also referred to as an acid, base, oxidizing agent, reducing agent, chemical initiator, or radical initiator. This refers to a group that is destroyed or cut when exposed to it.

[0136] As used herein, the term “chemiluminescent group” refers to a group resulting from a chemical reaction that does not involve heating. This refers to a group that emits light. For example, luminol (5-amino-2,3-dihydro-1 ,4-phthalazinedione) is converted to hydrogen peroxide (H2O2) in the presence of a base and a metal catalyst. It reacts with an oxidizing agent such as (3-aminophthalate (3-APA): It produces 3-aminophthalate.

[0137] As used herein, the term “chromophore” refers to a chromophore that emits light at visible wavelengths, ultraviolet wavelengths, or infrared wavelengths. This refers to molecules that absorb light of linear wavelengths.

[0138] As used herein, the term “cofactor” means an atom or part of a macromolecule that is essential for the function of the macromolecule. It refers to a molecule. A cofactor is an inorganic ion, coenzyme, protein, or other substance necessary for enzyme activity. This includes, but is not limited to, factors such as heme and chlorophyll in hemoglobin. Examples include magnesium and metal ions contained in proteins.

[0139] As used herein, "cofolding" refers to the interaction between two components. Using at least two molecules, unfolded or improperly folded Refolding converts folded molecules back into properly folded molecules. It refers to the process, reaction, or method of folding. For example, "cofolding" refers to the process of folding each other. Using at least two polypeptides that interact with each other, unfolded, or Improperly folded polypeptides are transformed into their original, properly folded polypeptides. Convert into polypeptides. Such polypeptides are derived from natural amino acids and / or small amounts of amino acids. It may contain at least one non-natural amino acid.

[0140] As used herein, the "comparison window" is used to determine the optimal alignment of the two arrays. After being processed, the array is used to compare it with a reference array consisting of the same number of consecutive positions. This refers to a segment consisting of any consecutive positions. Such consecutive positions can range from approximately 20 to 600. A sequence of units (containing approximately 50 to 200 sequence units, and approximately 100 to 150 sequence units) This includes, but is not limited to, groups composed of (including positions). For example, such a group The column contains polypeptides and polypeptides containing non-natural amino acids, and the continuous single The amino acids include, but are not limited to, natural and non-natural amino acids. Furthermore, As an example, such a sequence is a polynucleotide consisting of nucleotides corresponding to a continuous unit. Contains cydops. Methods for aligning sequences for comparison are well known in the art. The optimal alignment of sequences for comparison is determined by the local homology algorithm (Smith and Waterman). (1970) Adv. Appl. Math. 2:482c), Homologous 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), execution of these algorithms by computer (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) This can be done by (see plement), but is not limited to these methods.

[0141] For example, it is used to determine percent sequence identity and percent sequence similarity. Possible algorithms are Altschul et al. (1997) Nuc. Acids Res. 25:3389-3402 and A The BLAST algorithms described in Itschul et al. (1990) J. Mol. Biol. 215:403-410 are respectively This is the rhythm and BLAST2.0 algorithm. Software is from the National Center for Biotechnology Information (the National Center for Biotechnology Information). Publicly available via (inter for Biotechnology Information) It is available to the public. The variables W, T, and X of the BLAST algorithm are used to align... The sensitivity and speed of the signal are determined. The BLASTN program (for nucleotide sequences) is Initial settings include: word length (W) 11, expected value (E) 10, M=5, N=-4, and We will use a comparison of both strands. Regarding amino acid sequences, the BLASTP program will The default settings are: word length (W) 3, expected value (E) 10, and BLOSUM62 score. Ring matrix (Henikoff and Henikoff (1992) Proc. Natl. Acad. Sci. USA 89:109) (See 15) Alignment (B) 50, Expected value (E) 10, M=5, N=-4, and both strikes It uses land comparison. The BLAST algorithm described above is generally for "low complexity" This will be done with the filter 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. BLA One measure of similarity provided by the ST algorithm is that of two nucleotide sequences. The minimum sum probability (P( N)) For example, the minimum sum probability when comparing the test nucleic acid with the reference nucleic acid is less than approximately 0.2. If the value is less than approximately 0.01 or less than approximately 0.001, the nucleic acid is similar to the reference sequence. It is thought that they exist.

[0143] The term "conservatively modified variant" refers to both natural and non-natural amino acids, and This applies to non-natural nucleic acid sequences and combinations thereof. For specific nucleic acid sequences, "Conservatively modified variants" are identical or essentially identical natural and non-natural amino acids. This refers to natural and non-natural nucleic acids that encode sequences. Or, natural and non-natural nucleic acids are natural And in cases where it does not encode a non-natural amino acid sequence, a "conservatively modified mutant" is essential. It refers to essentially the same sequence. For example, due to the degeneracy of the gene code, multiple sequences with the same function may be used. A specific protein can be encoded by a number of nucleic acids. For example, codons GCA and GCC. GCG and GCU all encode the amino acid alanine. Therefore, by codon This involves modifying the encoded polypeptide at all locations where alanine is identified. The aforementioned codon can be modified to any of the corresponding codons described. Such nucleic acids The mutant is a type of "silent mutant" that has been conservatively modified. For example, all natural or non-natural polypeptides described herein Natural or non-natural nucleic acid sequences are all possible silent mutations in natural or non-natural nucleic acids. The differences are also described. Those skilled in the art will know each codon (AUG) of natural or unnatural nucleic acids. And TGG is excluded, and AUG is the only codon that normally codes for methionine. And TGG is the only codon that normally codes for tryptophan) modifying They would acknowledge that it is possible to produce functionally identical molecules. Therefore, Each silent mutation in natural and non-natural nucleic acids encoding natural and non-natural polypeptides is , which are latent within each of the sequences described.

[0144] Regarding amino acid sequences, a single amino acid or a few percent of an amino acid in the encoded sequence Nucleic acids, peptides, polypeptides, or other substances that cause modification, addition, or deletion of amino acids. Individual substitutions, deletions, or additions to an amino acid sequence are modifications that result in the loss of amino acids. Loss, addition, or substitution of a natural or unnatural amino acid with a chemically similar amino acid. When this happens, it becomes a "conservatively modified mutant." It is a related species that provides functionally similar amino acids. Substitution tables are well known in the art. Such conservatively modified mutants This refers to polymorphic variants, interspecific homologs, and alleles of the methods and compositions described herein. This is an addition to the existing set, and does not exclude them.

[0145] A conserved substitution table providing functionally similar amino acids is available to those skilled in the art. This is knowledge. The following eight groups contain amino acids that are conserved substitutions with each other. い: (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) (For example, Creighton, Proteins: Structures and Molecular Properties (WH Freeman) See & Co.; 2nd edition (December 1993).

[0146] The terms "cycloalkyl" and "heterocycloalkyl" can be used alone or in combination with other terms. When used together, unless otherwise specified, "alkyl" and "heteroalkyl" are used. These refer to the cyclic forms of each. Therefore, they are cycloalkyl or heterocycloalkyl. This includes ring bonds in saturated, partially saturated, and fully saturated states. Furthermore, heterocycloalkyl Regarding heteroatoms, heterocycles can occupy positions where they bond to the remaining part of the molecule. The heteroatom may contain, but is not limited to, oxygen, nitrogen, or sulfur. Examples of cycloalkyls include cyclopentyl, cyclohexyl, 1-cyclohexenyl, and 3. -Includes, but is not limited to, cyclohexenyl and cycloheptyl. Examples of rocycloalkyl groups include 1-(1,2,5,6-tetrahydropyridyl) and 1-piperyl. Dinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl, 3-morpholinyl, Tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrotiene 2-yl, tetrahydrothien-3-yl, 1-piperazinyl, and 2-piperazinyl This includes, but is not limited to, the above terms. Furthermore, the above terms refer to polycyclic structures (bicyclic or tricyclic). This includes, but is not limited to, ring structures of rings. Similarly, the term "heterocycloal" "Chilene" is a compound derived from heterocycloalkyls, either alone or as part of another molecule. This refers to a valence radical. The term "cycloalkylene" is used both individually and as part of another molecule. This refers to a divalent radical derived from a cycloalkyl group.

[0147] As used herein, the term "cyclodextrin" means at least a ring-formed It also refers to a cyclic carbohydrate composed of 6 to 8 glucose molecules. The outer part of the ring is water-soluble. It contains a sex group. The central part of the above ring is relatively nonpolar, capable of accepting small molecules. It is a sexual cavity.

[0148] As used herein, the term “cytotoxic” means a compound that causes damage to cells. To point.

[0149] As used herein, “modifier” is responsible for causing the reversible development of the polymer. It refers to a compound or material that causes the reversible decomposition of a protein. For example, a "denaturant" causes the reversible decomposition of a protein. It may be caused. The advantages of a denaturing agent are determined by both the characteristics and concentration of the specific denaturing agent. This will be done. For example, denaturants include chaotropes, cleaning agents, organic substances, and water-miscible solutions. This includes, but is not limited to, a medium, phospholipids, or combinations thereof. Non-limiting examples of p include urea, guanidine, and sodium thiocyanate, but these include Not limited. Examples of non-limiting detergents include sodium dodecyl sulfate or polyoxyethylene Strong cleaning agents such as ether (e.g., Tween or Triton cleaning agents), Sar kosyl, weak nonionic detergent (e.g., digitonin), N-2,3-(dioleyl Weak cationic cleaning agents such as oxy)-propyl-N,N,N-trimethylammonium Agents, weak ionic cleaning agents (e.g., sodium cholate or sodium deoxycholate) (Um), or amphoteric detergents (sulfobetaine (amphoteric detergent), 3-(3-chloro Amidopropyl)dimethylammonio-1-propane sulfate (CHAPS), and 3-(3-chloroamidopropyl)dimethylammonio-2-hydroxy-1-propane This includes, but is not limited to, insulfate (CHAPSO), and also includes, these. Not limited. Examples of non-limited organic water miscible solvents include acetonitrile, lower alkanols (specifically... In addition, ethanol or isopropanol (C2-C4 alkanols), or lower ammonium compounds. It contains alkanediols (C2-C4 alkanediols such as ethylene glycol), The above are not limited to these. Furthermore, even if the above-mentioned non-limiting examples of organic water miscible solvents are used as denaturants, Good. 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 synthetic phospholipid variant.

[0150] For use in this specification, the term “desired functional group” is selected from the following: Refers to any group: label; dye; polymer; water-soluble polymer; polyethylene glycol inducer Conductors; photocrosslinking agents; cytotoxic compounds; drugs; affinity labels; photoaffinity labels Labels; photoaffinity labels; reactive compounds; resins; second protein analogs, polyp Butyroid analogs or polypeptide analogs; antibodies or antibody fragments; metal chelating agents Cofactor; fatty acid; carbohydrate; polynucleotide; DNA; RNA; antisense poly Nucleotides; Polysaccharides; Water-soluble dendrimers; Cyclodextrins; Biocompatible substances; Nano Particles; spin-labeled; fluorescent probes; metal-containing parts; radioactive parts; novel functional groups; other molecules A group that interacts with another group, either co- or non-co-co-interacting; a photocaged moiety; a chemically excited moiety; a ligan D; Photoisomerizable moiety; Biotin; Biotin analogue; Biotin analogue; Combining heavy elements Enclosed portion; chemically cleavable group; photocleavable group; extended side chain; carbon bond type Sugars; redox active substances; aminothio acids; toxic parts; isotope-labeled parts; biophysical products -b; phosphorescent group; chemiluminescent group; electron-dense group; magnetic group; insertion group; chromophore; energy transfer Transfer of drugs; biologically active substances (in this case, biologically active substances include agents having therapeutic activity) This can be achieved, and non-natural amino acid polypeptides or modified non-natural amino acids can be used when the therapeutic agent adheres to them. It can act as a means of delivering a co-therapeutic agent or therapeutic agent to a desired location on an organism. ); detectable labels; small molecules; inhibitory ribonucleic acids; radioactive nucleotides; neutron capture materials Biotin derivatives; quantum dots; nanotransmitter; radiotransmitter; antibody enzyme; activation Complex activators; viruses; adjuvants; aggrecans; allergans; angiostatins; Antihormones; antioxidants; aptamers; inducing RNA; saponins; reciprocal vectors; macromolecules; Mimotopes; receptors; reverse micelles; and any combination thereof.

[0151] As used herein, the term “diamine” means having at least two amine functional groups This refers to the group / molecule it contains. The above groups / molecule include hydrazine group, amidine group, imine group, and 1,1- Contains diamine groups, 1,2-diamine groups, 1,3-diamine groups, and 1,4-diamine groups. However, it is not limited to these. Furthermore, such groups may be linear, segmented, or cyclic. It may be just a part of a molecule.

[0152] As used herein, the term "detectable label" refers to analytical techniques (fluorescence methods, chemiluminescence). methods, electron spin resonance methods, ultraviolet and visible absorbance spectroscopic methods, mass spectrometry methods, nuclear magnetic resonance Using methods including, but not limited to, magnetic resonance methods and electrochemical methods It refers to a marker that can be observed.

[0153] The term "dicarbonyl" refers to -C(O)-, -S(O)-, -S(O)2-, and -C This refers to a group comprising at least two parts selected from the group consisting of (S)-. ,2-dicarbonyl group, 1,3-dicarbonyl group, 1,4-dicarbonyl group, and a small At least one ketone group, at least one aldehyde group, and at least one ester group , comprising at least one carboxylic acid group and / or at least one thioester group It includes, but is not limited to, dicarbonyl groups such as diketones and ketones. It encapsulates toaldehyde, keto acid, keto ester, and ketothioester groups. Furthermore, Such groups may be part of linear, segmented, or cyclic molecules. The two parts of the bonyl group may be identical or different, and one of them may be, for example... It contains substituents that produce esters, ketones, aldehydes, thioesters, or amides. That's fine.

[0154] As used herein, the term “drug” means the suppression, diagnosis, relief, or treatment of a disease or condition. This refers to any substance used for treatment or healing.

[0155] As used herein, the term “dye” refers to a soluble colorant containing a chromophore.

[0156] As used herein, the term “effective dose” means one or more of the diseases or conditions being treated. This refers to the amount of administered drug or compound sufficient to alleviate the symptoms to some extent. to reduce the signs, symptoms, or causes of disease, or other desired changes in the biological system and / or Or it can be mitigated. For example, the administered drug or compound is a natural amino acid. Polypeptides, non-natural amino acid polypeptides, modified natural amino acid polypeptides, This includes, but is not limited to, modified non-amino acid polypeptides. Natural amino acid polypeptides, unnatural amino acid polypeptides, modified natural amino acid polypeptides , or compositions containing modified non-natural amino acid polypeptides for prophylactic treatment, augmentative treatment, and It may be administered for / or therapeutic purposes. The appropriate “effective” dose for each individual case. This may be determined using techniques such as dose-increase studies.

[0157] As used herein, the term “high electron density group” means when irradiated with a group electron beam. This refers to a group that causes electron scattering. Examples of such groups include ammonium molybdate and binitrate. Cadmium iodide, 99%, carbohydrazide, ferric chloride hexahydrate, hexamethyl Lentetramine, 98.5%, indium trichloride anhydrous, lanthanum nitrate, lead acetate trihydrate, Lead citrate trihydrate, lead nitrate, periodic acid, phosphomolybdic acid, phosphotungstic acid, phosphate Potassium lysyanide, potassium ferrocyanide, ruthenium red, silver nitrate, silver protein (Ag assay: 8.0~8.5%) "Strong", tetraphenyl porcine Silver tetraphenylprophin (S-TPPS), sodium chloride aurate, tan Sodium gustate, thallium nitrate, thiosemicarbazide (TSC), uranyl acetate, It may encapsulate, but is not limited to, uranyl nitrate and vanadyl sulfate.

[0158] As used herein, the term “energy transfer agent” means a molecule that provides energy to other molecules. This refers to molecules that either contribute energy or receive energy from other molecules. Fluorescence resonance is one example. Energy transfer (FRET) is when the energy of the excited state of a fluorescence donor molecule changes when the energy of the non-excitation acceptor molecule changes. The energy is transferred non-radiatively, and then this non-excitation-receiving molecule donates energy at longer wavelengths. This is a dipole coupling process that emits fluorescence.

[0159] The term "to improve" or "to enhance" means to increase the effectiveness or duration of a desired effect. This means to prolong. For example, "improving" the effect of a drug means that the disease, To increase the effectiveness or duration of the effect of a therapeutic agent acting during the treatment of a disease or condition. It refers to the ability to prolong. As used herein, “effective amount to improve” means disease, illness, and This refers to an appropriate amount to enhance the effectiveness of a drug in treating a disease. In some cases, the amount effective for the above uses depends on the severity and course of the disease, illness, or condition, and previous The treatment will be determined based on the treatment, the patient's health condition and response to the medication, and the judgment of the treating physician. It will become that.

[0160] As used herein, the term "eukaryote" refers to a phylogenetic eukaryote. This refers to organisms belonging to the Eukaryote domain. Organisms belonging to the Eukaryote domain include animals (mammals, insects, reptiles). This includes, but is not limited to, insects and birds, ciliates, and plants (monocotyledonous plants, twins). Leafy plants and algae (including, but not limited to, fungi, yeasts, flagellates, microsporids, This includes, but is not limited to, protists.

[0161] As used herein, the term “fatty acid” refers to a hydrocarbon whose side chain is C6 or more This refers to long-chain carboxylic acids.

[0162] As used herein, the term "fluorescent probe" refers to a device that emits photons when excited. This refers to fluorescent molecules.

[0163] As used herein, the terms “functional group,” “active moiety,” “activating group,” and “detachment” are used. The terms "group," "reaction site," "chemical reaction group," and "chemical reaction part" refer to the area where a chemical reaction occurs. This refers to a part or unit of a molecule. These terms have some synonymy in the field of chemistry. This is a term. In this specification, it refers to a substance that provides a certain function or activity and reacts with other molecules. It is used as a term to refer to a part of a molecule.

[0164] The term "halogen" encompasses fluorine, chlorine, iodine, and bromine.

[0165] As used herein, the term "haloacyl" refers to the halogen portion (-C(O)CH3 It includes, but is not limited to, -C(O)CF3 and -C(O)CH2OCH3. This refers to an acyl group that includes (not)

[0166] As used herein, the term "haloalkyl" refers to the halogen portion (-CF3 and This refers to alkyl groups that include, but are not limited to, -CH2CF3, etc.

[0167] As used herein, the term "heteroalkyl" refers to alkyl groups with O, N, Si It consists of a nitrogen atom and at least one heteroatom selected from the group consisting of nitrogen and S. The atoms and sulfur atoms may be oxidized as appropriate, and the nitrogen heteroatom may be quaternized as appropriate. This refers to linear, segmented, or cyclic hydrocarbon radicals, or combinations thereof. The tetraatoms O, N, S, and Si can be at any internal position of the heteroalkyl group or alkyl group. The group may be positioned to bond to the remaining part of the molecule. For example, -CH2-CH2 -O-CH3, -CH2-CH2-NH-CH3, -CH2-CH2-N(CH3)-C H3, -CH2-S-CH2-CH3, -CH2-CH2, -S(O)-CH3, -CH 2-CH2-S(O)2-CH3, -CH=CH-O-CH3, -Si(CH3)3, - It encompasses CH2-CH=N-OCH3 and -CH=CH-N(CH3)-CH3, but , but not limited to these. Furthermore, up to two heteroatoms (for example, -CH2) The groups may be consecutive (e.g., -NH-OCH3 and -CH2-O-Si(CH3)3).

[0168] The term "heterocyclic bond" or "heterocyclic bond" refers to a bond where the dicarbonyl group is connected to a diamine. This refers to the part formed by the reaction with the group. The resulting reaction product is heteroali It is a heterocycle containing a cycloalkyl group or a heterocycloalkyl group. The resulting heterocycle The group is a chemical bond between a non-natural amino acid or non-natural amino acid polypeptide and other functional groups. It functions as such. In one embodiment, the above heterocyclic bond is a nitrogen-containing heterocyclic bond (for example) , pyrazole linkage, pyrrole linkage, indole linkage, benzodiazepine linkage, and pyr It contains (including pyrazolone bonds).

[0169] Similarly, the term "heteroalkylene" is a heteroalkyl (-CH2-CH2-S-CH Examples include 2-CH2- and -CH2-S-CH2-CH2-NH-CH2-, but This refers to divalent radicals derived from (not limited to) heteroalkylene groups. These are identical or different heteroatoms (alkylene oxy, alkylenedioxy, alkylene This includes, but is not limited to, amino acids, alkylenediamino acids, and aminooxyalkylenes. (Not specified) may occupy one or both ends of the chain. Furthermore, alkylene bonding groups and Regarding heteroalkylene bonding groups, the orientation of the bonding group is determined by the direction in which the formula indicating the bonding group is written. Therefore, it is not suggested. For example, the formula -C(O)2R'- is -C(O)2R' This represents both - and -R'C(O)2-.

[0170] As used herein, the terms “heteroaryl” or “heteroaromatic” mean N This refers to an aryl group containing at least one heteroatom selected from O and S. The above aryl group may have its nitrogen and sulfur atoms oxidized as appropriate, and the nitrogen Elementary atoms may be quaternized as appropriate. Heteroaryl groups may be substituted, but may remain unsubstituted. It may be present. Also, the heteroaryl group is bonded to the rest of the molecule via the heteroatom. It may also be included. Non-limiting examples of the above heteroaryl group include 1-pyrrolyl, 2-pyrrolyl, 3 -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-Thiazoly Lu, 4-thiazolyl, 5-thiazolyl, 2-furyl, 3-furyl, 2-thienyl, 3-thi Enyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl , 5-benzothiazolyl, prinyl, 2-benzimidazolyl, 5-indolyl, 1- Soquinoryl, 5-Isoquinoryl, 2-Quinoxalinyl, 5-Quinoxalinyl, 3-Quinoli Contains 14, and 6-quinolyl.

[0171] As used herein, the term “homoalkyl” means an alkyl group which is a hydrocarbon group. To point.

[0172] As used herein, the term “identical” means two or more identical sequences or sub-arrangements. It refers to a column. Furthermore, as used herein, the term “substantially identical” is used in comparison to an alpha. The measurements were compared using a gyroscope or by manual alignment and visual inspection. When aligned to provide maximum coverage within the comparison window or specified area, This refers to two or more sequences that have a certain percentage of identical consecutive units. For example, The identity of continuous units across a specific domain is approximately 60%, 65%, 70%, and 75%. If the percentage is approximately 80%, 85%, 90%, or 95%, then two or more sequences are "real They can be "qualitatively identical". The percentage of such identity determines whether two or more sequences exist. This describes the "percent identity" of arrays. Array identity is determined by at least approximately 75-100 sequences. The entire region of continuous unit lengths, the entire region of approximately 50 continuous unit lengths, or unspecified In some cases, it can be present throughout the entire sequence. This definition also applies to the complementary strand of the test sequence. It also refers to a polypeptide sequence in which two or more polypeptide sequences have identical amino acid residues. In this case, the polypeptide sequence is identical. On the other hand, the amino acid residues throughout a specific region are identical. One gender accounts for approximately 60%, 65%, 70%, 75%, 80%, 85%, and 90%, and also In approximately 95% of cases, the above two or more polypeptide sequences are "substantially identical." Identity is determined by the entire region of at least approximately 75 to 100 amino acids, and approximately 50 amino acids. It is present throughout the entire length region of the no acid, or, if not specifically, throughout the entire polypeptide sequence. Furthermore, as an example, for two or more polynucleotide sequences, nucleic acids If the residues are the same, these polynucleotide sequences are identical. On the other hand, if the entire specific region is identical... The identity of nucleic acid residues is approximately 60%, 65%, 70%, 75%, 80%, and 85%. If the percentage is %, approximately 90%, or approximately 95%, then the two or more of the above-mentioned inucleotide sequences are "actual They are "qualitatively identical." The above identity is determined by the length of at least 75 to 100 nucleic acids. The entire region, the entire region of approximately 50 nucleic acids in length, or, if not specified, the entire region of nucleotides It can exist in the entire sequence of columns.

[0173] In sequence comparison, typically one sequence acts as a reference sequence for comparing the test sequences. When using a comparison algorithm, the test sequence and reference sequence are entered into the computer. Specify the sub-array configuration if necessary, and specify the variables for the array algorithm program. Alternatively, you can use the initialization variables of the array algorithm program. Substitute variables can also be specified. Subsequently, the above reference array is compared using the above array comparison algorithm. The percentage sequence identity of the above test sequence for each column is calculated based on the above program variable. ru.

[0174] As used herein, the term “immunogenicity” refers to an antibody response to the administration of a therapeutic agent. This refers to a quantitative analytical method for detecting anti-non-natural amino acid polypeptide antibodies in biological fluids. Qualitative analysis methods are used to determine the immunogenicity of non-natural amino acid polypeptides for therapeutic use. These analytical methods include radioimmunoassay (RIA) and enzyme immunoassay (EL). This includes ISA, luminescence immunoassay (LIA), and fluorescence immunoassay (FIA), but The detection of immunogenicity to therapeutic non-natural amino acid polypeptides is a therapeutic process. The antibody response after administration of therapeutic non-natural amino acid polypeptides is compared to therapeutic natural amino acid polypeptides. This includes comparing the antibody response after cytotoxicity administration.

