Solid phase native chemical ligation of unprotected or n-terminal cysteine protected peptides in aqueous solution

A method of producing an assembled peptide comprising:

• a) binding a partially or completely unprotected first peptide segment to a solid phase via a linker, wherein said partially or completely unprotected first peptide segment comprises an N-terminus and a thioacid of the formula —COSH at its C-terminus, wherein said linker comprises a cleavable moiety and said partially or completely unprotected first peptide segment is bound to said linker at said N-terminus;
• b) converting said C-terminal thioacid of said first peptide segment to a thioester of the formula —COSR, wherein R is a straight or branched C1-15 functionalized alkyl group, a C1-15 aromatic structure, or 1 to 4 amino acids or derivatives thereof;
• c) ligating a partially or completely unprotected second peptide segment to said solid phase-bound first peptide segment, wherein said second peptide segment comprises a cysteine at its N-terminus and a thioacid at its C-terminus, and wherein said N-terminal cysteine of said second peptide segment is capable of selectively ligating to said C-terminus of said solid phase-bound first peptide segment to form a solid phase-bound peptide comprising a thioacid at its C-terminus;
• d) converting said thioacid of said phase-bound peptide to a thioester of the formula —COSR to form a solid phase-bound peptide comprising a thioester at its C-terminus;
• e) ligating a partially or completely unprotected third peptide segment to said solid phase-bound peptide, wherein said third peptide segment comprises a cysteine at its N-terminus, to form said assembled peptide;
• and
• f) optionally repeating steps c), d), and e) with additional partially or completely unprotected peptide segments, wherein if said steps c) and d) are repeated, the peptide segment of step e) prior to such repetition comprises a thioacid at its C-terminus.

A method of preparing an assembled peptide comprising:

• a) binding a partially or completely unprotected first peptide segment to a solid phase via a linker, wherein said first peptide segment comprises an N-terminal cysteine and a C-terminal residue capable of binding to said linker, wherein said linker comprises a cleavable moiety and said first peptide segment is bound to said linker at said C-terminal residue;
• b) ligating a partially or completely unprotected second peptide segment to said solid phase-bound first peptide segment, wherein said second peptide segment comprises a thioester at its C-terminus, and wherein said C-terminal thioester of said second peptide segments binds to said N-terminal cysteine of said solid phase-bound first peptide segment to form a solid phase-bound peptide;
• c) removing said protecting groups;
• d) optionally repeating steps b) and c) at least once with another peptide segment, wherein if said steps b) and c) are repeated, the peptide segment of step b) prior to such repetition comprises a cysteine at its N-terminus, said cysteine being capable of binding to a protective group;
• and
• e) cleaving said cleavable moiety to release said assembled peptide from said solid phase.

A method of producing an assembled peptide in the C-terminal to N-terminal direction, said method comprising the steps:

• a) ligating a partially or completely unprotected first peptide segment to a partially or completely unprotected second peptide segment in aqueous solution, said first peptide segment having an unprotected N-terminal cysteine and a C-terminal group bonded to a solid phase via a linker adjoined to said C-terminal group, said second peptide segment having an unprotected C-terminal thioester and optionally a protected N-terminal cysteine;
• b) deprotecting said protected N-terminal cysteine of said second peptide segment, if present, and ligating a third peptide segment having an unprotected C-terminal thioester to said unprotected N-terminal cysteine of said second peptide segment;
• and
• c) recovering said produced assembled peptide.