主权项 |
1. A method for making a macrocyclic peptide, the method comprising:
(a) providing an artificial nucleic acid molecule encoding for a polypeptide of structure:
(AA)m-Z-(AA)n-Cys-(AA)p (I)or(AA)m-Cys-(AA)n-Z-(AA)p (II)or(AA)m-Cys-(AA)n-Z2-(AA)o-Cys-(AA)p (V) wherein:
(i) (AA)m is an N-terminal amino acid or peptide sequence,(ii) Z is a non-canonical amino acid carrying a side-chain functional group FG1, FG1 being a functional group selected from the group consisting of —(CH2)nX, where X is F, Cl, Br, or I and n is an integer number from 1 to 10; —C(O)CH2X, where X is F, Cl, Br, or I; —CH(R′)X, where X is F, Cl, Br, or I; —C(O)CH(R′)X, where X is F, Cl, Br, or I; —OCH2CH2X, where X is F, Cl, Br, or I; —C(O)CH═C═C(R′)(R″); —SO2C(R′)═C(R′)(R″); —C(O)C(R′)═C(R′)(R″); —C(R′)═C(R′)C(O)OR′; —C(R′)═C(R′)C(O)N(R′)(R″); —C(R′)═C(R′)—CN; —C(R′)═C(R′)—NO2; —C≡C—C(O)OR′; —C≡C—C(O)N(R′)(R″); unsubstituted or substituted oxirane; unsubstituted or substituted aziridine; 1,2-oxathiolane 2,2-dioxide; 4-fluoro-1,2-oxathiolane 2,2-dioxide; and 4,4-difluoro-1,2-oxathiolane 2,2-dioxide, where each R′ and R″ is independently H, an aliphatic, a substituted aliphatic, an aryl, or a substituted aryl group,(iii) Z2 is a non-canonical amino acid carrying two side-chain functional groups FG1 and FG2, wherein each of FG1 and FG2 is a functional group independently selected from the group consisting of —(CH2)nX, where X is F, Cl, Br, or I and n is an integer number from 1 to 10; —C(O)CH2X, where X is F, Cl, Br, or I; —CH(R′)X, where X is F, Cl, Br, or I; —C(O)CH(R′)X, where X is F, Cl, Br, or I; —OCH2CH2X, where X is F, Cl, Br, or I; —C(O)CH═C═C(R′)(R″); —SO2C(R′)═C(R′)(R″); —C(O)C(R′)═C(R′)(R″); —C(R′)═C(R′)C(O)OR′; —C(R′)═C(R′)C(O)N(R′)(R″); —C(R′)═C(R′)—CN; —C(R′)═C(R′)—NO2; —C≡C—C(O)OR′; —C≡C—C(O)N(R′)(R″); unsubstituted or substituted oxirane; unsubstituted or substituted aziridine; 1,2-oxathiolane 2,2-dioxide; 4-fluoro-1,2-oxathiolane 2,2-dioxide; and 4,4-difluoro-1,2-oxathiolane 2,2-dioxide, where each R′ and R″ is independently H, an aliphatic, a substituted aliphatic, an aryl, or a substituted aryl group,(iv) (AA)n is a target peptide sequence,(v) (AA)o is a second target peptide sequence, and(vi) (AA)p is a C-terminal amino acid or peptide sequence; (b) introducing the nucleic acid molecule into an expression system and expressing the nucleic acid molecule in the expression system, thereby producing the polypeptide; and (c) allowing the functional group FG1, and whenever present, FG2, to react with the side-chain sulfhydryl group (—SH) of the cysteine (Cys) residue(s), thereby producing the macrocyclic peptide. |