[0175] As used herein, the term “insertion agent” is also referred to as “insertion group,” and refers to an intramolecular space Alternatively, it refers to a chemical substance that can be inserted into the intermolecular space. For example, an inserting agent or inserting group is It may also be a DNA molecule. It is a molecule that is inserted between the stacked bases of double-stranded DNA. That's fine.

[0176] As used herein, the term “isolation” means separating an element from other elements. This refers to separating and removing the substance. The isolated substance can be dried or semi-dried, It can also be in a solution (including, but not limited to, aqueous solutions). The isolated element is homogeneous. It can also be in this state. Or, an additional pharmaceutically acceptable carrier and / or excipient. It can also be part of a pharmaceutical composition containing the agent. Purity and homogeneity are determined by analytical chemistry techniques. This includes polyacrylamide gel electrophoresis or high-performance liquid chromatography, but It may also be determined using (but not limited to) the elements in question. Furthermore, the elements in question may be isolated and this may be determined In cases where the dominant species is present in the product, the element is substantially purified in this specification. It is stated that it is at least pure. 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 The above can refer to the target elements. For example, nucleic acids or proteins are naturally associated with each other. If a cell does not contain at least some of its constituent cellular elements, it is considered "isolated." Alternatively, it may be concentrated to a higher level than that obtained by in vitro or in vivo production. In this case, the nucleic acid or protein mentioned above is in an "isolated" state. Furthermore, as an example, The gene is a read frame located on the side of the gene that codes for proteins other than the target gene. It is isolated when separated.

[0177] As used herein, the term “labeling” means that a compound is incorporated into and readily detectable. This refers to a substance. The physical distribution of the above 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. This refers to a bond or chemical part that is formed by a chemical reaction. Such bonds include covalent bonds and Non-covalent bonds may be included, but are not limited to them. Furthermore, such chemical parts are Esters, carbonates, imine phosphate esters, hydrazones, acetals, orthoesters This includes, but is not limited to, tel, peptide bonds, and oligonucleotide bonds. The hydrolysis-stable bond exhibits substantial stability in water and has a useful pH value (under physiological conditions). It exhibits a bond that does not react with water (for long periods or indefinitely). It is unstable or degradable by hydrolysis. A bond is defined as a bond that is decomposable in water or an aqueous solution (e.g., blood). This means that an enzyme-unstable or degradable bond is a bond that can be broken down by one or more enzymes. This means that it is solvable. For example, PEG and related polymers are highly Degradability within the molecular skeleton or within the bonding groups between the polymer skeleton and one or more terminal functional groups of the polymer. It may contain a bond. Such a degradable bond may be PEG carboxylic acid or activated PEG. Ester bonds are formed when a carboxylic acid reacts with an alcohol group on a physiologically active agent. This includes, but is not limited to, such ester groups. Below, hydrolysis releases the bioactive agent. Other bonds that can be broken down by hydrolysis are carbohydrates. Imine bond; imine bond formed from the reaction of amines and aldehydes; alcohol Phosphate ester bonds formed by reaction with phosphate groups; hydrazides and aldehydes Hydrazone bonds are reaction products of aldehydes and alcohols; Cetal bond; orthoester bond, which is a reaction product of formate and alcohol; amine. Group (including, but not limited to, amine groups located at the ends of polymers such as PEG) peptide bonds formed by the carboxyl groups of peptides; and phosphoramidite Groups (including, but not limited to, phosphoramidite groups at the ends of polymers) and oligos It contains an oligonucleotide bond formed from the 5' hydroxyl group of a nucleotide, It is not limited to this.

[0179] When used herein, the terms “culture medium” or “culture solution” refer to the growth of cells and and recovery, and / or expression and / or secretion of the product by such cells. This refers to any culture medium in which a host is present. Such a "culture medium" or "culture solution" can be any host. Cells (e.g., bacterial host cells, yeast host cells, insect host cells, plant host cells, eukaryotic host cells) Chief cells, mammalian host cells, CHO cells, prokaryotic host cells, E. coli host cells, or Pseudomonas host cells), and capable of retaining or containing cellular contents. Possible solutions, solids, semi-solids, or immobilization carriers include, but are not limited to, these. A "culture medium" or "culture solution" like this is a medium in which host cells are cultured and polypeptides are secreted. Examples include culture media or culture solution (culture media either before or after the growth stage). However, it is not limited to these. Furthermore, such "culture media" or "culture solution" may include: For example, polypeptides are produced within cells, causing the host cell to lyse or collapse. A buffer or reagent containing host cell lysates that are broken down and release polypeptides. These include, but are not limited to, the following:

[0180] As used herein, the term “metabolites” means, for example, natural amino acids Lipeptides, non-natural amino acid polypeptides, modified natural amino acid polypeptides, This refers to derivatives of compounds such as modified non-natural amino acid polypeptides, for example, Natural amino acid polypeptides, non-natural amino acid polypeptides, modified natural amino acid polypeptides When compounds such as peptides or modified non-natural amino acid polypeptides are metabolized The term "pharmaceutically active metabolite" or "active metabolite" is an example. Examples include natural amino acid polypeptides, non-natural amino acid polypeptides, and modified natural amino acids. The biology of compounds such as anoacid polypeptides, or modified non-natural amino acid polypeptides. This refers to naturally active derivatives, such as natural amino acid polypeptides and unnatural amino acid polypeptides. Peptides, modified natural amino acid polypeptides, or modified unnatural amino acid polypeptides It is produced when compounds such as peptides are metabolized.

[0181] As used herein, the term “metabolized” means that a particular substance is metabolized by an organism. It refers to the entire process of change. Such processes include hydrolysis reactions and enzyme-catalyzed reactions. Examples of such reactions include, but are not limited to, those mentioned above. Further details on metabolism can be found in The Ph See Armacological Basis of Therapeutics, 9th Edition, McGraw-Hill (1996). For example... Examples include natural amino acid polypeptides, non-natural amino acid polypeptides, and modified natural amino acids. Metabolites of amino acid polypeptides, or modified non-natural amino acid polypeptides, are natural amino acid polypeptides, non-natural amino acid polypeptides, modified natural amino acid polypeptides Administration of or modified non-natural amino acid polypeptides to a host and tissue from the host By analysis of the sample, or by identifying natural amino acid polypeptides, or unnatural amino acid polypeptides... , modified natural amino acid polypeptides, or modified non-natural amino acid polypeptides In vitro incubation using hepatocytes and analysis of the resulting compounds Therefore, it can be identified.

[0182] As used herein, the term "metal chelating agent" refers to a metal chelating agent that kills metal ions. This refers to molecules that form genus complexes. For example, such molecules have two central metal ions. The above coordination bonds can be formed, and a ring structure can be created.

[0183] When used herein, the term “metal-containing portion” means metal ions, atoms It refers to a group containing particles, or a part containing particles. Such parts include cisplatin, chelates Metal ions (nickel, iron, platinum, etc.), and metal nanoparticles (nickel, iron, platinum Examples include, but are not limited to, these.

[0184] When used herein, the term “parts incorporating heavy atoms” is usually This refers to a group that incorporates ions of atoms heavier than carbon. Such ions or atoms include Examples include, but are not limited to, silicon, tungsten, gold, lead, and uranium. .

[0185] As used herein, the term “modified” refers to natural amino acids, non-natural amino acids. The presence of changes to anoacids, natural amino acid polypeptides, or non-natural amino acid polypeptides. It refers to such changes or modifications of natural amino acids, unnatural amino acids, and natural amino acid polysaccharides. By post-synthetic modification of peptides or non-natural amino acid polypeptides, or by natural amino acids Acids, unnatural amino acids, natural amino acid polypeptides, or unnatural amino acid polypeptides It is obtained through simultaneous or post-translational modification. The “modified or unmodified” forms are: The terms "natural amino acids," "non-natural amino acids," "natural amino acid polypeptides," or "non-natural" are used to describe the natural amino acids, non-natural amino acids, natural amino acid polypeptides, or non-natural amino acids. Natural amino acid polypeptides are arbitrarily modified, that is, the natural amino acids mentioned Acids, unnatural amino acids, natural amino acid polypeptides, or unnatural amino acid polypeptides It means that it can be modified or cannot be modified.

[0186] When used herein, the term “modified half-life” refers to its unmodified form. In contrast, this refers to the positive or negative change in the circulating half-life of modified biologically active molecules. For example, modified biologically active molecules include natural amino acids and unnatural amino acids. Examples include ano acids, natural amino acid polypeptides, or unnatural amino acid polypeptides, This is not limited to these. For example, the half-life in the blood is the half-life of a biologically active molecule or modified molecule. Collecting blood samples at various time points after administration of a biologically active molecule. Time is measured by determining the concentration of molecules in each of the samples. The half-life in the blood can be calculated by its correlation with serum concentration. Adjusted blood half-life For example, increasing the blood half-life may allow for improvements in the administration plan, or it may be harmful. The effect can be avoided. Such serum enhancement is at least about 2 times, at least about 3 times. It is twice, at least about 5 times, or at least about 10 times. Method for evaluating the enhancement of the blood half-life. Examples are given in Example 33, but are not limited thereto. This method can be used with any poly It can be used to evaluate the half-life of peptides in the blood.

[0187] When used herein, the term “modified therapeutic half-life” refers to its unmodified form. Compared to the positive or negative half-life of the therapeutically effective dose of a modified biologically active molecule It signifies change. For example, modified biologically active molecules include natural amino acids. Examples include unnatural amino acids, natural amino acid polypeptides, or unnatural amino acid polypeptides. These are some examples, but are not limited to these. For example, the therapeutic half-life is related to various factors after administration. This is measured by measuring the pharmacokinetic and / or pharmacological properties of the molecule at a given time. The enhanced therapeutic half-life is determined to be particularly beneficial for dosing regimens or those that offer particular benefits. To enable or avoid undesirable effects by increasing the total dose. The enhanced therapeutic half-life, the enhanced efficacy of the modified molecule against its target Alternatively, reduced binding, enhancement or weakening of other elements or mechanisms of action of the unmodified molecule. Or, for example, by an increase or decrease in molecular breakdown by enzymes such as proteases. This occurs. An example of a method for evaluating the enhancement of the therapeutic half-life is given in Example 33, but this This method is not limited to the following. This method can be used to evaluate the therapeutic half-life of any polypeptide. can.

[0188] As used herein, the term "nanoparticles" refers to particles with a size of approximately 500 nm. This refers to particles approximately 1 nm in size.

[0189] As used herein, the term “nearly stoichiometric” refers to a chemical reaction. This refers to a molar ratio of approximately 0.75 to 1.5 of the compounds present.

[0190] As used herein, the term "non-eukaryote" refers to non-eukaryotic organisms. For example, non-eukaryotic organisms are eubacteria (Escherichia coli, The rmus thermophilus, or Bacillus stearother mophilus, Pseudomonas fluorescens, Pseudom Examples include onas aeruginosa and Pseudomonas putida. (Phylogenetic domains), or archaea (Methan ococcus jannaschii, Methanobacterium ther moautotrophicum, Archaeoglobus fulgidus, P yrococcus furiosus, Pyrococcus horikoshii , Aeuropyrum pernix, or Halobacterium (for example, NRC- 1) are some examples, but are not limited to these) (or belong to a systematic area) It is possible.

[0191] "Non-natural amino acids" are not one of the usual 20 amino acids, but rather pyrrolelysine or This refers to amino acids other than selenocysteine. It is synonymous with the term "unnatural amino acids." Other possible terms include "amino acids not naturally coded" and "non-natural." "Amino acids," "amino acids that do not exist in nature," and various hyphens connecting them There are forms with and without hyphens. The term "unnatural amino acids" is , naturally coded amino acids (the usual 20 amino acids or pyrrolidine and selenoside) A natural result of modification of stains (though not limited to these), and its translation Examples of amino acids that are not incorporated into the growing polypeptide chain by the complex include this This is not limited to those mentioned above. Examples of naturally occurring amino acids that are not naturally coded are also included. For example, N-acetylglucosaminyl-L-serine, N-acetylglucosaminyl-L- Examples include, but are not limited to, threonine and O-phosphotyrosine. The term "non-natural amino acid" refers to an amino acid that does not occur naturally and is obtained synthetically. Examples include amino acids obtained by modifying natural amino acids or non-natural amino acids, but It is not limited to them.

[0192] As used herein, the term "nucleic acid" refers to deoxyribonucleotides, deoxyribonucleotides. Xyribonucleosides, ribonucleosides, or ribonucleotides, and one of them This refers to polymers in the form of single-stranded or double-stranded polymers. Examples include nucleic acids and nucleic acid polymers. (i) It has binding properties similar to those of standard nucleic acids, and is a naturally occurring nucleotide. (ii) an analog of a natural nucleotide metabolized in a similar manner to (ii) oligonucleotides Otide analogs (PNA (peptide nucleic acid)) are used in DNA antisense technology. Analogues (such as phosphorothioates and phosphoramidates) are examples, but these (iii) These conservatively modified variants (including those with altered codon substitutions) (However, this is not limited to) and complementary sequences and clearly indicated sequences, These are some examples, but are not limited to them. For example, denaturing codon substitution is one or more choices. The third position of the (or all) codons is a mixed base and / or deoxyin This can be achieved by generating sequences in which syn residues are substituted (Batzer et al., Nucle 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] When used herein, "oxidizing agent" refers to a compound capable of removing electrons from the compound being oxidized. It refers to a compound or substance. Examples of oxidizing agents include glutathione oxide, cystine, and cysteine. Examples include stamin, dithiothreitol oxide, erythreitol oxide, and oxygen, This does not limit the use of a wide range of oxidizing agents in the methods and compositions described herein. It is suitable.

[0194] As used herein, the term "pharmaceutically acceptable" means the biological acceptability of a compound. This refers to salts, carriers, or diluents that do not inhibit activity or properties and are relatively non-toxic, but It is not limited to these; that is, without causing undesirable biological effects, or if It is administered to an individual without causing any interaction that degrades any of the components of the composition it contains. It refers to a substance.

[0195] As used herein, the term “optical affinity label” means that when light is shone upon it This refers to labeling by a group that forms a bond with a molecule that has affinity for that label. Therefore, such a bond is either a covalent bond or a non-covalent bond.

[0196] As used herein, the term “light-caged portion” refers to a portion illuminated by a specific wavelength. This refers to a group that forms covalent or noncovalent bonds with other ions or molecules.

[0197] As used herein, the term “photocleavable group” refers to a group that is cleaved when exposed to light. It refers to the base that is attached.

[0198] As used herein, the term “photocrosslinking agent” means a substance that reacts when exposed to light. Two or more functional groups that form covalent or non-covalent bonds with one or more monomer molecules or polymers. This refers to compounds that contain [the specified element].

[0199] As used herein, the term “photoisomerizable portion” means when exposed to light. This refers to the group that changes from one isomer to another.

[0200] As used herein, the term "polyalkylene glycol" means linear or This refers to branched-chain polymeric polyether polyols. The materials used include polyethylene glycol, polypropylene glycol, and polybutylene glycol. Examples include, but are not limited to, ru and their derivatives. Other exemplary implementations For example, the catalog of Shearwater Co., Ltd., "Polyethylene Glycol and De Commercial suppliers such as “rivatives for Biomedical Applications” (2001) It is listed in the catalog. For example, such high-molecular-weight polyether polyols are approximately 0 It has an average molecular weight between 0.1 kDa and approximately 100 kDa. For example, As for polymeric polyether polyols, the range is between approximately 100 Da and approximately 100,000 Da or more. Examples include, but are not limited to, those listed above. The molecular weight of the polymer ranges from approximately 100 Da to Between approximately 100,000 Da, for example, approximately 100,000 Da, approximately 95,000 Da, approximately 9,000 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, approximately 7000Da, approximately 6000Da, approximately 5000Da, approximately 4000Da, approximately 300 0Da, approximately 2000Da, approximately 1000Da, approximately 900Da, approximately 800Da, approximately 700Da Approximately 600Da, approximately 500Da, 400Da, approximately 300Da, approximately 200Da, and approximately 1 Examples include, but are not limited to, 00Da. In some embodiments, polymer The molecular weight of - is between approximately 100 Da and approximately 50,000 Da. In some embodiments, The molecular weight of the polymer is between approximately 100 Da and approximately 40,000 Da. Several implementations In terms of morphology, the molecular weight of the polymer is between approximately 1000 Da and approximately 40000 Da. In several embodiments, the molecular weight of the polymer ranges from approximately 2000 Da to approximately 50000 Da. It is in between. In some embodiments, the molecular weight of the polymer is about 5000 Da to about 40 It is between 000Da. In some embodiments, the molecular weight of the polymer is about 10000. It is between Da and approximately 40,000 Da. In some embodiments, poly(ethylene) The molecule (recall) is a branched polymer. The molecular weight of branched PEG is approximately 1000. Between Da and approximately 100,000 Da, approximately 100,000 Da, approximately 95,000 Da, approximately 9,000 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, approximately 7000Da, approximately 6000Da, approximately 5000Da, approximately 4000Da, approximately 300 Examples include, but are not limited to, 0 Da, approximately 2000 Da, and approximately 1000 Da. In some embodiments, the molecular weight of the branched PEG ranges from approximately 1000 Da to approximately 50 It is between 000Da. In some embodiments, the molecular weight of branched PEG is about 1 It is between 000Da and approximately 40000Da. In some embodiments, it is branched. The molecular weight of PEG is between approximately 5000 Da and approximately 40000 Da. Several embodiments In this context, the molecular weight of branched PEG is between approximately 5000 Da and approximately 20000 Da. In other embodiments, the molecular weight of the branched PEG ranges from approximately 2000 Da to approximately 50000. It is between Da.

[0201] As used herein, the term "polymer" consists of repeating subunits. This refers to molecules such as polypeptides, polynucleotides, or polysaccharides. Examples include, but are not limited to, polyalkylene glycols.

[0202] The terms “polypeptide,” “peptide,” and “protein” are used herein. It refers to a polymer of amino acid residues that is used interchangeably. In other words, a polypeptide The description of a molecule applies equally to the description of peptides and proteins, and vice versa. The same applies. The term refers to naturally occurring amino acid polymers and one or more amino acid residues. This applies to amino acid polymers whose base is a non-natural amino acid. Furthermore, such "polyp The terms "peptide," "peptide," and "protein" refer to any length (full-length protein). It contains amino acid chains (such as the 'k' component), where the amino acid residues are connected by covalent peptide bonds. They are joined together by [the other party].

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

[0204] When used herein, "prodrug" or "pharmaceutically acceptable prodrug" The term "g" refers to a substance that is converted to the parent drug in vivo or in vitro. This refers to a substance that does not inhibit the biological activity or properties of a drug and is relatively non-toxic; in other words, Without causing undesirable biological effects, or by rendering any component of a composition containing it inferior. This refers to a substance administered to an individual without causing any interaction that would alter it. Generally, prod A lag is a drug precursor that, following administration to the target and subsequent absorption, undergoes any process. It is converted into active or more active species through metabolic pathways (such as conversion). Some prodrugs have their activity reduced and / or their solubility in the drug or other It has a chemical group present in the prodrug that gives it the properties. When that chemical group cleaves... When modified by a prodrug, an active drug is produced. Prodrugs are, A prodrug is converted into an active drug through enzymatic or non-enzymatic reactions in the body. This involves improved solubility and targeted transport of specific cells, tissues, organs, or ligands. This leads to improvements in physiological and chemical properties, such as enhanced characteristics, and also increases the therapeutic value of the drug. The advantages of prodrugs such as (i) are easier to administer compared to the parent drug, (ii) Unlike the parent drug, the prodrug can be absorbed and utilized in the body through oral administration, and (iii) Compared to the parent drug, the prodrug also has higher solubility in the pharmaceutical composition. These are some of the points that can be mentioned, but are not limited to these. Prodrugs are pharmacologically impairing active drugs. Includes active or less active derivatives. Prodrugs have physiological and biopharmaceutical properties. or by manipulating the properties of the drug, such as its pharmacokinetic properties, to reach the site of action. The purpose is to regulate the amount of a drug or biologically active molecule. For example, as an ester ("prodrug"), water solubility negatively affects mobility. It is administered to facilitate transmission across the cell membrane, and is then metabolically hydrolyzed to produce carbohydrates. It becomes an acid (active substance) and is a non-natural amino acid polypeptide that has beneficial effects in water solubility within cells. Prodrugs are examples of drugs that target specific tissues. It is designed as a reversible drug derivative used as a modifier to improve drug transport.

[0205] As used herein, the term “preventive effective dose” refers to one or more symptoms of a disease. It is administered prophylactically to patients who are likely to experience some degree of the symptoms or ailments being treated. Each of the following is either one unnatural amino acid polypeptide or at least one modified unnatural amino acid This refers to the amount of a composition containing an acid polypeptide. In such preventative applications, the amount is the amount of the disease It depends on the individual's health condition and weight, etc. Routine experiments (e.g., gradual increase in dose) The determination of the preventive effective amount by means of floor testing (including, but not limited to, floor testing) is performed by those skilled in the art. It is considered to be well within the range.

[0206] As used herein, the term "protection" means a chemical reaction under certain reaction conditions. This refers to the presence of a "protecting group" or protective moiety that prevents the reaction of sexual functional groups. It depends on the type of chemically reactive group being used. For example, (i) chemically reactive When the group shown is an amine or hydrazide, the protecting group is tert-butyloxycal It consists of bonyl (t-Boc) and 9-fluorenyl methoxycarbonyl (Fmoc). (ii) When a group is selected from the group and the chemically reactive group is a thiol, the protecting group is O (iii) The chemically reactive group is butanoic acid or When the protecting group is a carboxylic acid such as propionic acid, or a hydroxyl group, the protecting group is It can be a methyl group or an alkyl group such as methyl, ethyl, or tert-butyl. .

[0207] For example, blocking groups / protecting groups are,

[0208] [ka]

[0209] It can be selected from the following.

[0210] Furthermore, as protecting groups, there are photosensitive groups such as Nvoc and MeVoc, and in the field of... Other known protecting groups include, but are not limited to, those mentioned by reference. The entire text of Greene and Wuts, Protective Groups in Organic Syntactic This is described in hesis, 3rd Ed., John Wiley & Sons, New York, NY, 1999.

[0211] As used herein, the term “radioactive portion” refers to alpha particles, beta particles , or a group having an atomic nucleus that spontaneously emits radiation such as gamma particles, alpha particles A helium nucleus is a helium core, a beta particle is an electron, and a gamma particle is a high-energy photon.

[0212] When used herein, the term “reactive compound” means that, under appropriate conditions, another atom This refers to a compound that reacts with a molecule or compound.

[0213] The term "recombinant host cell" is also referred to as "host cell," and refers to the method used for insertion. (For example, direct incorporation, transduction, f-mating, or recombinant host cell) Regardless of other known methods used to create cysts, exogenous polynucleotides This refers to cells containing a polynucleotide. For example, an exogenous polynucleotide can be found in the host genome. It is a vector that is not incorporated (e.g., a plasmid), or it is incorporated into the host genome. obtain.

[0214] As used herein, the term “redox-active substance” means that another molecule is acid-induced This refers to molecules that change or reduce, thereby reducing or oxidizing substances that have redox activity. Examples of substances with redox activity include ferrocene, quinone, and Ru 2+ / 3+ composite Body, Co 2+ / 3+ Complex, and Os 2+ / 3+ While complexes are a possibility, we are not limited to these. It will not be done.

[0215] As used herein, the term “reducing agent” means adding electrons to a compound in a reduced state. It refers to a compound or substance that can do so. For example, dithiothreate is a reducing agent. Dithioerythritol (DTT), 2-mercaptoethanol, dithioerythritol, cysteine, cysteine Examples include steamine (2-aminoethanethiol) and reduced glutathione, but It is not limited to these. For example, the reducing agent maintains a sulfidyl group in the reduced state. It is used to reduce intramolecular or intermolecular disulfide bonds.

[0216] When used herein, "refolding" means improperly folded From its unfolded or unfolded state, it returns to its original three-dimensional structure or is properly folded. This refers to any process, reaction, or method that deforms a structure into a three-dimensional form. For example, a refolding By improper folding, polypeptides containing disulfide bonds are subjected to From the folded or unfolded state, the original three-dimensional structure with respect to the disulfide bonds Or, to deform into a properly folded three-dimensional structure. Such disulfide bonds Polypeptides containing these include natural amino acid polypeptides or non-natural amino acid polypeptides. Lipeptides are one example.

[0217] As used herein, the term "resin" refers to high molecular weight insoluble polymer beads. This refers to the following. For example, these beads can be used as a support for solid-phase peptide synthesis, Alternatively, it is used as a site for molecules to adhere before purification.

[0218] As used herein, the term “monosaccharides” refers to a set of carbohydrates, including sugars, monosaccharides. Examples include, but are not limited to, sugars, oligosaccharides, and polysaccharides.

[0219] When used herein, the terms “safe” or “safety” refer to the administration of the drug. This refers to potential side effects related to the number of times a drug is administered. For example, the number of doses. Even if there are many side effects, drugs that cause only a few or no side effects are considered to have excellent safety. It can be said that it possesses this. An example of a safety evaluation method is given in Example 26, but this This method is not limited to any polypeptide and can be used to assess the safety of any polypeptide. .

[0220] When used herein, it means "to selectively hybridize with ~" or "to ~". In contrast, the expression "specifically hybridizes" means that the sequence is a complex mixture (of cells or This includes, but is not limited to, DNA or RNA in a library. If present, specific nucleotides in stringent hybridization conditions This refers to the bonding of molecules with a sequence, the double-chaining of molecules, or the hybridization of molecules.

