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 an unprotected first peptide segment to a solid phase via a linker, wherein said 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 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;
• c) ligating a second unprotected peptide segment to said first peptide segment bound to said solid phase, 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 C-terminal thioacid of said solid phase-bound peptide to an activated thioester of the formula —COSR,
• e) repeating steps c) and d) with a third unprotected peptide segment;
• and
• f) optionally repeating steps c) and d) with additional unprotected peptide segments.

A method of preparing an assembled peptide comprising:

• a) binding a 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 second peptide segment to said first peptide segment bound to said solid phase, wherein said second peptide segment comprises a cysteine at its N-terminus and a thioester at its C-terminus, and wherein said N-terminal cysteine of said second peptide segment is capable of binding to a protecting group;
• and wherein said C-terminal thioester of said second peptide segments binds to said N-terminal cysteine of solid phase-bound first peptide segment to form a solid phase-bound peptide comprising protected Cysteine at its N-terminus;
• c) removing said protecting group;
• d) repeating steps b) and c) at least once with another peptide segment;
• e) ligating a final peptide segment to said solid phase-bound peptide, wherein said final peptide segment is unprotected and comprises a C-terminal thioester;
• f) cleaving said cleavable moiety to release an assembled peptide from the solid phase.

A kit for preparing assembled polypeptides comprising:

• a) a first unprotected peptide segment, comprising a thioester at its C-terminus and an N-terminus, wherein said first unprotected peptide segment is bound to a solid phase via a linker comprising a cleavable moiety;
• b) a set of second unprotected peptide segments, each comprising a thioacid at their C-termini and a cysteine at their N-termini, wherein each of said second unprotected peptide segments have the same number of amino acids;
• and
• c) one or more sets of different unprotected peptide segments, each comprising a thioester at their C-termini and a cysteine residue at their N-termini, wherein each of the members of each set have the same number of amino acids.

An apparatus for producing assembled polypeptides, comprising:

• a) a solid support, having bound thereto a first unprotected peptide having a thioester at its C-terminus and a cleavable linker at its N-terminus, wherein said unprotected peptide is bound to said solid support via a linker;
• b) a set of second unprotected peptides, each comprising a thioacid at their C-termini and a cysteine residue at their N-termini;
• and
• c) one or more sets of different unprotected peptides, each comprising a thioacid at their C-termini and a cysteine residue at their N-termini.

A method of producing polypeptide libraries of solid phase sequentially ligated assembled peptides, comprising:

• a) covalently binding a set of unprotected first peptide segments to a solid support via a linker, wherein said linker comprises a cleavable moiety stable under ligating conditions and said unprotected first peptide segments are each bound to said linker at its N-terminus and has a thioester at its C-terminus;
• b) introducing a set of second unprotected peptide segments, wherein each of said second segments each comprise a cysteine residue at its N-terminus and a thioacid at its C-terminus, under conditions suitable to permit ligation between said first unprotected peptide segments and said second unprotected peptide segments to form a natively ligated polypeptide bound to said solid support, wherein said bound polypeptide comprises a thioacid at its C-terminus;
• c) converting said thioacid to a thioester;
• d) optionally repeating steps b) and c) with additional sets of unprotected peptide segments.