Methods and compositions for display of macrocyclic peptides

A macrocyclic peptide library display system, comprising at least one 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,
• vi. (AA)p is a C-terminal amino acid or peptide sequence;
• and
• vii. wherein at least one of (AA)p and (AA)m comprises an amino acid sequence of a polypeptide for presentation of the macrocyclic peptide on an outer surface of a cell or phage particle,
• wherein the functional group FG1, and whenever present, FG2, react with the side-chain sulfhydryl group (—SH) of the cysteine (Cys) residue(s), thereby producing the macrocyclic peptide, and further wherein the at least one nucleic acid molecule is incorporated into an expression system that allows for the incorporation of the non-canonical amino acid Z or Z2 into an expressed polypeptide.