[0221] As used herein, the term "spin-labeled" refers to electron spin resonance spectroscopy. Atoms or atoms that exhibit unpaired electron spins that can be detected by scoping and can attach to other molecules This refers to molecules containing a group (i.e., stable paramagnetic groups). These include nitrile radicals and nitroxides, and single-spin labeled or double-spin labeled These include, but are not limited to, the following:

[0222] As used herein, the term “stoichiometric” is used in reference to chemical reactions. This refers to a molar ratio of approximately 0.9 to 1.1 of the compounds.

[0223] As used herein, the term “stoichiometric” refers to a change in reaction conditions or additions. This refers to a chemical reaction that becomes stoichiometric or nearly stoichiometric depending on the presence or absence of a substance. Changes in pH include, but are not limited to, an increase in temperature or a change in pH. Examples of such additives include, but are not limited to, accelerators.

[0224] The expression "stringent hybridization conditions" refers to low ion intensity and DNA, RNA, PNA, or other nucleic acid mimetic under high temperature conditions, This refers to the hybridization of sequences of combinations of these. For example, Under harsh conditions, the probe is a complex mixture of nucleic acids (but not limited to, whole cells). The probe in the DNA library or RNA library targets It hybridizes with subsequences, but not with other sequences in this complex mixture. The stringent conditions depend on the array, and the stringent conditions will vary depending on the environment. The conditions also differ. For example, the longer the sequence, the more specific the requirements for hybridization. The temperature will increase. The stringent hybridization conditions are (i) normal ions The specific sequence's thermal fusion temperature (Tm) is approximately 5-10°C lower in terms of strength and pH. (ii) At approximately pH 7.0 to approximately pH 8.3, the salt concentration is approximately 0.01 M to approximately 1.0 M Therefore, temperature is important for short probes (not limited to, but approximately 10 to 50 nucleotides). For example, the temperature is at least about 30°C, and the probe is long (but not limited to) For probes longer than 50 nucleotides, at least approximately 60°C is required. (iii) The addition of an unstable agent such as formamide, (iv) Incubate in 50% formamide, 5×SSC, and 1% SDS at 42°C Incubate or incubate 5x SSC and 1% SDS at 65°C Then, in 0.2×SSC and approximately 0.1%SDS, at 65°C for approximately 5 minutes to approximately 120°C This includes, but is not limited to, washing for minutes. For example, selective and As a detection of specific hybridization, a positive signal is observed in the background. One example is that it is at least twice as many as the hybridization of Wound, but these Not limited. Extensive guidelines on nucleic acid hybridization can be found at Tijssen, Laboratory Te. 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” means the subject of a treatment, observation or experiment. It refers to animals. For example, mammals such as humans are included, but it is not limited to them. It will not be done.

[0226] When used herein, the term “substantially purified” means that the subject matter is subject to purification. The element that is associated with or interacts with another element substantially or almost entirely contains It does not refer to the element in question. For example, the preparation of the element in question is (dry weight and (and) less than approximately 30%, less than approximately 25%, less than approximately 20%, less than approximately 15%, less than approximately 10%, approximately 5 Contains impurities of less than %, approximately less than 4%, approximately less than 3%, approximately less than 2%, or approximately less than 1%. In this case, the element in question is considered to have been "substantially refined". The elements that have been "purified" are approximately 70%, 75%, 80%, 85%, and 90%. Having a purity of approximately 95%, 96%, 97%, 98%, 99%, or higher. For example, natural amino acid polypeptides or non-natural amino acid polypeptides are naturally occurring. Natural cells or natural amino acid polypeptides or non-natural amino acid polypeptides are recombinant It is purified from host cells when it is produced. For example, the preparation is (dry weight (As) less than approximately 30%, less than approximately 25%, less than approximately 20%, less than approximately 15%, less than approximately 10%, approximately Containing impurities of less than 5%, approximately less than 4%, approximately less than 3%, approximately less than 2%, or approximately less than 1% If present, preparations of natural amino acid polypeptides or non-natural amino acid polypeptides are " This means that the substance is "substantially purified." For example, natural amino acid polypeptides or non-natural ones. When amino acid polypeptides are recombinantly produced by host cells, natural amino acid polypeptides Lipeptides or non-natural amino acid polypeptides make up about 30% of the dry weight of the cells, about 25%. Approximately 20%, approximately 15%, approximately 10%, approximately 5%, approximately 4%, approximately 3%, approximately 2%, or approximately 1%, and They exist in even lower proportions. For example, natural amino acid polypeptides or non-natural amino acids. When amino acid polypeptides are recombinantly produced by host cells, natural amino acid polypeptides Butides or non-natural amino acid polypeptides are present in cells at approximately 5 g / L and approximately 4 g / L in dry weight. Approx. 3g / L, Approx. 2g / L, Approx. 1g / L, Approx. 750mg / L, Approx. 500mg / L, Approx. 250 mg / L, approximately 100 mg / L, approximately 50 mg / L, approximately 10 mg / L, or approximately 1 mg / L Or it is present in the culture medium at a lower proportion. For example, "substantially purified" natural oats. Amino acid polypeptides or non-natural amino acid polypeptides are processed by appropriate methods (e.g., SDS). PAGE analysis, RP-HPLC, SEC, and capillary electrophoresis are examples, When determined by (not limited to these), approximately 30%, approximately 35%, approximately 40%, approximately 45%, approximately 50%, approximately 55%, approximately 60%, approximately 65%, approximately 70%, approximately 75%, approximately 80%, approximately It has a purity of 85%, approximately 90%, approximately 95%, approximately 99%, or higher.

[0227] The term "substituent" is also called a "non-interfering substituent," and refers to a substituent that is placed on another base in a molecule. This refers to the group used for substitution. These groups include halos and C1-C12 10 Alkyl, C2~C 10 Alkenil, C2~ 10 Alkinyl, C1~C 10 Alkoxy, C5-C1 2 Aralkil, C3~C 12 Cycloalkyl, C4~C 12 Cycloalkenyl, Phenyl substituted phenyl, toluolyl, xylenyl, biphenyl, C2~C 12 Al Coxyalkyl, C5~C 12 Alkoxyaryl, C5~C 12 Aryloxyalkyl Ru, C7~C 12 Oxyaryl, C1-C6 alkylsulfinyl, C1-C 10 Al Cyrsulfonyl, -(CH2) m -O-(C1~C 10 Alkyl) (where m is 1 or (8) aryl, substituted aryl, substituted alkoxy, fluoroalkyl, heterocyclic Radical, substituted heterocyclic radical, nitroalkyl, -NO2, -CN, -NRC(O) -(C1~C 10Alkyl), -C(O)-(C1~C 10 Alkyl), C2~C 10 a alkthioalkyl, -C(O)O-(C1~C 10 Alkyl), -O H, -SO2, =S, -COOH, -NR2, carbonyl, -C(O)-(C1~C 10 Alkyl)-CF3, -C(O)-CF3, -C(O)NR2, -(C1~C 10 Ally Ru)-S-(C6~C 10 Aryl), -C(O)-(C6~C 10 Ariel), -(C H2) m -O-(CH2) m -O-(C1~C 10 Alkyl) (where each of m is (1 to 8), -C(O)NR2, -C(S)NR2, -SO2NR2, -NRC( Examples include, but are not limited to, O)NR2, -NRC(S)NR2, and their salts. No. Each of the R groups in the list above can be H, alkyl, or substituted alkyl. Examples include, but are not limited to, aryl or substituted aryl compounds, or alkaryl compounds. No. When substituents are identified by conventional chemical formulas written from left to right, they are By describing the structure from right to left, it equally includes chemically identical substituents. (For example, -CH2O- is equivalent to -OCH2-).

[0228] As an example, alkyl radicals and heteroalkyl radicals (alkylene, alkeni , heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl, heterocyclo Examples of these groups include alkyl, cycloalkenyl, and heterocycloalkenyl groups. The substituents for (can be) are -OR, =O, =NR, =N-OR, -NR2, -SR, -halogen, -SiR3, -OC(O)R, -C(O)R, -CO2R, -CONR2, -OC(O)NR2, -NRC(O)R, -NRC(O)NR2, -NR(O)2R, - NR-C(NR2)=NR, -S(O)R, -S(O)2R, -S(O)2NR2, -N Examples include, but are not limited to, RSO2R, -CN, and -NO2. Each of the R groups in the string can be hydrogen, substituted or unsubstituted heteroalkyl, or substituted. Alternatively, unsubstituted aryls (such as aryls substituted with halogens 1-3) are available, but this (Not limited to) substituted or unsubstituted alkyl, alkoxy, or thioalkoxy Examples include, but are not limited to, groups or aralkyl groups. Two R groups are from the same nitrogen. When bonded to an atom, they form 5-, 6-, and 7-membered rings with the nitrogen atom. They can be combined. For example, -NR2 encapsulates 1-pyrrolidinil and 4-morpholinil. It is intended to include, but is not limited to, these.

[0229] As an example, substituents for aryl and heteroaryl groups range from 0 to aromatic rings. In the range of the total number of available valences in a system, -OR, =O, =NR, =N- OR, -NR2, -SR, -halogen, -SiR3, -OC(O)R, -C(O)R, - CO2R, -CONR2, -OC(O)NR2, -NRC(O)R, -NRC(O)NR 2, -NR(O)2R, -NR-C(NR2)=NR, -S(O)R, -S(O)2R, -S(O)2NR2, -NRSO2R, -CN, -NO2, -R, -N3, -CH(Ph )2, fluoro(C1~C4)alkoxy and fluoro(C1~C4)alkyl These are some examples, but are not limited to them. Here, each of the R groups in the list above is Examples include hydrogen, alkyl, heteroalkyl, aryl, and heteroaryl, This is not limited to these.

[0230] When used herein, the term “therapeutic dose” means that a disease, illness, or For a patient who is feeling unwell, one of the symptoms of the disease, discomfort, or illness that should be treated is one of the symptoms of the disease. To treat the above, or at least partially slow its progression, or reduce it to some extent. At least one non-natural amino acid polypeptide and / or at least 1 This refers to the amount of a composition containing a modified non-natural amino acid polypeptide. The effectiveness depends on the patient's condition (disease, ailment, or severity and course of the illness, previous treatments, and the patient's health). These include the patient's health condition, response to medication, and the judgment of the treating physician, but It depends on the (not limited) effective dose. For example, the effective therapeutic dose depends on routine experiments (gradual increase in dose). This is determined by clinical trials (including, but not limited to, those listed below).

[0231] As used herein, the term "thioalkoxy" refers to a molecule via an oxygen atom. This refers to a sulfur-containing alkyl group that is bonded to it.

[0232] The term "thermal melting temperature" or Tm refers to (a given ionic strength, pH, and nucleic acid concentration) In this case, 50% of the probe complementary to the target hybridizes to the target sequence in equilibrium. It is that temperature.

[0233] When used herein, the terms “toxic part” or “toxic group” mean “damage” or “injury.” It refers to compounds that can cause death or other fatalities. The toxic components include auristatin and DNA. Small groove binding substance, DNA small groove alkylating substance, enediin, lexitropsin, zuokarma Icin, Taxane, Puromycin, Dorastatin, Mytansinoid, Vinca Alka Lloyd, AFP, MMAF, MMAE, AEB, AEVB, Auristatin E, Paclita Kiseru, docetaxel, CC-1065, SN-38, topotecan, morpholinodoxol Bicin, Rhizoxin, Cyanomorpholinodoxorubicin, Dorastatin-10, Echinoma Icin, combretatstatin, chalicheamycin cin), meitansine, DM-1, netropsin, podophyllotoxin (e.g., etopo (e.g., cytoside, teniposide), bacatin and its derivatives, antitubulin substances, cryptofants Cryptophysin, Combretastatin, Auristatin E, Vincristine, Blastine, vindesine, vinorelbine, VP-16, camptothece Epothiron A, Epothiron B, Nocodazole, Colchicine, Colcimid Estramustine, semadin, discodermolide, meitansine, eleuterobin Mechloretamine, cyclophosphamide, melphalan, calmastine, lomustine Semustine, streptozocin, chlorozotocin, uracil mustard, chlormethine Ifosfamide, chlorambucil, pipobromane, triethylenemelamine, triethylene Nthiophosphoramine, busulfan, dacarbazine, and temozolomide, itarabine (ytarabine), cytosine arabinoside, fluorouracil, floxiuridine, 6-cyan Oguanine, 6-mercaptopurine, pentostatin, 5-fluorouracil, methotrexate Xate, 10-propargyl-5,8-dideazafolate, 5,8-dideazatetrahydro Folic acid, leucovorin, fludarabine phosphate, pentostatin, gemcita Vinn, Ara-C, Paclitaxel, Docetaxel, Deoxycoformycin, Mitoma Isin-C, L-asparaginase, azathioprine, Brekinal, antibiotics (for example) Anthracyclines, gentamicin, cephalothin, vancomycin, teravancin , daptomycin, azithromycin, erythromycin, rocithromycin (rocithro mycin), furazolidone, amoxicillin, ampicillin, carbenicillin, flucloxacin Sacillin, methicillin, penicillin, ciprofloxacin, moxifloxacin, ofuro Xacin, doxycycline, minocycline, oxytetracycline, tetracycline Phosphorus, streptomycin, rifabutin, ethambutol, rifaximin, etc.) Antiviral drugs (e.g., abacavir, acyclovir, ampligen, cidofovir, delta) Virdin, didanosine, efavirenz, entecavir, phosphonet, gansic Robil, Ibasitabine, Immunovir, Idoxuridine, Inosine, Lopinavir, Methisa Zon, nexavir, nevirapine, oseltamivir, penciclovir, stabuzin, truffle Lurizine, Truvada, Valacyclovir, Zanamivir, etc.), Daunorubicin hydrochloride, Dow Nomycin, rubidomycin, seruvidine, idarubicin, doxorubicin, epirubicin and morpholino derivatives, phenoxyzomides cyclopeptides (e.g., dactinomycin) Syn), basic glycopeptides (e.g., bleomycin), anthraquinone glycosides (e.g., (e.g., Plicamycin, Mitramycin), Anthracendione (e.g., Mitoxantrone) ), azilinopyrroloindoledione (e.g., mitomycin), macrocyclic immunosuppressants (For example, cyclosporine, FK-506, tacrolimus, Prograf, rapamycin, etc.) (do), Navelben, CPT-11, Anastrazole, Letrazole, Ka Pecitabine, reloxafine, cyclophosphamide, ifosamide, Roloxafin, allocortisin, halichondrin B, colchicine, colchicine derivatives, Maytansine, lyzoxin, paclitaxel, paclitaxel derivatives, docetaxel, Okolchicine, Tritylcisterine, Vinblastine Sulfate, Vincristine Sulfate, Sysphate Latin, carboplatin, hydroxyurea, N-methylhydrazine, epidophyllotoxy Examples include procarbazine, mitoxantrone, leucovorin, and tegafur. However, it is not limited to these. "Taxane" can refer to any active taxane derivative or p In addition to rodrugs, paclitaxel is another example.

[0234] When used herein, “to treat,” “to treat,” or “treatment” refers to the same term. The term includes the act of alleviating, easing, or improving the symptoms of a disease or illness, and additional To prevent symptoms, to improve or prevent the underlying metabolic causes of symptoms, or to treat a disease or illness. To suppress energy, for example, to suppress the progression of the development of a disease or illness, disease or illness To soothe the mind, to reverse illness or disease, to treat abnormalities caused by illness or disease This includes alleviating or stopping the symptoms of a disease or illness. "To treat" The term "treatment" or "treatment" can refer to preventive and / or therapeutic treatments. Treatments are listed, but are not limited to these.

[0235] As used herein, the term "water-soluble polymer" refers to a polymer that is soluble in aqueous solvents. This refers to any polymer. Examples of such water-soluble polymers include polyethylene glycol. Polyethylene glycol propionaldehyde, mono C1-C 10 Alkoxy derivatives also Kuha Mono C1~C 10 Aryloxy derivatives (as incorporated herein by reference) Monomethoxypolyethylene glycol, (as described in License No. 5,252,714) Polyvinylpyrrolidone, polyvinyl alcohol, polyamino acids, divinyl ether maleate N-(2-hydroxypropyl)-methacrylamide, dextran, dextrin Strain derivatives (such as dextran sulfide), polypropylene glycol, polyp Polypropylene oxide / ethylene oxide copolymer, polyoxyethylated polyol, HEPA Phosphorus, heparin fragments, polysaccharides, oligosaccharides, glycans, cellulose and cellulose derivatives ( Examples include, but are not limited to, methylcellulose and carboxymethylcellulose. (None), serum albumin, starch and starch derivatives, polypeptides, polyalkylates Polyalkylene glycols and their derivatives, polyalkylene glycol copolymers and Derivatives, polyvinyl ethyl ether, and alpha-beta-poly[(2-hydroxy Examples include [(Ciethyl)-DL-aspartamide] or mixtures thereof, but These are not the only examples. Binding with natural or non-natural polypeptides brings about changes, including improved water solubility. Sex, extended or modified half-life, extended or modified compared to the unmodified form Extended therapeutic half-life, improved bioavailability, regulated biological activity, and extended Modified circulation time, regulated immunogenicity, regulated physical association characteristics (e.g., aggregation and Examples include, but are not limited to, multimerization, modified receptor binding, and one or more Altered binding with its binding partner, as well as altered receptor dimerization or polymerization. Mergerization is one example, but it is not limited to these. Furthermore, the water-soluble polymer itself It may or may not have biological activity.

[0236] Unless otherwise specified, mass spectrometry, NMR, HPLC, protein chemistry, etc., within the scope of the relevant technology. Traditional methods such as biochemistry, recombinant DNA technology, and pharmacology are used.

[0237] Compounds described herein (unnatural amino acids, unnatural amino acid polypeptides, modified Examples include unnatural amino acid polypeptides and reagents for producing the aforementioned compounds. However, these (and are not limited to these) include isotope-labeled compounds. The substance is the same compound as those shown by the various formulas and structures described herein, but one The above atoms have atomic weights or masses that differ from those found in nature. It is an atom that is replaced by a different atom. Examples of isotopes that can be incorporated into the compound of the present invention are For example, isotopes of hydrogen, carbon, nitrogen, oxygen, fluorine, and chlorine (for example, each of them) 2 H , 3 H, 13 C, 14 C, 15 N, 18 O, 17 O, 35 S, 18 F, 36 Cl) was mentioned This applies to certain isotope-labeled compounds described herein, for example, radioactive isotopes (e.g., , 3 H and 14 The isotope-labeled compound incorporating C) is a drug and / or substrate. It is useful in tissue distribution assays. Furthermore, deuterium (i.e., 2 Isotopes such as H Substitution with higher metabolic stability (e.g., extended in vivo half-life or low It may provide certain therapeutic benefits resulting from a reduced dosage requirement.

[0238] Some compounds described herein (unnatural amino acids, unnatural amino acid polypeptides, Examples include modified non-natural amino acid polypeptides and reagents for producing the aforementioned compounds. (However, these are not limited to) have an asymmetric carbon atom, and therefore enantio It can exist as a mer or diastereomer. Diastereomer Compounds are analyzed based on their physicochemical differences, for example, by chromatography and / or f The individual diastereomers can be separated by a method known as lactal crystallization. Yes, it is possible. Enantiomers can be formed by reaction with a suitable optically active compound (e.g., an alcohol). This involves separating the enantiomer mixture by converting it into a diastereomer mixture. This can be done, and in that case, the diastereomers can be divided and the individual diastereomers can be converted to the corresponding pure Convert to an enantiomer (e.g., by hydrolysis). Diastereomer, enantiomer, and all such isomers, including mixtures thereof, are one of the compositions described herein. It is considered a department.

[0239] In additional or further embodiments, the compounds described herein (non-natural) Amino acids, unnatural amino acid polypeptides, modified unnatural amino acid polypeptides, and Examples of reagents for producing the aforementioned compounds include, but are not limited to, those listed above. Used in the form of a rag. In additional or further embodiments, this specification The compounds described in the book (unnatural amino acids, unnatural amino acid polypeptides, modified unnatural amino acids) Examples include amino acid polypeptides and reagents for producing the aforementioned compounds, but these (Not limited to) When administered to an organism that needs to produce metabolites, it is metabolized, and then The metabolites produced are then utilized to achieve the desired effect (the desired therapeutic effect). This is brought about. In further or additional embodiments, non-natural amino acids and They are active metabolites of "modified or unmodified" non-natural amino acid polypeptides.

[0240] The methods and preparations described herein include non-natural amino acids, non-natural amino acid polypeptides, and N-oxides of modified non-natural amino acid polypeptides, crystalline forms (also known as polymorphs) This includes the use of (or pharmaceutically acceptable) salts. In certain embodiments, non-natural Amino acids, unnatural amino acid polypeptides, and modified unnatural amino acid polypeptides These may exist as tautomers. All tautomers are unnatural amino acids as described herein. Within the range of non-natural amino acid polypeptides and modified non-natural amino acid polypeptides. It is included in the following: Furthermore, the non-natural amino acids and non-natural amino acid polypeptides described herein. And modified non-natural amino acid polypeptides are pharmaceutically acceptable in solvents (e.g., water). (and ethanol, etc.) in both unsolvated and solvated forms It may exist. Non-natural amino acids, non-natural amino acid polypeptides, and modified amino acids as described herein. The solvated forms of decorated non-natural amino acid polypeptides are also described herein. They are considered to be present.

[0241] Some compounds described herein (unnatural amino acids, unnatural amino acid polypeptides, Examples include modified non-natural amino acid polypeptides and reagents for producing the aforementioned compounds. These may exist in several tautomer forms (but are not limited to these). All such tautomer forms are considered to be part of the compositions described herein. Furthermore, for example, any compound described herein (non-natural amino acids, non-natural amino acid poly To produce peptides, modified non-natural amino acid polypeptides, and the aforementioned compounds Examples of reagents include, but are not limited to, all forms of enol-keto compounds. It is considered to be part of the composition described in the details.

[0242] Some compounds described herein (unnatural amino acids, unnatural amino acid polypeptides, To produce modified non-natural amino acid polypeptides and any of the compounds mentioned above Examples of reagents include, but are not limited to, those that are acidic and pharmaceutically acceptable cathodic agents. Salts can be produced by ON. Some compounds described herein (non-natural amino acids, non Natural amino acid polypeptides, modified non-natural amino acid polypeptides, and the above-mentioned compounds Reagents for producing substances (though not limited to these) are basic, Therefore, salts can be produced by pharmaceutically acceptable anions. Such total salts including disalts The salts are included in the range of compositions described herein and can be prepared by conventional methods. Yes, it is possible. For example, salt can be added to aqueous, non-aqueous, or partially aqueous media. Salts can be prepared by contacting acidic and basic substances. It is recovered using at least one of the following methods: filtration, precipitation with a non-solvent, and subsequent Filtration, evaporation of the solvent, or, in the case of an aqueous solution, freeze-drying.

[0243] The pharmaceutically acceptable salts of the non-natural amino acid polypeptides described herein are related to non-natural amino acids. Acidic protons present in amino acid polypeptides are metal ions (for example, alkali metals). ON, alkaline earth ions, aluminum ions, or equivalent organic bases (coordinate It can be produced when replaced with either of the following: Furthermore, the salt form described herein The unnatural amino acid polypeptides listed can be prepared using salts of the starting material or intermediate. Yes, it is possible. The non-natural amino acid polypeptides described herein are in free base form. The non-natural amino acid polypeptides are reacted with pharmaceutically acceptable inorganic or organic acids. This process prepares a pharmaceutically acceptable acid-added salt (a type of pharmaceutically acceptable salt). It is possible. Alternatively, the non-natural amino acid polypeptides described herein may be in the free acid form. The non-natural amino acid polypeptide described herein in its state can be pharmaceutically acceptable to an inorganic base or By reacting with an organic base, a pharmaceutically acceptable base-added salt (pharmaceutically acceptable It can be prepared as a type of salt.

[0244] The types of pharmaceutically acceptable salts include (1) hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, and phosphoric acid. Acid-added salts produced by inorganic acids such as acetic acid, propionic acid, and hexanoic acid; or 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-hydrochloric acid) Cibenzoyl benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-Ethanedisulfonic acid, 2-Hydroxyethanesulfonic acid, Benzesulfonic acid, 2-Naphthalenesulfonic acid, 4-Methylbicyclo-[2.2.2]octa-2-ene-1 -carboxylic acid, glucoheptonic acid, 4,4'-methylenebis-(3-hydroxy-2 -en-1-carboxylic acid), 3-phenylpropionic acid, trimethylacetic acid, tertiary butyl acetic acid Acids, lauryl sulfate, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, (2) Acid-added salts produced by organic acids such as thearic acid and muconic acid; (3) Presence of the parent compound The acidic proton present is a metal ion (for example, alkali metal ions, alkaline earth ions, Replace with an aluminum ion or an equivalent organic base (coordinates). Examples include, but are not limited to, the salts produced when this occurs. Acceptable organic bases Examples include ethanolamine, diethanolamine, triethanolamine, and tromethamine. Examples include water and N-methylglucamine. Acceptable inorganic bases include water. Aluminum oxide, calcium hydroxide, potassium hydroxide, sodium carbonate, sodium hydroxide Examples include "um," etc.

[0245] The corresponding counterions of pharmaceutically acceptable salts of non-natural amino acid polypeptides vary. Methods (ion exchange chromatography, ion chromatography, capillary electrophoresis) Examples include inductively coupled plasma, atomic absorption spectroscopy, mass spectrometry, or any combination thereof. It can be analyzed and identified using, but is not limited to, the following: The therapeutic activity of pharmaceutically acceptable salts of such non-natural amino acid polypeptides is shown in the examples. The tests can be conducted using the techniques and methods described in 87-91.

[0246] Naturally, references to salts refer to the form of solvent addition or its crystalline form, in particular, the solvate. or includes polymorphs. Solvates contain stoichiometric or non-stoichiometric amounts of solvent. Often formed during the crystallization process using water and pharmaceutically acceptable solvents such as ethanol. When the solvent is water, a hydrate is produced, and when the solvent is alcohol, an alcohol is produced. A polymorph is produced. The polymorph contains different crystal-packed arrangements with the same component composition as the compound. Typically, Polymorphs exhibit different X-ray diffraction patterns, infrared spectra, melting points, densities, hardness, crystal shapes, and light. It possesses chemical and electrical properties, stability, and solubility. Recrystallization solvent, crystallization rate, Various factors, such as storage temperature, contribute to the formation of a single central crystal form.

[0247] Polymorphs and / or solvates of pharmaceutically acceptable salts of unnatural amino acid polypeptides This can be achieved using various techniques (such as thermal analysis, X-ray diffraction, spectroscopy, vapor sorption, and microscopy). Screening and characterization can be performed using (but not limited to) these. Analysis methods include thermochemical decomposition or thermophysical treatment (polymorphic transitions are an example, but are not limited to these). ) are dealt with, and such methods include analysis of the relationships between polymorphic phases, measurement of weight loss, and detection of glass transition temperature. It is used for excipient or excipient compatibility studies. Such methods include differential scanning thermal analysis. DSC, Modulated Differential Scanning Thermal Analysis (MDCS), Thermogravimetric Analysis (TGA), and Thermogravimetric Infrared Analysis (TG / IR) is one example, but it is not limited to these. X-ray diffraction methods include simple Examples include, but are not limited to, crystalline powder diffractometers and synchrotron radiation sources. Various spectroscopic techniques used include Raman, FTIR, UVIS, and NMR (liquid-like). Examples of microscopy techniques include, but are not limited to, solid materials. Polarized light microscopy and scanning electron microscopy (SEM) using energy-dispersive X-ray spectroscopy (EDX). ), Environmentally controlled scanning electron microscopy using EDX (in a gaseous or water vapor atmosphere), Examples include, but are not limited to, IR microscopy and Raman microscopy.

[0248] [Brief explanation of the drawing] Novel features of the present invention are described in detail in the appended claims. For further details and advantages, please refer to the following detailed description which outlines exemplary embodiments utilizing the principles of the present invention. This will lead to a better understanding. The attached diagram is as follows.

[0249] [Figure 1] This figure shows the binding of Her-tox to the Her2 receptor.

[0250] [Figure 2] This figure shows the expression of the Anti-Her2 mutant as determined by ELISA analysis. .

[0251] [Figure 3] This figure shows the expression of the Anti-Her2 mutant as determined by ELISA analysis. .

[0252] [Figure 4] Cell proliferation using the HCC1954 breast cancer cell line and drastatin linker derivatives This is a diagram of Issei.

[0253] [Figure 5] Cell proliferation using the HCC1954 breast cancer cell line and drastatin linker derivatives This is a diagram showing Issey's analysis.

[0254] [Figure 6] Cell proliferation using the SKOV-3 ovarian cancer cell line and drastatin linker derivatives This is a diagram showing Issey's analysis.

[0255] [Figure 7] Cell proliferation using the SKOV-3 ovarian cancer cell line and trastuzumab-tox complex This is a diagram showing the assay analysis.

[0256] [Figure 8] Cells using the MDA-MB-468 breast cancer cell line and drastatin linker derivatives This is a diagram showing the analysis of a proliferation assay.

[0257] [Figure 9] Microscopic examination using the MDA-MB-468 breast cancer cell line and trastuzumab-tox complex. This figure shows the analysis of the cyst proliferation assay.

[0258] [Figure 10] Single intracardiac administration of various trastuzumab-conjugated drastatin derivatives (3.3 Tumor volume measurement after doses of mg / kg, 10mg / kg, and 20mg / kg (unit: mm) 3 ) This is a diagram.

[0259] [Figure 11] Concentrations of various trastuzumab-conjugated drastatin derivatives in SD rat serum This figure shows the assay format used for measurement.

[0260] [Figure 12] Serum concentrations of various trastuzumab-conjugated drastatin derivatives after a single intravenous injection (units) This is a diagram showing the values ​​in ng / mL.

[0261] [Figure 13] Serum concentrations of various trastuzumab-conjugated drastatin derivatives after a single intravenous injection (units) This figure shows the antibody (:ng / mL). This assay involves the antibody bound to the ErbB2 receptor. Detects.

[0262] [Figure 14] Serum concentrations of various trastuzumab-conjugated drastatin derivatives after a single intravenous injection (units) This figure shows the concentration (ng / mL). In vivo stability measurement is performed on trastuzumab. The researchers detect at least two bound drastatin derivatives.

[0263] [Figure 15] Body weight and This figure shows the change in tumor volume.

[0264] [Figure 16] Definitive tumors of HCC1954 in SCID-bg mic mice Trastuzumab, Her2-HS122-NCD1, and Her2-HS122 / LK This figure shows the antitumor effect of 145-HCD1. Mice received a single intravenous injection on day 1 (indicated by the arrow). Note: The data points represent the group mean of tumor volume, and the error bars represent the standard error.

[0265] [Figure 17] Various drags in the MDA361DYT2 breast (2+) xenograft model This figure shows the antitumor effect of tatin linker derivatives.

[0266] [Figure 18] Various dras in the MDA361DYT2 breast (2+) xenograft model This figure shows the antitumor effect of tatin linker derivatives.

[0267] [Detailed description of the invention] In this specification, preferred embodiments of the present invention have been shown and described, but these embodiments It will be obvious to those skilled in the art that this is provided merely as an example. Numerous variations, alterations, and substitutions may be conceived without departing from the present invention. In carrying out the invention, various alternatives to the embodiments described herein may be adopted. It should be understood that the following claims define the scope of the present invention, and within the scope of these claims Methods and structures, as well as their equivalents, are intended to be covered herein. .

[0268] (I. Introduction) In recent years, it has become possible to overcome many limitations related to site-specific modification of proteins. A completely new technology in chemical science has been reported. Specifically, a new component is used in the original The nuclear organism Escherichia coli (E. coli) (for example, L. Wang, et al., (2001), Science 292:498-500), and eukaryotic yeast (Sacchromyces) cerevisiae)(S. cerevisiae)(e.g. J. Chin et al., Science 301:964-7 (2003) These components are added to the protein biosynthesis mechanism in vivo. This allows for the incorporation of genetically unencoded amino acids into proteins. Many novel amino acids with novel chemical, physical, or biological properties (photoaffinity Amino acids labeled by E-label, photoisomerizable amino acids, ketoamino acids, and (Examples include glycosylated amino acids), but using this methodology, in response to the amber codon TAG In E. coli and yeast, it exhibits efficient and high fidelity to proteins. It is incorporated into the journal. For example, JW Chin et al., (2002), Journal of the American Chemical 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, & PG Schultz, ( 2002), Chem. Comm., 1-11 (incorporated by reference). These studies are genetic. Chemically incompatible with all of the functional groups found in the 20 typical amino acids that are coded. It is active and can be used to react efficiently and selectively to form stable covalent bonds. This makes it possible to selectively and routinely introduce chemical functional groups not found in proteins. This proves something.

[0269] (II. Overview) At one level, at least one carbonyl, dicarbonyl, oxime, or hydroxy Silamines, aldehydes, protected aldehydes, ketones, protected ketones, thioes Tel, ester, dicarbonyl, hydrazine, azide, amidine, imine, diamine, ke Drasta containing toamines, ketoalkynes, alkynes, cycloalkynes, or enedions Tools (methods, compositions, techniques) for manufacturing and using tinlinker derivatives or analogs The technique is described herein. At another level, at least one non-natural amino acid Acids, or oximes, aromatic amines, heterocyclic compounds (e.g., indole, quinoxaline, f Drastat contains non-natural amino acids modified with enazine, pyrazole, triazole, etc. Tools (methods, compositions, technologies) for manufacturing and using linker derivatives or analogs. ) is described herein.

[0270] Such drastatin linker derivatives containing non-natural amino acids include further functionalities. It may have. Functionally, it may be a polymer, a water-soluble polymer, or polyethylene glycol. Derivatives, second proteins, polypeptides or polypeptide analogs, antibodies or antibodies Examples include, but are not limited to, fragments and any combination thereof. Furthermore, various of the above functionalities are classified as follows: one functionality is classified as a member of another functionality. This is not intended to suggest that it is impossible. In fact, depending on the circumstances, there may be overlap. Yes, they exist. As just one example, water-soluble polymers, while ranging from polyethylene glycol, are examples of induced polyethylene glycol. Although there is some overlap with the conductor function, this overlap is not complete, so both functions are listed above. .

[0271] In some embodiments, carbonyl, dicarbonyl, oxime, hydroxylamine aldehydes, protected aldehydes, ketones, protected ketones, thioesters, es Tel, dicarbonyl, hydrazine, azide, amidine, imine, diamine, ketoamine, Linker derivatives of toxic groups including ketoalkynes, alkynes, cycloalkynes, or enedions. The body is described herein. In some embodiments, the toxic group derivative is described herein. Includes any of the linkers disclosed in the book. In other embodiments, at least one non-natural Amino acids, or oximes, aromatic amines, heterocyclic compounds (e.g., indole, quinoxali) Toxicity including unnatural amino acids modified with (e.g., phenazine, pyrazole, triazole) The present invention relates to tools (methods, compositions, technologies) for manufacturing and using derivatives or analogs of a specific group. It is described in the details.

[0272] In some embodiments, such toxic group derivatives, including non-natural amino acids, further It may contain functional properties. Functional properties include polymers, water-soluble polymers, polyethylene, etc. Recall derivatives, second proteins, polypeptides or polypeptide analogs, antibodies This may include, but is not limited to, antibody fragments or any combination thereof. Not specified. In certain embodiments, the toxic group derivative is drastatin, or oli It is a statin. In a particular embodiment, the toxic group derivative is drastatin-10. Yes. Furthermore, various of the above functionalities are such that a member of one functionality is a member of another functionality. This is not intended to suggest that they cannot be classified. In fact, depending on the specific circumstances, Multiple types exist. Just as an example, water-soluble polymers include polyethylene glycol. Although it overlaps with the derivatives of the above, this overlap is not complete, so both functionalities are listed above. Yes, they are.

[0273] In one embodiment of the present invention, the preparation of a toxic portion having a linker is described, These linkers reduce the toxicity of the relevant part in the body, while the toxic part retains its pharmacological activity. In some embodiments, the toxicity of the bound toxic group, when administered to animals or humans, Compared to free toxic groups or toxic group derivatives containing unstable bonds, it retains pharmacological activity while, Reduced or removed. In some embodiments, the bound toxic group (e.g., drastach) Even if the dosage of linker derivatives (non-natural amino acid-bound drastatin derivatives) is increased, It can be administered to substances or humans with extreme safety. In one embodiment, it is bound to the toxic portion. The resulting non-natural amino acid polypeptides (e.g., drastatin derivatives) are used in vivo and in living organisms. Provides stability outdoors. In some embodiments, non-natural ami bonded to the toxic portion. No acid polypeptides (e.g., drastatin-10 derivatives) are effective and free-toxin It has lower toxicity compared to other functional groups (e.g., drastatin-10).

[0274] (III. Drastatin linker derivatives) At one level, at least one non-natural amino acid, or carbonyl, dicarbol Draco containing non-natural amino acids modified with a yl, oxime, or hydroxylamine group Tools (methods, compositions) for manufacturing and using statin linker derivatives or analogs The technology described herein includes non-natural amino acids such as drastatin The carb derivative may contain further functionalities. These functionalities may include polymers and water-soluble compounds. Polymers, polyethylene glycol derivatives, polypeptides, or polypeptide analogs Examples include the second protein, antibody or antibody fragment, and any combination thereof. These are some examples, but are not limited to these. Furthermore, various of the above functionalities are members of a particular functionality. This is not intended to suggest that it cannot be classified as a member of another functional group. In fact, there is overlap depending on the specific situation. As just one example, water-soluble polymers are, The area overlaps with the polyethylene glycol derivatives, but this overlap is not complete. Therefore, both functionalities are listed above.

[0275] In some embodiments, drastatin modified using these methods, compositions, and techniques Methods for selecting and designing novel drug derivatives are described herein. Statin linker derivatives are just one example of high-throughput screening methods. As part of this (in this case, designing, synthesizing, characterizing, and / or testing a large number of polypeptides), (They may be reviewed), or they may be newly designed based on the researchers' interests. These drastatin linker derivatives are known or partially characterized polypeptides. It may be designed based on [something]. Just as one example, drastatin is a major player in the scientific community. It is a subject of research, and novel compounds may be designed based on the structure of drastatin. The principles for selecting whether to substitute and / or modify the amino acids are described herein. The choice of which modification to adopt is also described in this specification, and the experiment It can be used according to the needs of the manufacturer or end user. Such needs include: Manipulating the therapeutic effects of lipeptides and improving the safety profile of polypeptides This includes regulating the pharmacokinetics, pharmacological effects, and / or pharmacokinetics of polypeptides. However, this is not limited to these, but includes modifying the pharmacological effects and / or pharmacokinetics, Just one example is improving water solubility and improving bioavailability. This involves increasing the blood half-life, increasing the therapeutic half-life, and regulating immunogenicity. This includes regulating biological activity or extending circulation time. Furthermore, such modifications, to name just a few, provide further functionality to polypeptides. These include incorporating antibodies and any combination of the modifications mentioned above.

[0276] Furthermore, having or containing an oxime, carbonyl, or hydroxylamine group. Drastatin linker derivatives that can be modified in this manner are described herein. This involves manufacturing, purifying, characterizing, and using such drastatin linker derivatives. This also includes methods of use.

[0277] The drastatin linker derivatives include at least one, at least two, and at least three. At least four, at least five, at least six, at least seven, at least eight, At least nine or more carbonyl or dicarbonyl groups, oxime groups, hydr It may contain a roxylamine group or a protected form thereof. Drastatin linker The derivatives may be the same or different. For example, the derivative may contain 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 There may be 13, 14, 15, 16, 17, 18, 19, or even 20 or more different parts.

[0278] (A. Structure and synthesis of drastatin linker derivatives: electrophilic and nucleophilic groups) A drastatin induction containing a hydroxylamine group (also called aminooxy) in the linker. The conductor reacts with various electrophiles (but is not limited to PEG and other water-soluble polymers) ) can form a complex. Hydrazine, hydrazide, and semicarbazide can Sea urchin contains carbonyl or dicarbonyl groups due to the enhanced nucleophilicity of the aminooxy group. This allows various molecules and aminooxy groups to react efficiently and selectively. Carbonyl or dicarbonyl groups include ketones, aldehydes, or similar chemical reactions. Other functional groups that exhibit responsiveness can be mentioned, but are not limited to these. 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). As a result of the reaction with the hydrazine group, the corresponding hydr A rhazon is obtained, but the oxime is generally the aminooxy group of a carbonyl or dicarbyl group. These are obtained by reacting with a nyl group. These carbonyl or dicarbonyl groups Examples include ketones, aldehydes, or other functional groups with similar chemical reactivity. These are not the only embodiments. In some embodiments, the linker contains azide, alkyne, or syl Drastatin derivatives containing chloralkynes undergo cycloaddition reactions (e.g., 1,3-bipolar addition). This enables the bonding of molecules via cyclization or azido-alkyne cycloaddition. U.S. Patent 7,807,619 is incorporated into this invention by reference to the scope relating to the reaction. (As stated in the document).

[0279] Therefore, in one embodiment, the linker contains hydroxylamine, aldehyde, and protective Protected aldehydes, ketones, protected ketones, thioesters, esters, dicarbonyls, Hydrazines, amidines, imines, diamines, ketoamines, ketoalkynes, and engioes (Having similar reactivity to the hydroxylamine group, hydroxyl Hydroxylamine-like groups (structurally similar to amine groups), (similar to hydroxylamine groups) (But it can be converted) Masked hydroxylamine group, or (hydroxylamine when deprotected) Drastarch containing a protected hydroxylamine group (which has similar reactivity to a hydroxylamine group) The derivatives described herein are drastatin linker derivatives of the formula ( This includes compounds having structures I), (III), (IV), (V), and (VI).

[0280] [ka] JPEG0007911040000058.jpg193169

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

[0282] [ka]

[0283] R5 is H, COR8, an alkyl group with 1 to 6 carbon atoms, or a thiazole. R8 is OH or -NH- (alkylene-O) n -NH2, R6 is OH or H. Ar is phenyl or pyridine. R7 is an alkyl group with 1 to 6 carbon atoms, or hydrogen. Y and V are hydroxylamine, methyl, aldehyde, and protected hydroxylamine, respectively. Hydro, ketone, protected ketone, thioester, ester, dicarbonyl, hydra Zin, amidine, imine, diamine, azide, ketoamine, ketoalkyne, alkyne, cy Selected from the group consisting of chloralkynes and enedions, L, L1, L2, L3, and L4 are, respectively, -alkylene-, -alkylene -C(O)-, -alkylene-J-, -(alkylene-O) n -Alkilen-, -(A Lukilen-O) n -Alkylene-C(O)-, -(Alkylene-O) n -J-,-(Alchire (n-O) n -J-alkylene, -(alkylene-O) n -(CH2) n’ -NHC(O)- (CH2) n’’ -C(Me)2-SS-(CH2) n’’’ -NHC(O)-(Al Kiren-O) n’’’’ -Alkylene, -(Alkylene-O) n -Alkilen-W-,- Alkylene-C(O)-W-,-(Alkylene-O) n -Alkilen-J-, -Alkilen' -J-(alkylene-O) n -Alkilen-, -(Alkilen-O) n -Alkilen-J-Alki Ren'-, -J-(Alkiren-O) n -Alkilen-, -(Alkilen-O) n -Alkilen- J-(alkylene-O) n '-Alkilen-J'-, -W-, -Alkilen-W-, Alkilen'- J-(alkylene-NMe) n -Alkilen-W-, -J-(Alkilen-NMe) n -Alkilen-W- ,-(alkylene-O) n -Alkylene-UC(O)-, -(Alkylene-O) n -a Lukilen-U-alkylene;-J-alkylene-NMe-alkylene'-NMe-alkylene''- W-, and -alkylene-J-alkylene'-NMe-alkylene''-NMe-alkylene'''- Selected from the group consisting of W-, W has the following structure:

[0284] [ka]

[0285] U has the following structure:

[0286] [ka]

[0287] Each J and J' independently has the following structure:

[0288] [ka]

[0289] n, n', n'', n''', and n'''' are each independently one or more integers. It is a number, Alternatively, L is absent, Y is methyl, R5 is COR8, and R8 is -NH(A Lukilen-O) n -NH2. Such drastatin linker derivatives may also take the form of salts, and may contain non-natural amino acids. It can be incorporated into polymers, polysaccharides, or polynucleotides, and optionally modified after translation. stomach.

[0290] In some embodiments of the compounds of formulas (I), (III), and (V), R5 is thiazo It is a carboxylic acid. Some compounds of formulas (I), (III), and (V) In the application form, R5 is hydrogen. Compounds of formulas (I), (III), and (V) In a modified form, R5 is methyl, ethyl, propyl, isopropyl, butyl, or isobutyl These are sec-butyl, tert-butyl, pentyl, or hexyl. Formula (I), In some embodiments of compounds (III) and (V), R5 is -NH-(alkylene -O) n -NH2, and the alkylene is -CH2-, -CH2CH2-, -CH2 CH2CH2-, -CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2- , -CH2CH2CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2CH 2CH2-, -CH2CH2CH2CH2CH2CH2CH2CH2-, -CH2CH2 CH2CH2CH2CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2C H2CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2CH2CH2CH 2CH2CH2CH2-or-CH2CH2CH2CH2CH2CH2CH2CH It is 2CH2CH2CH2CH2-. Certain embodiments of the compounds of formulas (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 The numbers are 4, 95, 96, 97, 98, 99, or 100.

[0291] In some embodiments, Y is azide. In other embodiments, Y is cycloal It is a cycloalkyne. In a particular embodiment, the cycloalkyne has the following structure.

[0292] [ka]

[0293] (In the formula, Each R 19 These are, independently, alkyl groups with 1 to 6 carbon atoms, alkoxy groups with 1 to 6 carbon atoms, and ester groups. ethers, thioethers, aminoalkyls, halogens, alkyl esters, aryls Esters, amides, arylamides, alkyl halides, alkylamines, alkyls Sulfonic acid, alkyl nitro, thioester, sulfonyl ester, halosulfonyl, nitrate Selected from the group consisting of lyl, alkylnitrile, and nitro, q is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11. In some embodiments of the compounds of formulas (I), (III), and (V), R6 is hydrogen. In some embodiments of the compounds of formulas (I), (III), and (V), R6 is It is hydroxyl.

[0294] In some embodiments of the compounds of formulas (I), (III), and (V), Ar is phenyl That is the case.

[0295] In some embodiments of the compounds of formulas (I), (III), (IV), (V), and (V), R7 is methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, isopropyl It is butyl, tert-butyl, pentyl, or hexyl. Formulas (I), (III), In some embodiments of compounds (IV), (V), and (V), R7 is hydrogen.

[0296] In some embodiments of the compounds of formulas (I), (III), and (V), Y is hydroxyl Luamine, aldehyde, protected aldehyde, ketone, protected ketone, thioeste Lu, ester, dicarbonyl, hydrazine, amidine, imine, diamine, ketoamine, These are ketoalkynes, alkynes, or engiones.

[0297] In some embodiments of the compounds of formulas (IV) and (VI), V is a hydroxylamine, Methyl aldehydes, protected aldehydes, ketones, protected ketones, thioesters Esters, dicarbonyl, hydrazine, amidine, imine, diamine, ketoamine, ke These are toalkines and engions.

[0298] In certain embodiments of the compounds of formulas (I), (III), (IV), (V), and (VI) L, L1, L2, L3, and L4 are each independently cuttable linkers or It is a non-cuttable linker. Formulas (I), (III), (IV), (V), and (VI) In some embodiments of the compound, L, L1, L2, L3, and L4 are each independently It is an oligo(ethylene glycol) derivatized linker.

[0299] In certain embodiments of the compounds of formulas (I), (III), (IV), (V), and (VI) The alkylene, alkylene', alkylene'', and alkylene''' are, Each independently, -CH2-, -CH2CH2-, -CH2CH2CH2-, -CH2CH 2CH2CH2-, -CH2CH2CH2CH2CH2-, -CH2CH2CH2CH2 CH2CH2-, -CH2CH2CH2CH2CH2CH2CH2-, -CH2CH2C H2CH2CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2CH2CH 2CH2CH2-, -CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2 - EGOCH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2GO EVA is -CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2- Therefore, equations (XIV), (XV), (XVI), (XVII), and (XVIII) In some 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, It is 96, 97, 98, 99, or 100.

[0300] (B. Structure and synthesis of drastatin linker derivatives: hydroxylamine group) Therefore, in one embodiment, the linker has a hydroxylamine group, (hydroxylamine (Having similar reactivity to the mine group and structurally similar to the hydroxylamine group) Amine-like group, masked hydroxyl (which can be converted to a hydroxylamine group at any time) Amine group, or protected (which has similar reactivity to a hydroxylamine group when deprotected) A drastatin derivative containing a hydroxylamine group is described herein. Such drastatin linker derivatives include compounds having the structure of formula (I).

[0301] [ka]

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

[0303] [ka]

[0304] R5 is H, COR8, an alkyl group with 1 to 6 carbon atoms, or a thiazole. R8 is OH, or -NH- (alkylene-O) n -NH2, R6 is OH or H. Ar is phenyl or pyridine. R7 is an alkyl group with 1 to 6 carbon atoms, or hydrogen. Y is NH2-O-, or methyl. L represents -alkylene-, -alkylene-C(O)-, and -(alkylene-O). n -Aruki Ren-, -(Alkiren-O) n-Alkylene-C(O)-, -(Alkylene-O) n - (CH2) n’ -NHC(O)-(CH2) 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-U-alkylene-C(O)-, and -(alkylene-O) n -Aruki A linker selected from the group consisting of lene-U-alkylenes, W has the following structure:

[0305] [ka]

[0306] U has the following structure:

[0307] [ka]

[0308] Alternatively, L is absent, Y is methyl, R5 is COR8, and R8 is -NH(A Lukilen-O) n -NH2, where n, n', n'', n''', and n'''' are (Each of these is an independent integer greater than or equal to 1.) Such drastatin linker derivatives may also take the form of salts, and may contain non-natural amino acids. It can be incorporated into polymers, polysaccharides, or polynucleotides, and optionally modified after translation. stomach.

[0309] In some embodiments of the compound of formula (I), R5 is thiazole. In one embodiment, R5 is hydrogen. In one embodiment of the compound of formula (I), R5 methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, It is tert-butyl, pentyl, or hexyl. A certain embodiment of the compound of formula (I). In this state, R5 is -NH-(alkylene-O) n -NH2, and the alkylene is - CH2-, -CH2CH2-, -CH2CH2CH2-, -CH2CH2CH2CH2- , -CH2CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2 CH2CH2CH2-, -CH2CH2CH2CH2CH2CH2CH2CH2CH2- , -CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2-, -CH2CH 2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2- or -CH2CH It is 2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2-. Equation (I) In some 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, The numbers are 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100.

[0310] In some embodiments of the compound of formula (I), R6 is hydrogen. In some embodiments, R6 is hydroxyl.

[0311] In some embodiments of the compound of formula (I), Ar is phenyl.

[0312] In some embodiments of the compound of formula (I), R7 is methyl, ethyl, propyl, or isopropyl. Pill, butyl, sec-butyl, isobutyl, tert-butyl, pentyl, or he It is xyl. In some embodiments of the compound of formula (I), R7 is hydrogen.

[0313] In some embodiments of the compound of formula (I), Y is a hydroxylamine, aldehyde, protective Modified aldehydes, ketones, protected ketones, thioesters, esters, dicarbonyl , hydrazine, amidine, imine, diamine, ketoamine, ketoalkyne, or ene It is a dione. In some embodiments of the compound of formula (I), V is hydroxylamine, methyl Aldehydes, protected aldehydes, ketones, protected ketones, thioesters, etc. Stell, dicarbonyl, hydrazine, amidine, imine, diamine, ketoamine, keto Lukin and Engineon.

[0314] In some embodiments of the compound of formula (I), each L is independently a cleavable linker or non It is a cleavable linker. In some embodiments of the compound of formula (I), each L is independently O It is a ligone (ethylene glycol) derivatized linker.

[0315] In some embodiments of the compound of formula (I), each alkylene is -CH2-, -CH2C H2-, -CH2CH2CH2-, -CH2CH2CH2CH2-, -CH2CH2CH 2CH2CH2-, -CH2CH2CH2CH2CH2CH2-, -CH2CH2CH2 CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2CH2CH2CH2- , -CH2CH2CH2CH2CH2CH2CH2CH2CH2-, -CH2CH2CH 2CH2CH2CH2CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2 CH2CH2CH2CH2CH2CH2----CH2CH2CH2CH2CH2 It is CH2CH2CH2CH2CH2CH2CH2-. A certain embodiment of the compound of formula (I) In the state, n, n', n'', n''', and n'''' are 0, 1, 2, and 3, 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 one embodiment, the drastatin 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 It is a kylene. In some embodiments, each alkylene is -CH2CH2- In some embodiments of the compound of formula (II), n=3 and R7 is methyl. Each alkylene is -CH2CH2-, and R7 is methyl or hydrogen. In some embodiments of the compound of formula (II), L is -(alkylene-O) n -a It is ruquilen-C(O)-. In some embodiments of the compound of formula (II), each alke n is -CH2CH2-, n=4, and R7 is methyl. The chemical formula of equation (II) In some embodiments of the compound, L is -(alkylene-O) n -(CH2) n’ -NH C(O)-(CH2) n’’ -C(Me)2-SS-(CH2) n’’’ -NHC(O )-(alkylene-O) n’’’’ -It is alkylene. (II) Some compounds In the embodiment, each alkene is -CH2CH2-, n=1, and n'=2. n''=1, n'''=2, n''''=4, and R7 is methyl. Such drastatin linker derivatives may also take the form of salts and non-natural amino acids. Incorporated into acids, polymers, polysaccharides, or polynucleotides, and optionally modified post-translation. That's good too.

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

[0320] In some embodiments of the compound of formula (II), R7 is methyl, ethyl, propyl, or isopropyl. Ropil, butyl, sec-butyl, isobutyl, tert-butyl, pentyl, or It is hexyl. In some embodiments of the compound of formula (II), R7 is hydrogen.

[0321] In some embodiments of the compound of formula (II), each alkylene is -CH2-, -CH2 CH2-, -CH2CH2CH2-, -CH2CH2CH2CH2-, -CH2CH2C H2CH2CH2-, -CH2CH2CH2CH2CH2CH2-, -CH2CH2CH 2CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2CH2CH2CH2 -, -CH2CH2CH2CH2CH2CH2CH2CH2CH2-, -CH2CH2C H2CH2CH2CH2CH2CH2CH2CH2-, -CH2CH2CH2CH2CH 2CH2CH2CH2CH2CH2CH2- or -CH2CH2CH2CH2CH It is 2CH2CH2CH2CH2CH2CH2CH2-. A real compound of formula (II) In the application form, n, n', n'', n''', and n'''' are 0, 1, and 2, respectively. , 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 It is 8, 99, or 100.

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

[0323] [ka] JPEG0007911040000070.jpg169169 JPEG0007911040000071.jpg108169

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

[0325] [ka]

[0326] R5 is H, COR8, an alkyl group with 1 to 6 carbon atoms, or a thiazole. R8 is OH, R6 is OH or H. Ar is phenyl or pyridine. R7 is an alkyl group with 1 to 6 carbon atoms, or hydrogen. Y is NH2-O-, V is O-NH2, L1, L2, L3, and L4 are each independently linked, -alkylene-, -(A Lukilen-O) n -alkylene-J-, -alkylene'-J-(alkylene-O) n -Alkilen -, -J-(alkylene-O) n -Alkylene-, -(Alkylene-O) n -Alkilen-J-( Alkylene-O) n '-Alkilen-J'-, -(Alkilen-O) n -Alkilen-J-Alki Len'-, -W-, -Alkilen-W-, Alkilen'-J-(Alkilen-NMe) n -Archile N-W-, -J-(alkylene-NMe) n -Alkilen-W-, -J-Alkilen-NMe-Alki Len'-NMe-alkylene''-W-, and -alkylene-J-alkylene'-NMe-alkylene Selected from the group consisting of n''-NMe-alkylene'''-W-, W has the following structure:

[0327] [ka]

[0328] Each J and J' independently has the following structure:

[0329] [ka]

[0330] Each n and n' is an integer greater than or equal to 1, independently. The derivatives may take the form of a salt, a non-natural amino acid, a polymer, a polysaccharide, or a poly It can be incorporated into nucleotides and optionally modified after translation.

[0331] In some embodiments of the compounds of formula (III), (IV), (V), or (VI), R5 It is a thiazole. Compounds of formula (III), (IV), (V), or (VI) In this embodiment, R5 is hydrogen. Formulas (III), (IV), (V), or (VI) In some embodiments of the compound, Ar is phenyl. Formula (III), (IV), ( In some embodiments of the compound of formula (V) or (VI), R7 is methyl. Formula (III) In some embodiments of the compounds of (IV), (V), or (VI), n and n' are, It is an integer between 0 and 20. The compound of formula (III), (IV), (V), or (VI) In this embodiment, n and n' are integers from 0 to 10. Equations (III), (IV), ( In some embodiments of compound V) or (VI), n and n' are integers from 0 to 5. ru.

[0332] In some embodiments of the compounds of formulas (III) and (V), R5 is thiazole or ka It is a rubonate. In some embodiments of the compounds of formulas (III) and (V), R5 is methyl ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert -Butyl, pentyl, or hexyl. Compounds of formulas (III) and (V) In this embodiment, R5 is -NH-(alkylene-O) n -NH2, and the aforementioned alkyl N is -CH2-, -CH2CH2-, -CH2CH2CH2-, -CH2CH2CH2 CH2-, -CH2CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2C H2-, -CH2CH2CH2CH2CH2CH2CH2-, -CH2CH2CH2CH 2CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2CH2CH2CH2 CH2-, -CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2-, -C H2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2- or -C It is H2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2. In some 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 ru.

[0333] In some embodiments of the compounds of formulas (III), (IV), (V), and (VI), R6 is hydrogen. Some of the compounds of formulas (III), (IV), (V), and (VI) In this embodiment, R6 is hydroxyl.

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

[0335] In some embodiments of the compounds of formulas (III), (IV), (V), and (VI), R7 These include methyl, ethyl, propyl, isopropyl, butyl, sec-butyl, isobutyl, It is tert-butyl, pentyl, or hexyl. Formulas (III), (IV), (V In some embodiments of the compounds of (VI), R7 is hydrogen.

[0336] In some embodiments of the compounds of formulas (III) and (V), Y is a hydroxylamine, Aldehydes, protected aldehydes, ketones, protected ketones, thioesters, esters Lu, dicarbonyl, hydrazine, amidine, imine, diamine, ketoamine, ketoalkyl It is an ion or an engion. In some embodiments of the compounds of formulas (IV) and (VI), V is hydroxylamine, methyl, aldehyde, protected aldehyde, ketone, protected Protected ketones, thioesters, esters, dicarbonyls, hydrazines, amidines, imi These include diamines, ketoamines, ketoalkynes, and enedions.

[0337] Some compounds of formulas (XIV), (XV), (XVI), (XVII), (XVII) In the configuration, L1, L2, L3, and L4 are each independently cuttable linkers. or is a non-cuttable linker. Formulas (XIV), (XV), (XVI), (XVII), And in some embodiments of compound (XVIII), L1, L2, L3, and L4 are Each is independently an oligo(ethylene glycol) derivatized linker.

[0338] In some embodiments of the compounds of formulas (III), (IV), (V), and (VI), the above Each alkylene is -CH2-, -CH2CH2-, -CH2CH2CH2-, -CH2C H2CH2CH2-, -CH2CH2CH2CH2CH2-, -CH2CH2CH2CH 2CH2CH2-, -CH2CH2CH2CH2CH2CH2CH2-, -CH2CH2 CH2CH2CH2CH2CH2CH2-, -CH2CH2CH2CH2CH2CH2C H2CH2CH2-, -CH2CH2CH2CH2CH2CH2CH2CH2CH2CH 2-, -CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2CH2-, tahashigoch2ch2ch2ch2ch2ch2ch2ch2ch2ch2ch2ch2 - is the case in certain embodiments of the compounds of formulas (III), (IV), (V), and (VI). The alkylenes mentioned above are methylene, ethylene, propylene, butylene, pentylene, hex It is either silene or heptylene.

[0339] In some embodiments of the compounds of formulas (III), (IV), (V), and (VI), n, n' and n' are 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, respectively. 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, The numbers are 92, 93, 94, 95, 96, 97, 98, 99, or 100.

[0340] In one embodiment, the drastatin linker derivative is a compound having the structure of formula (II). Includes.

[0341] [ka]

[0342] In some embodiments of the compound of formula (VII), L1 is -(alkylene-O) n -Alkilen- It is J-, and L2 is -alkylene'-J'-(alkylene-O) n '-Alkilen-, L3 is- J''-(alkylene-O) n ''-alkylene-, alkylene is -CH2CH2-, and Chilen' is -(CH2)4-, n is 1, and n' and n'' are 3. J has the following structure:

[0343] [ka]

[0344] J' and J'' have the following structure:

[0345] [ka]

[0346] R7 is methyl. In some embodiments of the compound of formula (VII), L1 is -J-(A Lukilen-O) n -Alkylene-, and L2 is -(Alkylene-O) n' -Alkilen-J'- It is alkylene'-, and L3 is -(alkylene-O). n'' -alkylene-J''-, and Alky Len is -CH2CH2-, alkylene' is -(CH2)4-, and n is 1. n' and n'' are 4, and J, J', and J'' have the following structure:

[0347] [ka]

[0348] Such drastatin linker derivatives may also take the form of salts and non-natural amino acids. They can be incorporated into polymers, polysaccharides, or polynucleotides, and optionally modified after translation. good.

[0349] In one embodiment, the compounds of formulas (I) to (VII) are expressed in aqueous solution under weakly acidic conditions. It remains stable for at least one month. In one embodiment, the compounds of formulas (I) to (VII) It is stable in aqueous solution under weakly acidic conditions for at least two weeks. In one embodiment, 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 contain at least one carbon A dicarbonyl group, oxime group, hydroxylamine group, or their protection It contains a polypeptide comprising a drastatin linker derivative containing a masked form. By introducing at least one reactive group into the drastatin linker derivative, While it does not react with certain amino acids, it does not react with one or more drastatin linker derivatives. The application of conjugated chemistry involved in chemical reactions becomes possible, but is not limited to these. The drastatin linker derivative side chains are as described herein, or drastatin Utilizing chemical methodologies suitable for specific functional groups or substituents present in tachin linker derivatives. It can be modified in this way.

[0351] The methods and compositions of drastatin linker derivatives described herein are various The present invention provides a complex of a substance having a functional group, substituent, or part with another substance. Other substances include polymers, water-soluble polymers, polyethylene glycol derivatives, and 2. Protein or polypeptide or polypeptide analog, antibody or antibody fragment Examples include, but are not limited to, the nt and any combination thereof.

[0352] In some embodiments, the compounds described herein include compounds of formulas (I) to (VII). The drastatin linker derivative, linker, and reagents are tested in aqueous solution under weakly acidic conditions (pH 2 In other embodiments, Such compounds are stable in aqueous solution under weakly acidic conditions for at least one month. In one embodiment, such a compound is at least 2 in an aqueous solution under weakly acidic conditions. It is stable for a week. In one embodiment, such a compound is stable in aqueous solution under weakly acidic conditions. It remains stable for at least 5 days.

[0353] In other embodiments of the compositions, methods, techniques, and strategies described herein, the above-mentioned " Studying either modified or unmodified non-natural amino acid drastatin linker derivatives or There are ways to use them. This aspect includes, but is just one example, "modified or unmodified" non-celestial phenomena. Benefit from drastatin linker derivatives containing natural amino acid polypeptides or proteins Therapeutic use, diagnostic use, assay-based use, industrial use, cosmetic use , plant biological uses, applications in environmental problems, energy production applications, consumer product applications, and This includes / or military use.

[0354] Examples of drastatin linker derivatives include, but are not limited to, the following. do not have.

[0355] [ka] JPEG0007911040000080.jpg225169 JPEG0007911040000081.jpg212169 JPEG0007911040000082.jpg214169 JPEG0007911040000083.jpg216169 JPEG0007911040000084.jpg159169

[0356] (IV. Non-natural amino acid derivatives) The non-natural amino acids used in the methods and compositions described herein are the following four: It has at least one of the following properties: (1) In the side chain of a non-natural amino acid At least one functional group is one of the 20 normally genetically coded amino acids (that is, Alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, Glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, Phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and Polyparticles containing the above-mentioned non-natural amino acids that are orthogonal to the chemical reactivity of valine. The chemical reactivity of naturally occurring amino acids present in butyl is at least orthogonal to that of a small amount. (2) Introduced These non-natural amino acids are chemically synthesized compared to the 20 genetically encoded amino acids mentioned above. (3) The above non-natural amino acids are preferably naturally occurring It has stability equivalent to that of the amino acids, or is stable as a polypeptide under typical physiological conditions. It can be incorporated into. More preferably, such integration can be done via an in vivo system. (4) The above non-natural amino acids contain an oxime functional group. Or, preferably, the biological characteristics of the polypeptide containing the above-mentioned non-natural amino acids. Under conditions that do not destroy sex (however, such destruction of biological characteristics is not an objective modification / transformation) (Of course, this does not apply if it is a typical case) or under aqueous conditions with a pH between approximately 4 and 8. Under conditions that allow the above transformation to occur, or the reaction of the above non-natural amino acids Under conditions where the sexual portion is an electrophilic portion, it is transformed into an oxime group by reacting with a reagent. It contains functional groups that can be added to the polypeptide. Any number of non-natural amino acids can be added to the polypeptide. It can be introduced. Non-natural amino acids are protected oximes or masked oximes. It is possible to transform into an oxime group, or an oxime group after deprotection or unmasking. It may further contain protected or masked groups. Non-natural amino acids are , after deprotection or unmasking, it is transformed into a carbonyl group or a dicarbonyl group. This allows it to react with hydroxylamine or oxime to form an oxime group. A protected carbonyl group or a protected dicarbonyl group or It may further contain masked carbonyl groups or masked dicarbonyl groups. stomach.

[0357] The non-natural amino acids that can be used in the methods and compositions described herein include: Amino acids with novel functional groups, amino acids that interact with other molecules covalently or non-covalently. Modified by acids, glycosylated amino acids (e.g., sugar-substituted serine), and other carbohydrates. amino acids, keto-containing amino acids, aldehyde-containing amino acids, polyethylene glycol, This includes amino acids containing other polyethers, amino acids substituted with heavy elements, and chemical Amino acids that are chemically and / or photocleavable, compared to natural amino acids. Extended side chains (polyethers or long-chain hydrocarbons (containing approximately 5 or more carbon atoms)) Examples include, but are not limited to, those that have, but are not limited to, amino acids, amino acids containing carbon-bonded sugars, amino acids with oxidation-reduction activity, amino acids Examples include thioacid-containing amino acids, as well as amino acids containing one or more toxic parts. , but not limited to these.

[0358] In some embodiments, the non-natural amino acids contain a sugar chain moiety. Examples of ano acids include N-acetyl-L-glucosaminyl-L-serine, N-acetyl-L -Galactosaminyl-L-serine, N-acetyl-L-glucosaminyl-L-threonine , N-acetyl-L-glucosaminyl-L-asparagine and O-mannosaminyl-L -Serine is one example, but is not limited to it. This refers to naturally occurring N-type or O-type bonds between amino acids and sugar chains, which are not typically found in nature. Covalent bonds that cannot be formed (examples include alkenes, oximes, thioethers, and amides) Examples of cases where it is replaced by (but not limited to these) are given. An example of an amino acid is a sugar chain (e.g., 2-) not normally found in naturally occurring proteins. It contains deoxyglucose and 2-deoxygalactose.

[0359] Incorporated into the polypeptide via the incorporation of non-natural amino acids into the polypeptide The chemical moiety provides various advantages and operational benefits for polypeptides. For example, (keto functional groups) Intrinsic reactions of carbonyl functional groups or dicarbonyl functional groups (such as aldehyde functional groups) The properties were determined using either a hydrazine-containing reagent or a hydroxyamine-containing reagent. Enables selective modification of proteins in vivo and in vitro. Natural amino acids can be useful, for example, for phase alignment of X-ray structural data. Also, non-natural amino acids... Site-specific introduction of heavy atoms using amino acids offers selectivity in the choice of positions for the heavy atoms. It provides a degree of freedom. Photoreactive unnatural amino acids (benzophenone and aryl azide) Examples include amino acids having side chains (phenyl azides, but not limited to these). (However, it is not limited to these) For example, in vivo and in vitro of polypeptides This enables efficient photocrosslinking in [the region]. An example of a photoreactive amino acid is p-azido-f Examples include phenylalanine and p-benzoyl-phenylalanine, but are not limited to these. Polypeptides having photoreactive amino acids consequently provide the photoreactive group. It can be freely photobridged by excitation of a supplied temporary control. In one non-limiting example... The methyl group of non-natural amino acids is analyzed using nuclear magnetic resonance and vibration microscopy (without limiting it to...). However, these are some examples, and as probes for local structure and dynamics, radiation-labeled It can be substituted with a methyl group (but is not limited to this).

[0360] (A. Structure and synthesis of unnatural amino acid derivatives: carbonyl group, carbonyl-like group, mass (Removed carbonyl group, and protected carbonyl group) Amino acids with electrophilic reactive groups are capable of various reactions and can undergo various chemical reactions (nucleophilic). Molecules can be bonded via addition reactions (though not limited to these). The reactive groups include carbonyl groups or dicarbonyl groups (keto groups or aldehydes) (Examples include groups), carbonyl-like groups or dicarbonyl-like groups (carbonyl group or di It has similar reactivity to a carbonyl group, and its structure is similar to that of a carbonyl group or a dicarbonyl group. (Similar in nature), masked carbonyl group or masked dicarbonyl group ( (Can be immediately converted to a carbonyl group or dicarbonyl group), or protected carbon A nyl group or a protected dicarbonyl group (when deprotected, it becomes a carbonyl group or dicarbonyl group) Examples of such amino acids include those having similar reactivity to the rubonyl group. Examples of amino acids having the structure XXXVII) are given.

[0361] [ka]

[0362] (In the formula, A is optional, and if present, it may be a lower alkylene, substituted lower alkylene, or lower alkylene. Roalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, Alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene Len, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene N, substituted heteroarylene, alkalilene, substituted alkalilene, aralkylene, or It is a substituted aralkylene, B is optional and, if present, may be a lower alkylene, a substituted lower alkylene, or a lower alkylene. Kenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene, or substituted alkylene)-, -S-, -S-(alkylene (N, or substitution alkylene)-, -S(O) k -(where k is 1, 2, or 3 (There is), -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 phosphorus selected from the group consisting of -C(R')2-N(R')-N(R')- It is a ker (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'' can independently be H, alkyl, substituted alkyl, or a protecting group, or two or more. If the above R'' group is present, the two R'' groups can optionally form a heterocycloalkyl group. R1 is H, amino protecting group, resin, amino acid, polypeptide, or polynucleotide It is a do, R2 is an OH group, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide. Ochido is R3 and R4 are independently H, halogen, lower alkyl, or substituted lower alkyl. It is either a cycloalkyl group, or R3 and R4, or two R3 groups are cycloalkyl groups. Kill, or a heterocycloalkyl group may be optionally formed, or - Both ABKR groups contain at least one carbonyl group (including a dicarbonyl group). ), protected carbonyl groups (including protected dicarbonyl groups), or masked Bicyclic or tricyclic compounds containing carbonyl groups (including masked dicarbonyl groups) It forms a cycloalkyl or heterocycloalkyl, or - Both KR groups have at least one carbonyl group (including dicarbonyl groups), protecting A protected carbonyl group (including a protected dicarbonyl group), or a masked carbonyl group. Monocyclic or bicyclic cyclo containing a nyl group (including a masked dicarbonyl group) Forms alkyl or heterocycloalkyl groups. However, if A is phenylene and each R3 is H, then B exists and A is -(CH2 )4- and if each R3 is H, then B is not -NHC(O)(CH2CH2)- If A and B are absent and each R3 is H, then R is not methyl. The ano acid may be in the form of a salt, or a polypeptide, polymer, of a non-natural amino acid. They may be incorporated into polysaccharides or polynucleotides, and may be further optionally modified after translation. (It's okay to do that.) In one embodiment, the compound represented by formula (XXXVII) is at least under weakly acidic conditions. It remains stable in aqueous solution for one month. In one embodiment, it is represented by formula (XXXVII). The compound is stable for at least two weeks under weakly acidic conditions. In one embodiment, formula (XX The compounds shown in XVII) are stable for at least 5 days under weakly acidic conditions. In terms of application methods, the acidic conditions in question are a pH of 2 to 8.

[0365] In some embodiments of the compound shown in formula (XXXVII), B is a 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, also (substituted alkylene)-, -S(alkylene, or substituted alkylene)-, -S(O) (Alkylene, or substituted alkylene)-, or -S(O)2(alkylene, also (where is a substituted alkylene)-. In some embodiments of the compound shown in formula (XXXVII) B is -O(CH2)-, -CH=N-, -CH=N-NH-, -NHCH2-, -N HCO-, -C(O)-, -C(O)-(CH2)-, -CONH-(CH2)-, -S It is CH2-, -S(=O)CH2-, or -S(O)2CH2-. Equation (XXXV II) In some embodiments of the compounds shown, R is an alkyl or cyclo having 1 to 6 carbon atoms. It is alkyl. In some embodiments of the compound represented by formula (XXXVII), R is -CH 3, -CH(CH3)2, or cyclopropyl, as shown in formula (XXXVII) In one embodiment of the compound, R1 is H, tert-butyloxycarbonyl (Boc) , 9-Fluorenylmethoxycarbonyl (Fmoc), N-acetyl, tetrafluoro It is cetyl (TFA) or benzyloxycarbonyl (Cbz). Formula (XXXV II) In some embodiments of the compounds shown, R1 is a resin, amino acid, polypeptide, or anti It is a compound or polynucleotide. An implementation of the compound shown in formula (XXXVII) Morphologically, R2 is OH, O-methyl, O-ethyl, or Ot-butyl. Formula ( In one embodiment of the compounds shown in XXXVII), R2 is a resin, amino acid, polypeptide It is a nucleotide, antibody, or polynucleotide. The compound shown in formula (XXXVII) In one embodiment, R2 is a polynucleotide. The compound shown in formula (XXXVII) In one embodiment, R2 is ribonucleic acid (RNA).

[0366] In one embodiment of the compound represented by formula (XXXVII),

[0367] [ka]

[0368] The following group is selected: (i) A is a substituted lower alkylene, a 4-carbon arylene, a substituted arylene, a heteroarylene. N, substituted heteroarylene, alkalilene, substituted alkalilene, aralkylene, or It is a substituted aralkylene, B is optional and, if present, may be a lower alkylene, a substituted lower alkylene, or a lower alkylene. Kenylene, substituted lower alkene, -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')- (alkilene, also These are substituted alkylenes -, -CSN(R')-, -N(R')CO-(alkylenes), also (Substitution alkylene)-, -N(R')C(O)O-, -N(R')C(S)-, -S( O)N(R'), -S(O)2N(R'), -N(R')C(O)N(R')-, -N( R')C(S)N(R')-, -N(R')S(O)N(R')-, -N(R')S(O )2N(R')-, -N(R')-N=, -C(R')=NN(R')-, -C(R' )=NN=, -C(R')2-N=N-, and -C(R')2-N(R')-N( A divalent linker selected from the group consisting of R'-, (ii) A is optional, and if present, it may be a substituted lower alkylene, a carbon-4 arylene, or Substituted arylene, heteroarylene, substituted heteroarylene, alkalilene, substituted alkali These are ylene, aralkylene, or substituted aralkylene. B is a lower alkylene, a substituted lower alkylene, a lower alkenylene, 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)2N(R')-, -N(R' )-N=, -C(R')=NN(R')-, -C(R')=NN=, -C(R')2 Selected from the group consisting of -N=N- and -C(R')2-N(R')-N(R')- It is a divalent linker, (iii) A is a lower alkylene, B is optional and, if present, may be a lower alkylene, a substituted lower alkylene, or a lower alkylene. Kenylene, substituted lower alkene, -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')-, -CSN(R')-, -CON(R') -(alkylene, or substituted alkylene)-, -N(R')C(O)O-, -N(R' )C(S)-, -S(O)N(R'), -S(O)2N(R'), -N(R')C(O) N(R')-, -N(R')C(S)N(R')-, -N(R')S(O)N(R')- , -N(R')S(O)2N(R')-, -N(R')-N=, -C(R')=NN( R')-, -C(R')=NN=, -C(R')2-N=N-, and -C(R') A divalent linker selected from the group consisting of 2-N(R')-N(R')-, (iv) A is phenylene, B is a lower alkylene, a substituted lower alkylene, a lower alkenylene, 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)2N(R')-, -N(R' )-N=, -C(R')=NN(R')-, -C(R')=NN=, -C(R')2 Selected from the group consisting of -N=N- and -C(R')2-N(R')-N(R')- It is a divalent linker, K is

[0369] [ka]

[0370] And, Each R' is independently H, alkyl, or substituted alkyl. R1 is optional and, if present, H, amino protecting group, resin, amino acid, polypeptide It is a nucleotide, or polynucleotide. R2 is optional and, if present, can be an OH group, an ester protecting group, a resin, an amino acid, or a polyp It is a nucleotide, or polynucleotide. Each of R3 and R4 can independently be H, halogen, lower alkyl, or substituted lower alkyl It was a kill, R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl. be.

[0371] It also contains amino acids having the structure of formula (XXXVIII).

[0372] [ka]

[0373] (In the formula, A is optional, and if present, it may be a lower alkylene, substituted lower alkylene, or lower alkylene. Roalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, Alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene Len, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene N, substituted heteroarylene, alkalilene, substituted alkalilene, aralkylene, or It is a substituted aralkylene, B is optional and, if present, may be a lower alkylene, a substituted lower alkylene, or a lower alkylene. Kenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene, or substituted alkylene)-, -S-, -S-(alkylene (N, or substitution alkylene)-, -S(O) k-(where k is 1, 2, or 3 (There is), -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 phosphorus selected from the group consisting of -C(R')2-N(R')-N(R')- It is a ker (where each R' is independently H, alkyl, or substituted alkyl). , R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl. can be, R1 is H, amino protecting group, resin, at least one amino acid, polypeptide, or , it is a polynucleotide, R2 is OH, ester protecting group, resin, at least one amino acid, polypeptide, or is a polynucleotide, However, if A is phenylene, then B exists, and if A is -(CH2)4-, then B is -N If A and B are not present, rather than HC(O)(CH2CH2)-, then R is methyl. No. The above non-natural amino acids may be in the form of salts, or polysaccharides of non-natural amino acids. They may be incorporated into peptides, polymers, polysaccharides, or polynucleotides, and further (This may be modified as needed after translation.) It also contains 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, a substituted lower alkenylene n, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene, Alternatively, (substituted alkylene)-, -S-, -S-(alkylene, or substituted alkylene) -, -S(O) k -(where k is 1, 2, or 3), -S(O) k (Aruki Len, 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 Len, or substitution alkylene)-, -C(O)N(R')-, -CON(R')-(A Lucilen, or substituted alkylene)-, -CSN(R')-, -CSN(R')-( (Lucilene, or substituted alkylene)-, -N(R')CO-(alkylene, or substituted alkylene) (Converting 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- A linker selected from the group consisting of 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, R1 is H, amino protecting group, resin, amino acid, polypeptide, or polynucleotide It is a do, R2 is an OH group, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide. Ochido is Each R a 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 Call, -S(O) k It is independently selected from the group consisting of R' (where each R' is independently (H, alkyl, or substituted alkyl). The above unnatural amino acids are available in salt form. Alternatively, a polypeptide, polymer, polysaccharide, or polynucleotide of a non-natural amino acid may be used. It may be incorporated into the leotide, and may also be optionally modified after translation. It also contains the following amino acids:

[0376] [ka]

[0377] The above non-natural amino acids are optionally protected groups, carboxyl protecting groups, and / Alternatively, it may be in the form of a salt, or a polypeptide, polymer, of a non-natural amino acid. They may be incorporated into polysaccharides or polynucleotides, and may be optionally modified after translation. That's good too.

[0378] Furthermore, it contains the following amino acids having the structure of formula (XXXX).

[0379] [ka]

[0380] (In the formula, B is -NS(O)2-, -OS(O)2-, or any other, and if present, a lower alpha Chelen, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, lower hetero alkylene, substituted lower heteroalkylene, -O-, -O-(alkylene, or substituted alkylene) -S-(alkylene, or substituted alkylene)-, -S(O) k -(where k is 1, 2, or 3), -S(O) k (Alkylene, or place (Converted alkylene)-, -C(O)-, -NS(O)2-, -OS(O)2-, -C(O)- (Alkylene, or substituted alkylene)-, -C(S)-, -C(S)-(alkylene) , or substitution alkylene)-, -N(R')-, -NR'-(alkylene, or substitution 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( A linker selected from the group consisting of R')- (where each R' is independently H, A (Lucyl, or substituted alkyl) R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl. can be, R1 is H, amino protecting group, resin, amino acid, polypeptide, or polynucleotide It is a do, R2 is an OH group, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide. Ochido is Each R a 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 Call, -S(O) k Selected independently from the group consisting of R' (where each R' is independently, H is alkyl or substituted alkyl, and n is 0 to 8. However, if A is -(CH2)4-, then B is -NHC(O)(CH2CH2)- There is none. The above non-natural amino acids may be in the form of salts, or the non-natural amino acids They may be incorporated into lipeptides, polymers, polysaccharides, or polynucleotides, and further (This may be modified as needed after translation.) It also contains the following amino acids:

[0381] [ka]

[0382] (In the formula, the above 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, These are incorporated into polypeptides, polymers, polysaccharides, or polynucleotides of non-natural amino acids. (It may be included, and may be further modified as desired after translation.) Furthermore, it contains the following amino acids having the structure of formula (XXXXI).

[0383] [ka]

[0384] (In the formula, A is optional, and if present, it may be a lower alkylene, substituted lower alkylene, or lower alkylene. Roalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, Alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene Len, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene N, substituted heteroarylene, alkalilene, substituted alkalilene, aralkylene, or It is a substituted aralkylene, B is optional and, if present, may be a lower alkylene, a substituted lower alkylene, or a lower alkylene. Kenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene, or substituted alkylene)-, -S-, -S-(alkylene (N, or substitution alkylene)-, -S(O) k -(where k is 1, 2, or 3 (There is), -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 phosphorus selected from the group consisting of -C(R')2-N(R')-N(R')- It is a ker (where each R' is independently H, alkyl, or substituted alkyl). , R1 is H, amino protecting group, resin, amino acid, polypeptide, or polynucleotide It is a do, R2 is an OH group, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide. Ochido is The above-mentioned non-natural amino acids may be in the form of salts, or polypeptides of non-natural amino acids. They may be incorporated into nucleotides, polymers, polysaccharides, or polynucleotides, and further translated (This may be modified later as desired.) Furthermore, it contains the following amino acids having the structure of formula (XXXXII).

[0385] [ka]

[0386] (In the formula, B is optional and, if present, may be a lower alkylene, a substituted lower alkylene, or a lower alkylene. Kenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene, or substituted alkylene)-, -S-, -S-(alkylene (N, or substitution alkylene)-, -S(O) k -(where k is 1, 2, or 3 (There is), -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 phosphorus selected from the group consisting of -C(R')2-N(R')-N(R')- It is a ker (where each R' is independently H, alkyl, or substituted alkyl). , R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl. can be, R1 is H, amino protecting group, resin, amino acid, polypeptide, or polynucleotide It is a do, R2 is an OH group, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide. Ochido is Each R a 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 Call, -S(O) k It is independently selected from the group consisting of R' (where each R' is independently (H, alkyl, or substituted alkyl).

[0387] The above-mentioned non-natural amino acids may be in the form of salts, or polypeptides of non-natural amino acids. They may be incorporated into nucleotides, polymers, polysaccharides, or polynucleotides, and further translated (This may be modified later as desired.) It also contains the following amino acids:

[0388] [ka]

[0389] (In the formula, the above 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, These are incorporated into polypeptides, polymers, polysaccharides, or polynucleotides of non-natural amino acids. (It may be included, and may be further modified as desired after translation.) Furthermore, it contains the following amino acids having the structure of formula (XXXXIV).

[0390] [ka]

[0391] (In the formula, B is optional and, if present, may be a lower alkylene, a substituted lower alkylene, or a lower alkylene. Kenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene, or substituted alkylene)-, -S-, -S-(alkylene (N, or substitution alkylene)-, -S(O) k -(where k is 1, 2, or 3 (There is), -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 phosphorus selected from the group consisting of -C(R')2-N(R')-N(R')- It is a ker (where each R' is independently H, alkyl, or substituted alkyl). , R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl. can be, R1 is H, amino protecting group, resin, amino acid, polypeptide, or polynucleotide It is a do, R2 is an OH group, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide. Ochido is Each R a 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 Call, -S(O) k Selected independently from the group consisting of R' (where each R' is independently, H is an alkyl or substituted alkyl group, and n is between 0 and 8.

[0392] The above-mentioned non-natural amino acids may be in the form of salts, or polypeptides of non-natural amino acids. They may be incorporated into nucleotides, polymers, polysaccharides, or polynucleotides, and further translated (This may be modified later as desired.) It also contains the following amino acids:

[0393] [ka]

[0394] (In the formula, the above 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, These are incorporated into polypeptides, polymers, polysaccharides, or polynucleotides of non-natural amino acids. (It may be included, and may be further modified as desired after translation.) The non-natural amino acids described herein, in addition to the monocarbonyl structure, also have a dicarbonyl group. Dicarbonyl-like groups, masked dicarbonyl groups, and protected dicarbonyl groups It may contain any of the groups.

[0395] For example, the following amino acids have the structure of formula (XXXXV).

[0396] [ka]

[0397] (In the formula, A is optional, and if present, it may be a lower alkylene, substituted lower alkylene, or lower alkylene. Roalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, Alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene Len, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene N, substituted heteroarylene, alkalilene, substituted alkalilene, aralkylene, or It is a substituted aralkylene, B is optional and, if present, may be a lower alkylene, a substituted lower alkylene, or a lower alkylene. Kenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene, or substituted alkylene)-, -S-, -S-(alkylene (N, or substitution alkylene)-, -S(O) k -(where k is 1, 2, or 3 (There is), -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 phosphorus selected from the group consisting of -C(R')2-N(R')-N(R')- It is a ker (where each R' is independently H, alkyl, or substituted alkyl). , R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl. can be, R1 is H, amino protecting group, resin, amino acid, polypeptide, or polynucleotide It is a do, R2 is an OH group, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide. Ochido is The above-mentioned non-natural amino acids may be in the form of salts, or polypeptides of non-natural amino acids. They may be incorporated into nucleotides, polymers, polysaccharides, or polynucleotides, and further translated (This may be modified later as desired.) Furthermore, it contains the following amino acids having the structure of formula (XXXXVI).

[0398] [ka]

[0399] (In the formula, B is optional and, if present, may be a lower alkylene, a substituted lower alkylene, or a lower alkylene. Kenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene, or substituted alkylene)-, -S-, -S-(alkylene (N, or substitution alkylene)-, -S(O) k -(where k is 1, 2, or 3 (There is), -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 phosphorus selected from the group consisting of -C(R')2-N(R')-N(R')- It is a ker (where each R' is independently H, alkyl, or substituted alkyl). , R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl. can be, R1 is H, amino protecting group, resin, amino acid, polypeptide, or polynucleotide It is a do, R2 is an OH group, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide. Ochido is Each R a 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 Call, -S(O) k It is independently selected from the group consisting of R' (where each R' is independently (H, alkyl, or substituted alkyl).

[0400] The above-mentioned non-natural amino acids may be in the form of salts, or polypeptides of non-natural amino acids. They may be incorporated into nucleotides, polymers, polysaccharides, or polynucleotides, and further translated (This may be modified later as desired.) It also contains the following amino acids:

[0401] [ka]

[0402] (In the formula, the above compound may optionally consist of an amino protecting group and a carboxyl protecting group, or These are salts. The above non-natural amino acids may be in the form of salts, or non-natural amino acids The acid may be incorporated into a polypeptide, polymer, polysaccharide, or polynucleotide. (Further modifications may be made as desired after translation.) Furthermore, it contains the following amino acids having the structure of formula (XXXXVII).

[0403] [ka]

[0404] (In the formula, B is optional and, if present, may be a lower alkylene, a substituted lower alkylene, or a lower alkylene. Kenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene, or substituted alkylene)-, -S-, -S-(alkylene (N, or substitution alkylene)-, -S(O) k -(where k is 1, 2, or 3 (There is), -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 phosphorus selected from the group consisting of -C(R')2-N(R')-N(R')- It is a ker (where each R' is independently H, alkyl, or substituted alkyl). , R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl. can be, R1 is H, amino protecting group, resin, amino acid, polypeptide, or polynucleotide It is a do, R2 is an OH group, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide. Ochido is Each R a 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 Call, -S(O) k Selected independently from the group consisting of R' (where each R' is independently, H is an alkyl or substituted alkyl group, and n is between 0 and 8.

[0405] The above-mentioned non-natural amino acids may be in the form of salts, or polypeptides of non-natural amino acids. They may be incorporated into nucleotides, polymers, polysaccharides, or polynucleotides, and further translated (This may be modified later as desired.) It also contains the following amino acids:

[0406] [ka]

[0407] (In the formula, the above compound may optionally consist of an amino protecting group and a carboxyl protecting group, or These are salts, or polypeptides, polymers, polysaccharides, or polymers of unnatural amino acids. It may be incorporated into the creotide, and may also be optionally modified after translation. Furthermore, it contains the following amino acids having the structure of formula (XXXXVIII).

[0408] [ka]

[0409] (In the formula, A is optional, and if present, it may be a lower alkylene, substituted lower alkylene, or lower alkylene. Roalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, Alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene Len, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene N, substituted heteroarylene, alkalilene, substituted alkalilene, aralkylene, or It is a substituted aralkylene, R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl. can be, R1 is H, amino protecting group, resin, amino acid, polypeptide, or polynucleotide It is a do, R2 is an OH group, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide. Ochido is X1 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 above-mentioned non-natural amino acids may be in the form of salts, or polypeptides of non-natural amino acids. They may be incorporated into nucleotides, polymers, polysaccharides, or polynucleotides, and further translated (This may be modified later as desired.) Furthermore, it contains the following amino acids having the structure of formula (XXXXIX).

[0410] [ka]

[0411] (In the formula, A is optional, and if present, it may be a lower alkylene, substituted lower alkylene, or lower alkylene. Roalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, Alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene Len, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene N, substituted heteroarylene, alkalilene, substituted alkalilene, aralkylene, or It is a substituted aralkylene, R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl. can be, R1 is H, amino protecting group, resin, amino acid, polypeptide, or polynucleotide It is a do, R2 is an OH group, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide. Ochido is L stands for alkylene, substituted alkylene, N(R')(alkylene), or N(R') (substituted alkylene) (Note that R' is H, alkyl, substituted alkyl, cycloalkyl (or substituted cycloalkyl) The above-mentioned non-natural amino acids may be in the form of salts, or polypeptides of non-natural amino acids. They may be incorporated into nucleotides, polymers, polysaccharides, or polynucleotides, and further translated (This may be modified later as desired.) Furthermore, it contains the following amino acids having the structure of formula (XXXXX).

[0412] [ka]

[0413] (In the formula, A is optional, and if present, it may be a lower alkylene, substituted lower alkylene, or lower alkylene. Roalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, Alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene Len, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene N, substituted heteroarylene, alkalilene, substituted alkalilene, aralkylene, or It is a substituted aralkylene, R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl. can be, R1 is H, amino protecting group, resin, amino acid, polypeptide, or polynucleotide It is a do, R2 is an OH group, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide. Ochido is L stands for alkylene, substituted alkylene, N(R')(alkylene), or N(R') (substituted alkylene) (Note that R' is H, alkyl, substituted alkyl, cycloalkyl (It is either a cycloalkyl or a substituted cycloalkyl.)

[0414] The above-mentioned non-natural amino acids may be in the form of salts, or polypeptides of non-natural amino acids. They may be incorporated into nucleotides, polymers, polysaccharides, or polynucleotides, and further translated (This may be modified later as desired.) Furthermore, it contains the following amino acids having the structure of formula (XXXXXI).

[0415] [ka]

[0416] (In the formula, A is optional, and if present, it may be a lower alkylene, substituted lower alkylene, or lower alkylene. Roalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, Alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene Len, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene N, substituted heteroarylene, alkalilene, substituted alkalilene, aralkylene, or It is a substituted aralkylene, R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl. can be, R1 is H, amino protecting group, resin, amino acid, polypeptide, or polynucleotide It is a do, R2 is an OH group, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide. Ochido is X1 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 base 8 and R 9 H, alkoxy, alkylamine, halo Independently selected from the group consisting of gen, alkyl, and aryl, or any R 8 oh Call R9 Both can form =O or a cycloalkyl group, or R 8 Any group adjacent to the group can together form a cycloalkyl group.

[0417] The above-mentioned non-natural amino acids may be in the form of salts, or polypeptides of non-natural amino acids. They may be incorporated into nucleotides, polymers, polysaccharides, or polynucleotides, and further translated (This may be modified later as desired.) Furthermore, it contains the following amino acids having the structure of formula (XXXXXII).

[0418] [ka]

[0419] (In the formula, A is optional, and if present, it may be a lower alkylene, substituted lower alkylene, or lower alkylene. Roalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, Alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene Len, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene N, substituted heteroarylene, alkalilene, substituted alkalilene, aralkylene, or It is a substituted aralkylene, R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl. can be, R1 is H, amino protecting group, resin, amino acid, polypeptide, or polynucleotide It is a do, R2 is an OH group, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide. Ochido is n is 0, 1, 2, 3, 4, or 5, and each CR 8 R9 Each R in the base 8 and R 9 It consists of H, alkoxy, alkylamine, halogen, alkyl, and aryl. Either independently selected from the group or any R 8 and R 9 Both are = O or cycloal Can you form a kill, or R 8 Any group adjacent to the base is cycloal It can form a kill.

[0420] The above-mentioned non-natural amino acids may be in the form of salts, or polypeptides of non-natural amino acids. They may be incorporated into nucleotides, polymers, polysaccharides, or polynucleotides, and further translated (This may be modified later as desired.) Furthermore, it contains the following amino acids having the structure of formula (XXXXXIII).

[0421] [ka]

[0422] (In the formula, A is optional, and if present, it may be a lower alkylene, substituted lower alkylene, or lower alkylene. Roalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, Alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene Len, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene N, substituted heteroarylene, alkalilene, substituted alkalilene, aralkylene, or It is a substituted aralkylene, R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl. can be, R1 is H, amino protecting group, resin, amino acid, polypeptide, or polynucleotide It is a do, R2 is an OH group, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide. Ochido is n is 0, 1, 2, 3, 4, or 5, and each CR 8 R 9 Each R in the base 8 and R 9 It consists of H, alkoxy, alkylamine, halogen, alkyl, and aryl. Either independently selected from the group or any R 8 and R 9 Both are = O or cycloal Can you form a kill, or R 8 Any group adjacent to the base is cycloal It can form a kill.

[0423] The above-mentioned non-natural amino acids may be in the form of salts, or polypeptides of non-natural amino acids. They may be incorporated into nucleotides, polymers, polysaccharides, or polynucleotides, and further translated (This may be modified later as desired.) Furthermore, it contains the following amino acids having the structure of formula (XXXXXIV).

[0424] [ka]

[0425] (In the formula, A is optional, and if present, it may be a lower alkylene, substituted lower alkylene, or lower alkylene. Roalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, Alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene Len, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene N, substituted heteroarylene, alkalilene, substituted alkalilene, aralkylene, or It is a substituted aralkylene, R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl. can be, R1 is H, amino protecting group, resin, amino acid, polypeptide, or polynucleotide It is a do, R2 is an OH group, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide. Ochido is X1 is C, S, or S(O), and L is alkylene, substituted alkylene, N( R')(alkylene) or N(R')(substituted alkylene) (Note that R' is, H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl ).

[0426] The above-mentioned non-natural amino acids may be in the form of salts, or polypeptides of non-natural amino acids. They may be incorporated into nucleotides, polymers, polysaccharides, or polynucleotides, and further translated (This may be modified later as desired.) Furthermore, it contains the following amino acids having the structure of formula (XXXXXV).

[0427] [ka]

[0428] (In the formula, A is optional, and if present, it may be a lower alkylene, substituted lower alkylene, or lower alkylene. Roalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, Alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene Len, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene N, substituted heteroarylene, alkalilene, substituted alkalilene, aralkylene, or It is a substituted aralkylene, R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl. can be, R1 is H, amino protecting group, resin, amino acid, polypeptide, or polynucleotide It is a do, R2 is an OH group, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide. Ochido is L stands for alkylene, substituted alkylene, N(R')(alkylene), or N(R') (substituted alkylene) (Note that R' is H, alkyl, substituted alkyl, cycloalkyl (It is either a cycloalkyl or a substituted cycloalkyl.)

[0429] The above-mentioned non-natural amino acids may be in the form of salts, or polypeptides of non-natural amino acids. They may be incorporated into nucleotides, polymers, polysaccharides, or polynucleotides, and further translated (This may be modified later as desired.) Furthermore, it contains the following amino acids having the structure of formula (XXXXXVI).

[0430] [ka]

[0431] (In the formula, A is optional, and if present, it may be a lower alkylene, substituted lower alkylene, or lower alkylene. Roalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, Alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene Len, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene N, substituted heteroarylene, alkalilene, substituted alkalilene, aralkylene, or It is a substituted aralkylene, R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl. can be, R1 is H, amino protecting group, resin, amino acid, polypeptide, or polynucleotide It is a do, R2 is an OH group, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide. Ochido is L stands for alkylene, substituted alkylene, N(R')(alkylene), or N(R') (substituted alkylene) (Note that R' is H, alkyl, substituted alkyl, cycloalkyl (It is either a cycloalkyl or a substituted cycloalkyl.)

[0432] The above-mentioned non-natural amino acids may be in the form of salts, or polypeptides of non-natural amino acids. They may be incorporated into nucleotides, polymers, polysaccharides, or polynucleotides, and further translated (This may be modified later as desired.) It also contains amino acids having the structure of formula (XXXXXVII).

[0433] [ka]

[0434] (In the formula, A is optional, and if present, it may be a lower alkylene, substituted lower alkylene, or lower alkylene. Roalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, Alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene Len, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene N, substituted heteroarylene, alkalilene, substituted alkalilene, aralkylene, or It is a substituted aralkylene, M is

[0435] [ka]

[0436] And, (a) shows the bond to the A group, while (b) shows the bond to each carbonyl group. So, R3 and R4 are H, halogen, alkyl, substituted alkyl, cycloalkyl, if R3 and R4 are either independently selected from substituted cycloalkyl groups, or two groups R3 and R4. The R3 group, or the two R4 groups, can be either cycloalkyl or heterocycloalkyl. Formed as, R is H, halogen, alkyl, substituted alkyl, cycloalkyl, or substituted cyclo It is alkyl, T3 is a bond, C(R)(R), O, or S, where R is H, halogen, alkyl It is a substituted alkyl, cycloalkyl, or substituted cycloalkyl, R1 is H, amino protecting group, resin, amino acid, polypeptide, or polynucleotide It is a do, R2 is an OH group, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide. Ochido is The above-mentioned non-natural amino acids may be in the form of salts, or polypeptides of non-natural amino acids. They may be incorporated into nucleotides, polymers, polysaccharides, or polynucleotides, and further translated (This may be modified later as desired.) It also contains 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 each carbonyl group. So, R3 and R4 are H, halogen, alkyl, substituted alkyl, cycloalkyl, if R3 and R4 are either independently selected from substituted cycloalkyl groups, or two groups R3 and R4. The R3 group, or the two R4 groups, can be either cycloalkyl or heterocycloalkyl. Formed as, R is H, halogen, alkyl, substituted alkyl, cycloalkyl, or substituted cyclo It is alkyl, T3 is a bond, C(R)(R), O, or S, where R is H, halogen, alkyl It is a substituted alkyl, cycloalkyl, or substituted cycloalkyl, R1 is H, amino protecting group, resin, amino acid, polypeptide, or polynucleotide It is a do, R2 is an OH group, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide. Ochido is Each R a 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 Call, -S(O) kIt is independently selected from the group consisting of R' (where each R' is independently (H, alkyl, or substituted alkyl).

[0441] The above-mentioned non-natural amino acids may be in the form of salts, or polypeptides of non-natural amino acids. They may be incorporated into nucleotides, polymers, polysaccharides, or polynucleotides, and further translated (This may be modified later as desired.) It also contains amino acids having the structure of formula (XXXXXIX).

[0442] [ka]

[0443] (In the formula, R is H, halogen, alkyl, substituted alkyl, cycloalkyl, or substituted cyclo It is alkyl, T3 is either O or S.

[0444] The above-mentioned non-natural amino acids may be in the form of salts, or polypeptides of non-natural amino acids. They may be incorporated into nucleotides, polymers, polysaccharides, or polynucleotides, and further translated (This may be modified later as desired.) It also contains amino acids having the structure of formula (XXXXXX).

[0445] [ka]

[0446] (In the formula, R is H, halogen, alkyl, substituted alkyl, cycloalkyl, or substituted cyclo It is alkyl. Furthermore, it contains the following amino acids having the structure of formula (XXXXXX).

[0447] [ka]

[0448] The above-mentioned non-natural amino acids may be in the form of salts, or polypeptides of non-natural amino acids. They may be incorporated into nucleotides, polymers, polysaccharides, or polynucleotides, and further translated It may be modified later as desired.

[0449] Carbonyl functional groups or dicarbonyl functional groups are found in aqueous solutions under mild conditions. It can be selectively reacted with droxylamine-containing reagents, thereby forming the corresponding oxime bond. 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. See Soc. 117(14):3893-3899 (1995). Furthermore, carbonyl group or dical The unique reactivity of the bonyl group allows for selective modification in the presence of other amino acid side chains. For example, Cornish, VW, et al., J. Am. Chem. Soc. 118:8150-8151 (1996); Geog hegan, KF & Stroh, JG, Bioconjug. Chem. 3:138-146 (1992); Mahal, LK, et See al., Science 276:1125-1128 (1997).

[0450] p-acetyl-(+ / -)-phenylalanine and m-acetyl-(+ / -)-phenylalanine The synthesis of nyalanin is referenced by Zhang, Z. et al, Biochemistry 42: 673. It is described in 5-6746 (2003). Other carbonyl-containing amino acids or dicarbonyl-containing amino acids. Amino acids can be prepared in a similar manner.

[0451] In some embodiments, polypeptides containing non-natural amino acids are chemically modified. This reaction generates a reactive carbonyl or dicarbonyl functional group. For example, conjugation reaction The aldehyde functional group that is particularly useful has adjacent amino and hydroxyl groups. It can be generated from functional groups. When the biologically active molecule is a polypeptide, for example, N-terminal serine or threonine (normally present or chemically or enzymatically) (which can be exposed through digestion) under mild oxidative cleavage conditions using periodate It can be used to generate rudehyde functional groups. For example, Gaertner, et al., Bioconjug. Chem. 3: 262-268 (1992); Geoghegan, K. & Stroh, J., Bioconjug. Chem. 3:138-146 ( See Gaertner et al, J. Biol. Chem. 269:7224-7230 (1994). Furthermore, methods known in this technology involve the amino at the N-terminus of a peptide or protein. It is limited by acids.

[0452] Furthermore, as an example, non-natural amino acids having adjacent hydroxyl and amino groups. No acids can be incorporated into polypeptides as "masked" aldehyde functional groups. For example. Furthermore, 5-hydroxylysine has a hydroxyl group adjacent to the amine at the ε position. The reaction conditions for generating dehyde are designed to avoid oxidation at other sites in the polypeptide. Therefore, it includes the addition of excessive molar concentrations of sodium metaperiodate under mild conditions. The pH of the oxidation reaction is typically around 7.0. The normal reaction involves the slow oxidation of polypeptides. Adding an excess of approximately 1.5 molars of sodium metaperiodate to the saturation solution, and then darkening the solution. This includes incubating for approximately 10 minutes at the location. For example, U.S. Regulation 6, Refer to issues 423 and 685.

[0453] (B. Structure and synthesis of unnatural amino acids: dicarbonyl group, dicarbonyl-like group, mask) (Dicarbonyl groups that have been modified, and protected dicarbonyl groups) Amino acids with electrophilic reactive groups can undergo various reactions, particularly nucleophilic addition reactions. This allows molecules to be bonded. The electrophilic reactive group is a dicarbonyl group (diketone group, ke Examples include toaldehyde groups, keto acid groups, keto ester groups, and ketothioester groups. ), carbonyl-like group or dicarbonyl-like group (having similar reactivity to a dicarbonyl group) (It is structurally similar to a dicarbonyl group), masked dicarbonyl group (dicarbonyl) (can be immediately converted to a bonyl group), or a protected dicarbonyl group (when deprotected, Examples include amino acids that have similar reactivity to a dicarbonyl group. Examples of amino acids having the structure of formula (XXXVII) include the following.

[0454] [ka]

[0455] (In the formula, A is optional, and if present, it may be a lower alkylene, substituted lower alkylene, or lower alkylene. Roalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, Alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene Len, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene N, substituted heteroarylene, alkalilene, substituted alkalilene, aralkylene, or It is a substituted aralkylene, B is optional, and if present, it is a phosphorus bonded to the diamine-containing portion at one end. The linker is a lower alkylene, a substituted lower alkylene, or 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)-( Here, k is 1, 2, or 3), -C(O)-(alkylene, or substitution a (alkylene)-, -C(S)-(alkylene or substituted alkylene)-, -NR”-( Alkylene, or substituted alkylene)-, -CON(R")-(alkylene, or Substituting alkylene)-, CSN(R")-(alkylene, or substituted alkylene)-, And from the group consisting of -N(R")-CO-(alkylene or substituted alkylene)- Selected (where each R'' is independently H, alkyl, or substituted alkyl), K is

[0456] [ka]

[0457] And, Here, T1 is a bond, an optionally substituted alkylene with 1 to 4 carbon atoms, an optionally substituted carbon atom 1-4 alkenylenes, or optionally substituted heteroalkyls, Each optional substituent is a lower alkylene, substituted lower alkylene, lower cycloalkylene, or substituted lower alkylene. Substituting lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkylene, low Lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heteroalkylene Telocycloalkylene, arylene, substituted arylene, heteroarylene, substituted hetero Reylene, alkaliylene, substituted alkaliylene, aralkylene, or substituted aralkylene Selected more independently, T2 is a lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenyl Len, 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 Chilen, 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'-(al Chilen, 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 Substitution 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 Selected from the group consisting of -N(R')-N(R')- (where each R' is independently H, Alkyl, or substituted alkyl, T3 is

[0458] [ka]

[0459] And, Here, Each X1 is -O-, -S-, -N(H)-, -N(R)-, -N(Ac)-, and X2 is independently selected from the group consisting of -N(OMe)-, and X2 is -OR, -OAc, -SR -N(R)2, -N(R)(Ac), -N(R)(OMe), or N3 ( Each R' is independently H, alkyl, or substituted alkyl. R is H, halogen, alkyl, substituted alkyl, cycloalkyl, or substituted cyclo It is alkyl, R1 is H, amino protecting group, resin, amino acid, polypeptide, or polynucleotide It is a do, R2 is an OH group, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide. It is Ochido, or, - Both ABKR groups contain at least one carbonyl group (including a dicarbonyl group). ), protected carbonyl groups (including protected dicarbonyl groups), or masked Bicyclic or tricyclic compounds containing carbonyl groups (including masked dicarbonyl groups) It forms a cycloalkyl or heterocycloalkyl, or - Both KR groups have at least one carbonyl group (including dicarbonyl groups), protecting A protected carbonyl group (including a protected dicarbonyl group), or a masked carbonyl group. Monocyclic or bicyclic cyclo containing a nyl group (including a masked dicarbonyl group) (Forms alkyl or heterocycloalkyl groups.) Non-limiting examples of dicarbonyl amino acids having the structure of formula (XXXVII) include: The following can be listed.

[0460] [ka] JPEG0007911040000124.jpg63169

[0461] The following amino acids, which have the structure of formula (XXXVII), are also included.

[0462] [ka]

[0463] The above-mentioned non-natural amino acids may be in the form of salts, or polypeptides of non-natural amino acids. They may be incorporated into nucleotides, polymers, polysaccharides, or polynucleotides, and further translated It may be modified later as desired.

[0464] (C. Structure and synthesis of unnatural amino acids: ketoalkyne group, ketoalkyne-like group, ma Screened ketoalkyne groups, protected ketoalkyne groups (Groupk), alkyne groups, and (cycloalkyne group) Amino acids containing a reactive group with dicarbonyl-like reactivity are divided via nucleophilic addition reactions. They can bond to other organisms. Examples of such electrophilic groups include ketoalkyne groups and ketoalkyne-like groups. (It has similar reactivity to the ketoalkyne group and is structurally similar to the ketoalkyne group.) ), masked ketoalkyne groups (which can be immediately converted to ketoalkyne groups), or protected The ketoalkyne group (which, when deprotected, exhibits similar reactivity to a ketoalkyne group) is an example. In some embodiments, terminal alkynes, internal alkynes or cycloal Amino acids containing a reaction group with a kin can undergo cycloaddition reactions (e.g., 1,3-bipolar cycloaddition). Molecules can be joined via azido-alkyne cycloaddition (Huisgen), etc. Such amino acids include those with the chemical formula (XXXXXXI-A) or (XXXXXXI- Examples of amino acids having structure B) include amino acids.

[0465] [ka]

[0466] (In the formula, A is optional, and if present, it may be a lower alkylene, substituted lower alkylene, or lower alkylene. Roalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, Alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene Len, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene N, substituted heteroarylene, alkalilene, substituted alkalilene, aralkylene, or It is a substituted aralkylene, B is optional, and if present, is a linker with one end bonded to the diamine-containing portion. Yes, the linker is a lower alkylene, substituted lower alkylene, lower alkenylene, 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) - (here (where k is 1, 2, or 3), -C(O)-(alkylene, or substituted alkylene) -C(S)-(alkylene, or substituted alkylene)-, -NR"-(alkyl -CON(R)-(alkylene, or substituted alkylene) alkylene)-, -CSN(R")-(alkylene, or substituted alkylene)-, and Selected from the group consisting of -N(R")CO-(alkylene or substituted alkylene)- (Note that each R'' is independently H, alkyl, or substituted alkyl.) G is optional, and if it exists,

[0467] [ka]

[0468] And, T4 is a carbonyl protecting group, and the following are examples of such carbonyl protecting groups. However, it is not limited to this.

[0469] [ka]

[0470] (In the formula, each X1 is independently -O-, -S-, -N(H)-, -N(R)-, -N(A c) Selected from the group consisting of - and -N(OMe)-, X2 is -OR, -OAc -SR, -N(R)2, -N(R)(Ac), -N(R)(OMe), or N3 (and each R' is independently H, alkyl, or substituted alkyl), R is H, halogen, alkyl, substituted alkyl, cycloalkyl, or substituted cyclo It is alkyl, R1 is H, amino protecting group, resin, amino acid, polypeptide, or polynucleotide It is a do, R2 is an OH group, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide. Ochido is R3 and R4 can independently be H, halogen, lower alkyl, or substituted. It is either a lower alkyl group, or R3 and R4, or two R3 groups are cycloa Lukyl or heterocycloalkyl groups may be optionally formed. R 19 These are, independently, alkyl groups having 1 to 6 carbon atoms, alkoxy groups having 1 to 6 carbon atoms, and esters. ethers, thioethers, aminoalkyls, halogens, alkyl esters, aryl esters Steryl amides, arylamides, alkyl halides, alkylamines, alkyl sulfates Phosphate, alkyl nitro, thioester, sulfonyl ester, halosulfonyl, nitrile Selected from the group consisting of nitriles, alkylnitriles, 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 group, ketoamine-like group, mask) (Ketoamine groups that have been modified, and protected ketoamine groups) Amino acids containing a reactive group with dicarbonyl-like reactivity are divided via nucleophilic addition reactions. It can bind to its offspring. Such reactive groups include ketoamine groups and ketoamine-like groups. It has similar reactivity to the ketoamine group and is structurally similar to the ketoamine group, and is masked. A ketoamine group (which can be immediately converted to a ketoamine group), or a protected ketoamine. Examples include groups (which, when deprotected, exhibit similar reactivity to ketoamine groups). Examples of amino acids include those having the chemical formula (XXXXXXII).

[0471] [ka]

[0472] (In the formula, A is optional, and if present, it may be a lower alkylene, substituted lower alkylene, or lower alkylene. Roalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, Alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene Len, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene N, substituted heteroarylene, alkalilene, substituted alkalilene, aralkylene, or It is a substituted aralkylene, B is optional, and if present, is a linker with one end bonded to the diamine-containing portion. Yes, the linker is a lower alkylene, substituted lower alkylene, lower alkenylene, 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) - (here (where k is 1, 2, or 3), -C(O)-(alkylene, or substituted alkylene) -C(S)-(alkylene, or substituted alkylene)-, -NR"-(alkyl -CON(R)-(alkylene, or substituted alkylene) alkylene)-, -CSN(R")-(alkylene, or substituted alkylene)-, and Selected from the group consisting of -N(R")CO-(alkylene or substituted alkylene)- (Note that each R'' is independently H, alkyl, or substituted alkyl.) G is

[0473] [ka]

[0474] And, T1 is an alkylene with 1 to 4 carbon atoms that is arbitrarily substituted, The alkenylene, or optionally substituted heteroalkyl, T4 is a carbonyl protecting group, and the following are examples of such carbonyl protecting groups. However, it is not limited to this.

[0475] [ka]

[0476] (In the formula, each X1 is independently -O-, -S-, -N(H)-, -N(R')-, -N( X2 is selected from the group consisting of Ac)- and -N(OMe)-, and X2 is -OR, -OA c, -SR', -N(R')2, -N(R')(Ac), -N(R')(OMe), and It is N3, and each R' is independently H, alkyl, or substituted alkyl. ru), R is H, halogen, alkyl, substituted alkyl, cycloalkyl, or substituted cyclo It is alkyl, R1 is H, amino protecting group, resin, amino acid, polypeptide, or polynucleotide It is a do, R2 is an OH group, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide. Ochido is R3 and R4 can independently be H, halogen, lower alkyl, or substituted. It is either a lower alkyl group, or R3 and R4, or two R3 groups are cycloa (A lukyl or heterocycloalkyl group may be optionally formed.) Amino acids having the structure of chemical formula (XXXXXXII) include those with the chemical formula (XXXXXX Examples include amino acids having the structure of (III) and chemical formula (XXXXXXIV).

[0477] [ka]

[0478] (In the formula, each R a These are independently H, halogen, alkyl, substituted alkyl, and -N(R')2 , -C(O) k R'(where k is 1, 2, or 3), -C(O)N(R') 2, -OR', and -S(O) k Selected from the group consisting of R' (Note that each R' is, They 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 with nucleophilic reactive groups bind molecules together through electrophilic addition reactions with other molecules. This enables various reactions. The nucleophilic reaction group is a diamine group (hydrazine group, a Midine group, imine group, 1,1-diamine group, 1,2-diamine group, 1,3-diamine group, (and, 1,4-diamine groups are examples), diamine-like groups (similar reactivity to diamine groups) (It has a masked diamine group (diamine group) and is structurally similar to a diamine group) (can be immediately converted to a diamine group), or a protected diamine group (when deprotected, it becomes a diamine group) Examples include those having similar reactivity to the group. In some embodiments, azide is present. Amino acids containing the reactive group can undergo cycloaddition reactions (e.g., 1,3-bipolar cycloaddition, azido- Molecules can be joined via alkyne huisgen addition (e.g., cycloaddition).

[0479] In another aspect, for the derivatization of carbonyl-substituted drastatin derivatives A method for the chemical synthesis of molecules substituted with dragine is described. In one embodiment, The hydrazine-substituted molecule may be a drastatin-bonded derivative. (In one embodiment) The method for preparing molecules substituted with hydrazine is described. This preparation method is, for example, Furthermore, it is suitable for the derivatization of carbonyl-containing non-natural amino acid polypeptides, and carbonyl-containing As just one example of a non-natural amino acid polypeptide, ketone-containing non-natural amino acids are... Examples include polypeptides, or aldehyde-containing non-natural amino acid polypeptides. In other embodiments, the non-natural amino acid is used in vivo in the protein. It is incorporated site-specifically during translation in. In further or additional embodiments The hydrazine-substituted drastatin derivative then exhibits site-specific nitrogen-containing hete Each Site-specific derivatives of carbonyl-containing unnatural amino acids via nucleophilic attack of the carbonyl group. It can be transformed. In further or additional embodiments, dras substituted with hydrazine The method for producing tatin derivatives allows for a wide variety of site-specifically derivatized polypeptides. This can be obtained. In further or additional embodiments, hydrazine vessel Method for synthesizing drastatin derivatives to which polyethylene glycol (PEG) is bonded. This will be written.

[0480] Such amino acids include those with chemical formulas (XXXVII-A) or (XXXVII-B). Examples of amino acids having the structure of ) are given.

[0481] [ka]

[0482] (In the formula, A is optional, and if present, it may be a lower alkylene, substituted lower alkylene, or lower alkylene. Roalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, Alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene Len, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene N, substituted heteroarylene, alkalilene, substituted alkalilene, aralkylene, or It is a substituted aralkylene, B is optional, and if present, is a linker with one end bonded to the diamine-containing portion. Yes, the linker is a lower alkylene, substituted lower alkylene, lower alkenylene, 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 alkylene) Chilen)-(where k is 1, 2, or 3),-C(O)-(alkylene, or substituted alkylene)-, -C(S)-(alkylene, or substituted alkylene)-, -NR”-(alkylene, or substituted alkylene)-, -CON(R”)-(alkylene (-, or substituted alkylene)-, -CSN(R)-(alkylene, or substituted alkylene) -N(R")CO-(alkylene or substituted alkylene) Selected from the following group (wherein each R'' is independently H, alkyl, or substituted alkyl) (It is a lu) K is

[0483] [ka]

[0484] (In the formula, R8 and R9 are independently H, alkyl, substituted alkyl, cycloalkyl, substituted cycloalkyl Selected from chloroalkyl or amine protecting groups, T1 is a bond, an optionally substituted alkylene with 1 to 4 carbon atoms, an optionally substituted carbon atom 1-4 alkenylenes, or optionally substituted heteroalkyls, T2 is an alkylene with 1 to 4 carbon atoms that can be arbitrarily substituted, alkenylenes, optionally substituted heteroalkyls, optionally substituted aryls, or , an arbitrarily substituted heteroaryl, Here, each arbitrary substituent is independently a lower alkyl, a substituted lower alkyl, or a lower cyclo Alkyl, substituted lower cycloalkyl, lower alkenyl, substituted lower alkenyl, alkynyl , lower heteroalkyl, substituted heteroalkyl, lower heterocycloalkyl, substituted lower hetero Rocycloalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, a (Selected from lucaryl, substituted alkalyl, aralkyl, or substituted aralkyl), R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl. can be, R1 is H, amino protecting group, resin, amino acid, polypeptide, or polynucleotide It is a do, R2 is an OH group, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide. Ochido is R3 and R4 can independently be H, halogen, lower alkyl, or substituted. It is either a lower alkyl group, or R3 and R4, or two R3 groups are cycloa Lukyl or heterocycloalkyl groups may be optionally formed, or - Both ABKR groups contain at least one diamine group, a protected diamine group, Alternatively, a bicyclic or tricyclic cycloalkyl or hemopropyl alcohol having a masked diamine group. Forms a telocycloalkyl (where at least one amine group on -ABKR). (is an optionally protected amine), or, - The BKR group together has at least one diamine group, a protected diamine group, and This refers to a bicyclic or tricyclic cycloalkyl or cyclo having a masked diamine group. Forms an aryl or heterocycloalkyl group, or - Both KR groups have at least one diamine group, a protected diamine group, or Monocyclic or bicyclic cycloalkyl or heterocyclic having a masked diamine group (Forms a rhalkyl group.) That is the case.

[0485] In one embodiment, there is a compound having either structure 1 or structure 2 below.

[0486] [ka]

[0487] (In the formula, A is optional, and if present, it may be a lower alkylene, substituted lower alkylene, or lower alkylene. Roalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, Alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene Len, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene N, substituted heteroarylene, alkalilene, substituted alkalilene, aralkylene, or It is a substituted aralkylene, B is optional, and if present, is a linker with one end bonded to the diamine-containing portion. Yes, the linker is a lower alkylene, substituted lower alkylene, lower alkenylene, 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) - (here (where k is 1, 2, or 3), -C(O)-(alkylene, or substituted alkylene) -C(S)-(alkylene, or substituted alkylene)-, -NR"-(alkyl -CON(R)-(alkylene, or substituted alkylene) alkylene)-, -CSN(R")-(alkylene, or substituted alkylene)-, and Selected from the group consisting of -N(R")CO-(alkylene or substituted alkylene)- (Note that each R'' is independently H, alkyl, or substituted alkyl.) T1 is a bond or CH2, and T2 is CH. Here, each arbitrary substituent is independently a lower alkyl, a substituted lower alkyl, or a lower cyclo Alkyl, substituted lower cycloalkyl, lower alkenyl, substituted lower alkenyl, alkynyl , lower heteroalkyl, substituted heteroalkyl, lower heterocycloalkyl, substituted lower hetero Rocycloalkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, a Selected from lucaryl, substituted alkalyl, aralkyl, or substituted aralkyl, R1 is H, amino protecting group, resin, amino acid, polypeptide, or polynucleotide It is a do, R2 is an OH group, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide. Ochido is R3 and R4 can independently be H, halogen, lower alkyl, or substituted. It is either a lower alkyl group, or R3 and R4, or two R3 groups are cycloa Lukyl or heterocycloalkyl groups may be optionally formed, or -AB-diamine-containing moieties each have at least one diamine group, protected diamine Bicyclic cycloalkyl or heterocyclic compounds having a mine group or a masked diamine group. Forms a cycloalkyl group (where at least one amine of the -AB-diamine-containing moiety) The amine group is optionally a protected amine, or an active metabolite, salt, or the same. (These are pharmaceutically acceptable prodrugs or solvates of the same drug), or - The group consisting of the β-diamine-containing moiety together has at least one diamine group, protected Bicyclic or tricyclic cycloa having a diamine group, or a masked diamine group (Forms cyclic or cycloaryl or heterocycloalkyl groups.) The following are non-limiting examples of amino acids having the structure of chemical formula (XXXVII): It can be listed.

[0488] [ka]

[0489] The above-mentioned non-natural amino acids may be in the form of salts, or non-natural amino acid polypeptides. Incorporated into nucleotides, polymers, polysaccharides, or polynucleotides, and / or optional It may be modified after translation.

[0490] In one embodiment, the compound of chemical formula (XXXVII) is found to be less acidic under weakly acidic conditions. It remains stable in aqueous solution for at least one month. In one embodiment, the chemical formula (XXXVI The compound in I) is stable for at least two weeks under weakly acidic conditions. In one embodiment... Furthermore, the compound of chemical formula (XXXVII) is stable for at least 5 days under weakly acidic conditions. In one embodiment, the above acidic conditions are such that the pH is approximately 2 to 8.

[0491] In one embodiment of the compound of chemical formula (XXXVII), B is a lower alkylene, Substituting lower alkylene, O-(alkylene, or substituted alkylene)-, C(R')=NN (R')-, -N(R')CO-, C(O)-, -C(R')=N-, C(O)-(Al (Chilene, or substituted alkylene)-, CON(R')(alkylene, or substituted alkyl -S(alkylene, or substituted alkylene)-, -S(O)(alkylene) , or substituted alkylene)-, or -S(O)2(alkylene, or substituted alkylene) It is (cheline). In some embodiments of the compound of chemical formula (XXXVII), B is - O(CH2)-, -CH=N-, CH=NNH-, -NHCH2-, -NHCO-, C( O)-, C(O)(CH2)-, CONH(CH2)-, -SCH2-, -S(=O)C It is H2- or -S(O)2CH2-. A compound of the chemical formula (XXXVII) In the embodiment, R is an alkyl or cycloalkyl group having 1 to 6 carbon atoms. In one embodiment of compound (XXXVII), R is -CH3, -CH(CH3) 2, or cyclopropyl. In one embodiment of the compound of chemical formula (XXXVII) In this case, R1 is H, tert-butyloxycarbonyl (t-Boc), 9-fluore Nylmethoxycarbonyl (Fmoc), N-acetyl, tetrafluoroacetyl (TFA) ), or benzyloxycarbonyl (Cbz). Chemical formula (XXXVII) In one embodiment of the compound, R1 is a resin, amino acid, polypeptide, or polynucleate. It is a creotide. In one embodiment of the compound of chemical formula (XXXVII), R1 is It is an antibody, antibody fragment, or monoclonal antibody. It is a compound of chemical formula (XXXVII). In one embodiment, R2 is OH, O-methyl, O-ethyl, or Ot- It is chill. In one embodiment of the compound of chemical formula (XXXVII), R2 is a resin. It is at least one amino acid, polypeptide, or polynucleotide. Chemical formula ( In one embodiment of compound XXXVII), R2 is an antibody, antibody fragment, or mo It is a noclonal antibody.

[0492] The following are non-exclusive examples of amino acids having the structure of chemical formula (XXXVII): It can be listed.

[0493] [ka]

[0494] As a non-limiting example of a protected amino acid having the structure of chemical formula (XXXVII), The following are some examples.

[0495] [ka]

[0496] (F. Structure and synthesis of unnatural amino acids: Aromatic amines) Unnatural amino acids with nucleophilic groups can bond molecules through various reactions. This makes it possible. Various reactions include aldehyde-containing drastatin linker derivatives. Examples include, but are not limited to, reductive alkylation reactions using such nucleophiles. As just one example of non-natural amino acids that have reactive groups, aromatic amine groups (secondary amino acids) are Examples include tertiary amine groups, masked aromatic amine groups (directly connected to aromatic amine groups) (can be converted to), or protected aromatic amine group (when deprotected, aromatic amine) Examples include those having similar reactivity to the 'n' group. Such non-natural amino acid-containing aromatic amino acids. Examples include amino acids having the chemical formula (XXXXXXV).

[0497] [ka]

[0498] (In the formula,

[0499] [ka]

[0500] These include monocyclic aryl rings, bicyclic aryl rings, polycyclic aryl rings, and monocyclic heteroaryl rings. Selected from the group consisting of bicyclic heteroaryl rings and polycyclic heteroaryl rings, A is independent, CR a , or N, B is independent of CR a , N, O, or S, Each R a These are independently H, halogen, alkyl, -NO2, -CN, substituted alkyl, - N(R')2, -C(O) k R', -C(O)N(R')2, -OR', and -S( O) k Selected from the group consisting of R' (where k is 1, 2, or 3), n is It is 0, 1, 2, 3, 4, 5, or 6. R1 is H, amino protecting group, resin, at least one amino acid, polypeptide, or , it is a polynucleotide, R2 is OH, ester protecting group, resin, at least one amino acid, polypeptide, or is a polynucleotide, R3 and R4 can independently be H, halogen, lower alkyl, or substituted. It is either a lower alkyl group, or R3 and R4, or two R3 groups are cycloa Lukyl or heterocycloalkyl groups may be optionally formed. M is H, or -CH2R5, or the MNC(R5) part is 4 They may also form a 7-membered ring structure. R5 is alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted a Lukinyl, alkoxy, substituted alkoxy, alkylalkoxy, substituted alkylalkoxy , polyalkylene oxide, substituted polyalkylene oxide, cycloalkyl, substituted cyclo Alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic Substituting heterocycle, alkalil, substituted alkalil, 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 Substituting alkenylene)-N(R")2,-(alkylene, or substituted alkylene)-(A (Reel, 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 It is ylene, or substituted alkylene)-SS-(aryl, or substituted aryl) (Here, each R'' is independently hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkyl Kenyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, substituted heterocyclic, alkalil, substituted alkalil, aralkyl Substitutional aralkyl (or -C(O)OR'), or The two R5 groups can optionally form a cycloalkyl or heterocycloalkyl group. Also, R5 and any R a And, arbitrarily form cycloalkyl or heterocycloalkyl It's okay to do it, Each R' is independently H, alkyl, or substituted alkyl. Also, the above non Natural amino acids may be in the form of salts, or non-natural amino acid polypeptides, poly Mer, polysaccharide, or polynucleotide is incorporated and optionally reductively alkylated. It's okay to be there. The following structure (as shown in all examples in this specification):

[0501] [ka]

[0502] These are "A", "B", "NH-M", and "R a The relative orientation of " is not shown, These four components of the structure are as shown in the examples herein (of the structure It can be oriented in any chemically appropriate manner (along with other components).

[0503] As for non-natural amino acids containing an aromatic amine moiety having the structure of chemical formula (A), Examples of unnatural amino acids having the following structure include:

[0504] [ka]

[0505] (In the formula, each A' is independently CR a , N, or,

[0506] [ka]

[0507] Selected from, up to two A's,

[0508] [ka]

[0509] And the remaining A' is CR a (Or, it may be selected from N.) Furthermore, the above-mentioned non-natural amino acids may be in the form of salts, or non-natural amino acid polysaccharides. Incorporated into peptides, polymers, polysaccharides, or polynucleotides, and optionally reductively... It's fine if he's turned into Rukia.

[0510] A non-natural amino acid containing an aromatic amine moiety with the chemical formula (XXXXXXV). As non-restrictive examples, consider the chemical formula (XXXXXXVI) and the chemical formula (XXXXXXV Examples include unnatural amino acids having the structure of (II).

[0511] [ka]

[0512] (In the formula, G is an amine protecting group, and includes, but is not limited to, the following.) do not have.)

[0513] [ka]

[0514] The above-mentioned non-natural amino acids may be in the form of salts, or non-natural amino acid polypeptides. Incorporated into polymers, polysaccharides, or polynucleotides, and optionally reductively alkylated. It's okay if it's been transformed.

[0515] Non-natural amino acids containing an aromatic amine moiety have the following structure:

[0516] [ka]

[0517] (In the formula, each Ra These are independently H, halogen, alkyl, -NO2, -CN, and substituted alkyl Ru, -N(R')2, -C(O) k R', -C(O)N(R')2, -OR', and - S(O) k Selected from the group R' (where k is 1, 2, or 3), M is H, or -CH2R5, or the MNC(R5) part is 4 They may also form a 7-membered ring structure. R1 is H, amino protecting group, resin, amino acid, polypeptide, or polynucleotide It is a do, R2 is an OH group, an ester protecting group, a resin, an amino acid, a polypeptide, or a polynucleotide. Ochido is R5 is alkyl, substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substituted a Lukinyl, alkoxy, substituted alkoxy, alkylalkoxy, substituted alkylalkoxy , polyalkylene oxide, substituted polyalkylene oxide, cycloalkyl, substituted cyclo Alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic Substituting heterocycle, alkalil, substituted alkalil, 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 Substituting alkenylene)-N(R")2,-(alkylene, or substituted alkylene)-(A (Reel, 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 It is ylene, or substituted alkylene)-SS-(aryl, or substituted aryl) (Here, each R'' is independently hydrogen, alkyl, substituted alkyl, alkenyl, substituted alkyl Kenyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, substituted heteroaryl, heterocyclic, substituted heterocyclic, alkalil, substituted alkalil, aralkyl Substitutional aralkyl (or -C(O)OR'), or R5 and any R a And, arbitrarily form cycloalkyl or heterocycloalkyl It's okay to do it, Each R' is independently H, alkyl, or substituted alkyl. Furthermore, the above-mentioned non-natural amino acids may be in the form of salts, or non-natural amino acid polysaccharides. They may be incorporated into peptides, polymers, polysaccharides, or polynucleotides.

[0518] The above unnatural amino acid with chemical formula (XXXXXXV) is the aromatic portion of the unnatural amino acid. It may be formed by the reduction of a protected or masked amine moiety. Examples of protected or masked amine moieties include imines, hydrazines, and nitrates. Examples include, but are not limited to, azide substituents. The above protected, and The reducing agents used to reduce the masked amine portion include TCEP and Na2 Examples include S, Na2S2O4, LiAlH4, NaBH4, or NaBCNH3. However, it is not limited to these.

[0519] (V. Non-natural amino acid-bonded drastatin derivative) Other embodiments described herein involve at least one of the above-mentioned drastatin linker inductions Methods, strategies, and technologies for incorporating the body into non-natural amino acids. This embodiment includes at least one... The above-mentioned drastatin linker derivatives containing the above-mentioned non-natural amino acids are manufactured, purified, and specially prepared. This includes marking and methods of use. Furthermore, the embodiment includes at least one non-natural mysid shrimp. To produce a drastatin linker derivative containing an acid, at least partially. A composition of oligonucleotides (including DNA and RNA) that can be present, This includes methods for producing, purifying, characterizing, and using the oligonucleotide in question. Furthermore, this This embodiment includes a drastatin linker derivative containing at least one non-natural amino acid. The above-mentioned oligonucleotides can be used to manufacture, at least partially. A composition of cells that can be expressed, and the production, purification, characterization, and use of such cells. The method is included.

[0520] Therefore, carbonyl, dicarbonyl, alkyne, cycloalkyne, azide, oxy M, or at least one unnatural amino acid having a hydroxylamine group, Drastatin linker derivatives having modified non-natural amino acids are, in this specification, Provided and described. In one embodiment, carbonyl, dicarbonyl, alkyne, Cycloalkynes, azides, oximes, or at least having a hydroxylamine group Drastatin linka containing one non-natural amino acid, or a modified non-natural amino acid. The derivative includes at least one post-translational modification at a specific position on the polypeptide. In some embodiments, the post-translational modification is performed by a cellular mechanism (e.g., globulin). Lycosylation, acetylation, acylation, lipid modification, palmitoylation, palmitic acid addition, ly It occurs via (oxidation, glycosphingolipid binding modification, etc.). In most cases, it is based on the cellular mechanisms described above. Translational modifications, or post-translational modifications, occur at naturally occurring amino acid sites on polypeptides. However, in some embodiments, translational modifications based on the above cellular mechanism, or translation Post-modification occurs at non-natural amino acid sites on polypeptides.

[0521] In another embodiment, the above post-translational modification does not utilize a cellular mechanism, but instead , the first reactive group (ketone, aldehyde, acetal, hemiacetal, alkyne, cyclo Alkynes, azides, oximes, or non-natural amino acids containing hydroxylamine functional groups at least one unnatural amino acid having (no acids, but not limited to these) In contrast, molecules having a second reactive group (polymers, water-soluble polymers, polyethylene glyco) Derivatives of proteins, second proteins or polypeptides or polypeptide analogs, antibodies or antibody fragments, and any combination thereof, as described herein by chemical treatment Alternatively, by using the appropriate reactant for the specific reactive group mentioned above, Sensory properties are imparted. In one embodiment, the above-mentioned translational or post-translational modifications are This is performed in vivo in eukaryotic or non-eukaryotic cells. In one embodiment, the above Post-translational modification occurs in vivo without utilizing cellular mechanisms. , at least one of the above translationally modified or post-translationally modified non-natural amino acids The method includes the production, purification, characterization, and use of the above-mentioned drastatin linker derivatives contained herein. Born.

[0522] Furthermore, the scope of the methods, compositions, strategies, and techniques described herein includes, To produce any of the post-translational modifications of the molecule, the polypeptide is part of the (carb) Nyl or dicarbonyl group, oxime group, alkyne, cycloalkyne, azide, hydro Contains xylamine groups, or masked or protected forms thereof. )The reagents may also react with the drastatin linker derivative.In one embodiment, The post-translationally modified drastatin linker derivative obtained as an effect contains at least one O It contains an oxime group. And the resulting modified oxime-containing drastatin phosphate The ker derivative may be subsequently modified. In this embodiment, the above-mentioned drasta The above reagents, capable of any post-translational modification of tin linker derivatives, are prepared, purified, and characterized. This also includes instructions on how to use it.

[0523] In one embodiment, the polypeptide or non-natural amino acid-linked drastatin induction described above. The body, by one host cell, has at least one translational modification made in vivo, This includes post-translational modifications, which are not typically performed by a different host cell species. In one embodiment, the polypeptide is produced by eukaryotic cells in vivo. It includes at least one translational modifier or post-translational modifier, and the translational modifier or translation Post-modification is not usually performed by non-eukaryotic cells. Translational modifications, or post-translational modifications... Examples include glycosylation, acetylation, acylation, fatty modification, palmitoylation, and palmi Examples include, but are not limited to, tinification, phosphorylation, and glycosphingolipid binding modification. In one embodiment, the above-mentioned translation-time or post-translation modification is GlcNAc-aspa Laginate bond (oligosaccharide is (GlcNAc-Man)2-Man-GlcNAc-Glc This includes, but is not limited to, cases containing NaAc, etc., and is used in relation to aspartic acid. It includes the bonding of oligosaccharides. In another embodiment, the above-mentioned post-translational modification or modification The adjectives are GalNAc-serine, GalNAc-threonine, GlcNAc-serine or GlcNAc-Threonine linkage to serine or threonine, oligosaccharide (Ga Examples include l-GalNAc and Gal-GlcNAc, but are not limited to these. ) includes binding. In one embodiment, the protein or polypeptide is secretory Column or localized sequence, epitope tag, FLAG tag, polyhistidine tag, and / Alternatively, it may include GST fusion, etc. Furthermore, in this embodiment, at least one of the above The polypeptides containing translational or post-translational modifications are manufactured, purified, and characterized. Methods of use are included. In another embodiment, glycosylated non-natural amino acids The lipeptides are produced in a non-glycosylated form. The nonglycosylated form of natural amino acids is obtained by the following steps, or any combination thereof. It may be manufactured by a method including: separated or substantially purified Alternatively, oligosaccharides derived from unpurified, glycosylated, non-natural amino acid polypeptides. Chemical or enzymatic removal of sugar groups; the above non-natural amino acid polypeptides are not glycosylated. The host (such a host is modified so as not to glycosylate the above polypeptide) Non-natural amino acids in (including mutated or altered prokaryotes or eukaryotes) Production of; or, the above non-natural amino acid polypeptide is usually glycated with the above polypeptide. Glycosylation inhibitors for polypeptides produced by cosylated eukaryotes into cell culture media Introduction of quality. Furthermore, in this specification, normally glycosylated non-natural amino acid polypeptides The above non-glycosylated forms of cydo are described (usually glycosylated, by When naturally occurring polypeptides are produced in a glycosylated state, glycosylation (This refers to polypeptides that are likely to be glycosylated.) Naturally, they are usually glycosylated non-polypeptides. Natural amino acid polypeptides (or any polypeptide actually described herein) The above non-glycosylated forms include unpurified forms, substantially purified forms, or fractions. They may be in separate forms.

[0524] In one embodiment, the non-natural amino acid polypeptide is used in the presence of a reaction accelerator. It includes one post-translational modification, and the post-translational modification is stoichiometric, stoichiometric, or It is almost stoichiometric. In another embodiment, the polypeptide is reacted in the presence of a reaction accelerator. Below, the reagent of chemical formula (XIX) is brought into contact with the reagent. In another embodiment, the reaction accelerator is brought into contact with the reagent of chemical formula (XIX). The following are selected from the group:

[0525] [ka]

[0526] (A. Chemical synthesis of non-natural amino acid-bonded drastatin derivatives: bonded oxime-containing (Lastatin derivatives) Non-natural amino acid drastatin-bonded derivatives containing oxime groups are reactive carbo Nyl group, or dicarbonyl group (ketone, aldehyde, or similarly reactive) Reactions using various reagents containing other groups (but not limited to these) This makes it possible to form new unnatural amino acids having new oxime groups. The xime exchange reaction enables further functionalization of drastatin-bound derivatives. The original drastatin bond derivative containing an oxime group has an oxime bond that polypeptides amino acids. As long as it is stable under the conditions necessary for incorporation into the cido (e.g., in vivo as described herein), It will be useful in terms of its capabilities (in vitro, chemical synthesis methods).

[0527] Therefore, in some embodiments described herein, oxime group, oxime-like group (It has similar reactivity to the oxime group and is structurally similar to the oxime group), Mass A protected oxime group (which can be immediately converted to an oxime group), or a protected oxime group. Unnatural side chains having a group (which, when deprotected, exhibits similar reactivity to an oxime group) There are amino acid drastatin-bound derivatives.

[0528] Such non-natural amino acid drastatin-bonded derivatives include those with chemical formula (VIII) and Examples include drastatin-bonded derivatives having the structure of (IX).

[0529] [ka]

[0530] (In the formula, A is optional, and if present, it may be a lower alkylene, substituted lower alkylene, or lower alkylene. Roalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, Alkynylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene Len, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene N, substituted heteroarylene, alkalilene, substituted alkalilene, aralkylene, or It is a substituted aralkylene, B is optional and, if present, may be a lower alkylene, a substituted lower alkylene, or a lower alkylene. Kenylene, substituted lower alkenylene, lower heteroalkylene, substituted lower heteroalkylene, -O-, -O-(alkylene, or substituted alkylene)-, -S-, -S-(alkylene (N, or substitution alkylene)-, -S(O) k -(where k is 1, 2, or 3 (There is), -S(O) k (Alkylene, or substituted alkylene)-, -C(O)-, -C (O)-(alkylene, or substituted alkylene)-, -C(S)-, -C(S)-(A Alkilen, or substituted alkylene)-, -N(R')-, -NR'-(alkylene, For example, substitution alkylene)-, C(O)N(R')-, -CON(R')-(alkylene, Alternatively, substitution alkylene)-, -CSN(R')-, -CSN(R')-(alkylene, Alternatively, (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')2N(R')-N A linker selected from the group consisting of (R')- (where each R' is independently H, Alkyl, or substituted alkyl, R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl. can be, R1 is H, amino protecting group, resin, at least one amino acid, polypeptide, or , it is a polynucleotide, R2 is OH, ester protecting group, resin, at least one amino acid, polypeptide, or is a polynucleotide, R3 and R4 are independently H, halogen, lower alkyl, or substituted lower alkyl. It is either a cycloalkyl group, or R3 and R4, or two R3 groups are cycloalkyl groups. Kill or, optionally, heterocycloalkyl groups may be formed. Z has the following structure:

[0531] [ka]

[0532] R5 is H, COR8, an alkyl group with 1 to 6 carbon atoms, or a thiazole. R8 is OH, R6 is OH or H. Ar is phenyl or pyridine. R7 is an alkyl group with 1 to 6 carbon atoms, or hydrogen. L stands for alkylene-, -alkylene-C(O)-, -(alkylene-O) n -Archile Hmm, -(Alkilen-O) n -Alkylene-C(O)-, -(Alkylene-O) n -( CH2) n’ -NHC(O)-(CH2) n’’ -C(Me)2-SS-(CH2) n ’’’ -NHC(O)-(alkylene-O) n’’’’ -Alkilen,-(Alkilen- O) n -alkylene-W-, -alkylene-C(O)-W-, -(alkylene-O) n - Alkylene-U-alkylene-C(O)-, and -(alkylene-O) n -Archile A linker selected from the group consisting of n-U-alkylenes, W has the following structure:

[0533] [ka]

[0534] U has the following structure:

[0535] [ka]

[0536] n, n', n'', n''', and n'''' are each independently one or more integers. It is a number, or, An active metabolite or an activatably acceptable prodrug, or a solvate thereof be.

[0537] In some embodiments of the compounds of chemical formulas (VIII) and (IX), R5 is thiazo It is a compound of chemical formulas (VIII) and (IX), in some embodiments, R6 is H. In some embodiments of compounds of chemical formulas (VIII) and (IX), A r is phenyl. In some embodiments of the compounds of chemical formulas (VIII) and (IX) And R7 is methyl. Certain implementations of compounds of chemical formulas (VIII) and (IX) In this state, n is an integer between 0 and 20. Compounds of chemical formulas (VIII) and (IX) In one embodiment, n is an integer from 0 to 10. Chemical formulas (VIII) and (I In one embodiment of compound X), n is an integer between 0 and 5.

[0538] In some embodiments of the compounds of chemical formulas (VIII) and (IX), R5 is thiazo It is a compound of chemical formulas (VIII) and (IX), in some embodiments, R5 is hydrogen. In some embodiments of the compounds of chemical formulas (VIII) and (IX), R5 contains methyl, ethyl, propyl...

Claims

1. Compounds represented by formula (VIII) or (IX), or their pharmaceutically acceptable solvates: 【Chemistry 1】 During the ceremony, A is optional and, if present, is a lower alkylene, substituted lower alkylene, lower cycloalkylene, substituted lower cycloalkylene, lower alkenylene, substituted lower alkenylene, alkylylene, lower heteroalkylene, substituted heteroalkylene, lower heterocycloalkylene, substituted lower heterocycloalkylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkalilene, substituted alkalilene, aralkylene, or substituted aralkylene. B is optional and, if present, may be 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 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 A linker selected from the group consisting of -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 an OH group, an ester protecting group, a resin, at least one amino acid, a polypeptide, or a polynucleotide. 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 groups may optionally form cycloalkyl or heterocycloalkyl, Z has the following structure: 【Chemistry 2】 R ５ H, COR ８ , an alkyl group having 1 to 6 carbon atoms, or a thiazole, R ８ It is OH, R ６ is OH or H, Ar is phenyl or pyridine. R ７ This is an alkyl group having 1 to 6 carbon atoms, or hydrogen. L represents -alkylene-, -alkylene-C(O)-, and -(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 A linker selected from the group consisting of -alkylene-U-alkylene-, W has the following structure: 【Transformation 3】 U has the following structure: 【Chemistry 4】 n, n', n'', n''', and n'''' are independent integers greater than or equal to 1.

2. The compound is represented by formula (VIII), The compound according to claim 1.

3. R １ and R ２ It is an antibody. The compound according to claim 1 or 2.

4. The antibody in question is trastuzumab. The compound according to claim 3.

5. A is optional and, if present, is an alkylene with 1 to 8 carbon atoms, a heteroalkylene with 1 to 8 carbon atoms, or an arylene with 5 to 20 ring atoms. The compound according to any one of claims 1 to 4.

6. A is an arylene with 5 to 20 ring atoms. The compound according to claim 5.

7. A is phenylene. The compound according to claim 6.

8. B is optional and, if present, can be an alkylene with 1 to 8 carbon atoms, a substituted alkylene with 1 to 8 carbon atoms, -O-, -O-(alkylene with 1 to 10 carbon atoms)-, -S-, -S-(alkylene with 1 to 10 carbon atoms)-, -C(O)-, -C(O)-(alkylene with 1 to 10 carbon atoms, or a substituted alkylene with 1 to 10 carbon atoms)-, -N(R')-, -NR'-(alkylene with 1 to 1 carbon atoms) The linker is selected from the group consisting of -0 alkylene-, -C(O)N(R')-, -CON(R')-(alkylene with 1 to 10 carbon atoms)-, -N(R')CO-(alkylene with 1 to 10 carbon atoms)-, -C(R')=N-, and -C(R')=N-N(R')- (wherein R' is independently H or an alkyl group with 1 to 10 carbon atoms). The compound according to any one of claims 1 to 7.

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

10. B is either absent or consists of alkylenes with 1 to 8 carbon atoms, -C(O)-(alkylenes with 1 to 10 carbon atoms), and -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 - A linker selected from the group consisting of The compound according to claim 9.

11. B does not exist. The compound according to claim 10.

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

13. R is H, an alkyl group having 1 to 6 carbon atoms, or a cycloalkyl group having 3 to 6 carbon atoms. The compound according to claim 12.

14. R is -CH ３ , -CH(CH ３ ) ２ , or cyclopropyl, The compound according to claim 13.

15. R ３ H is The compound according to any one of claims 1 to 14.

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

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

18. R ６ H is 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. R ７ It is methyl, The compound according to any one of claims 1 to 19.

21. L is -(alkylene-O with 1 to 3 carbon atoms) ｎ - Alkylenes with 1 to 3 carbon atoms - The compound according to any one of claims 1 to 20.

22. L is - (CH ２ CH ２ -O) ｎ -CH ２ CH ２ - is, The compound according to 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 - and R １ and R ２ This is an antibody, and this antibody is trastuzumab. The compound according to any one of claims 1 to 23.

25. A method for derivatizing the drastatin analog shown in formula (I), The method includes contacting the drastatin analog with the reagent of formula (XXXVII), Equation (I) corresponds to the following: 【Transformation 5】 During the ceremony, Z has the following structure: 【Transformation 6】 R ５ H, COR ８ , an alkyl group having 1 to 6 carbon atoms, or a thiazole, R ８ This is OH, or -NH- (alkylene-O) ｎ -NH ２ And, R ６ is OH or H, Ar is phenyl or pyridine. R ７ This is an alkyl group having 1 to 6 carbon atoms, or hydrogen. Y is NH ２ -O- or methyl, L represents -alkylene-, -alkylene-C(O)-, and -(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 A linker selected from the group consisting of -alkylene-U-alkylene-, W has the following structure: 【Transformation 7】 U has the following structure: 【Transformation 8】 Or, L is absent, Y is methyl, and R ５ is COR ８ and R ８ is -NH(alkylene-O) ｎ -NH ２ and Each of n, n', n'', n''', and n'''' is an independent integer greater than or equal to 1; Formula (XXXVII) corresponds to the following method: 【Chemistry 9】 During the ceremony, A is optional and, if present, is a lower alkylene, substituted lower alkylene, lower alkenylene, substituted lower alkenylene, arylene, substituted arylene, heteroarylene, substituted heteroarylene, alkalilene, substituted alkalilene, aralkylene, or substituted aralkylene. 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 -(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 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')- and is a linker selected from the group consisting of (where each R' is independently H, alkyl, or substituted alkyl), K is one of the following: 【Chemistry 10】 R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl. R １ This is H, an amino protecting group, a resin, at least one amino acid, or a polynucleotide. R ２ This is an OH group, an ester protecting group, a resin, at least one amino acid, or a polynucleotide. R ３ and R ４ These are independently H, halogen, lower alkyl, or substituted lower alkyl, or R ３ and R ４ , or two R's ３ The group may optionally form a cycloalkyl or heterocycloalkyl group.

26. The derivatized drastatin analog is an amino acid containing at least one oxime having the structure of formula (VIII) or formula (IX). The method according to claim 25: 【Chemistry 11】

27. The derivatized drastatin analog has the structure shown in formula (VIII), The method according to claim 26.

28. K is one of the following: The method according to any one of claims 25 to 27: 【Chemistry 12】

29. In an aqueous solution under weakly acidic conditions, the drastatin analog is brought into contact with the reagent of formula (XXXVII). The method according to any one of claims 25 to 28.

30. 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 29.

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