Process for production of peptide thioester
A process for chemically converting a peptide chain into a peptide thioester includes, when a —C(═X)—R 1 group is introduced to the thiol group of the cysteine residue and then the resulting peptide is reacted with a compound having a leaving group represented by the formula: —NH—C(═Y)NHR 3 in an organic solvent, the —NH—C(═Y)NHR 3 group binds via addition reaction to the carboxyl group of the N-terminal-side peptide bond of the cysteine residue, whereby the peptide bond is cleaved and the C-terminal-side peptide fragment is cut off. Further, when the resulting peptide chain having the —NH—C(═Y)NHR 3 group is reacted with a thiol in a buffer solution, a thiol exchange reaction occurs, namely, the thiol group of the thiol binds to the carbonyl carbon to which the —NH—C(═Y)NHR 3 group has bound, whereby the —NH—C(═Y)NHR 3 group is eliminated.
1. A process for producing a peptide thioester comprising the following steps (a) to (c):
(a) a step of producing a first intermediate by reacting a compound A represented by the following formula (I) with a thiol group of a cysteine residue to eliminate R 2 in a peptide chain having the cysteine residue:
wherein X is a sulfur atom or an oxygen atom, and R 1 and R 2 are leaving groups;
(b) a step of reacting a compound B represented by the following formula (II) with said first intermediate in an organic solvent to add a —NH—C(═Y)NHR 3 group to a carboxyl group forming a peptide bond between the cysteine residue and an amino acid adjacent to an N-terminal-side of said cysteine residue, and cleaving said peptide bond, thereby obtaining a peptide fragment from the N-terminal-side closer to the N-terminal-side than the cleaved peptide bond as a second intermediate:
wherein Y is an oxygen atom, a sulfur atom or an NH group and R 3 is a hydrogen atom, an acyl group or an alkoxycarbonyl group; and
(c) a step of thioesterifying a C-terminal of the second intermediate by reacting thiol with the second intermediate to exchange the —NH—C(═Y)NHR 3 group at the C-terminal for the thiol group.
2. The process according to claim 1 , wherein X is the sulfur atom.
3. The process according to claim 1 , wherein R 1 is a —O—C 6 aryl group.
4. The process according to claim 1 , wherein R 2 is a halogen atom, or a substituted or unsubstituted —S—C 6-10 aryl group.
5. The process according to claim 1 , wherein Y is an NH group.
6. The process according to claim 1 , wherein R 3 is an acetyl group.
7. The process according claim 1 , wherein the thiol in the step (c) is thiol represented by the following formula (III):
R 4 —SH (Formula III)
wherein R 4 is any one selected from a substituted or unsubstituted benzyl group, a substituted or unsubstituted aryl group, and a substituted or unsubstituted alkyl group.
8. The process according to claim 1 , wherein the peptide chain is a recombinant protein.
9. The process according to claim 1 , wherein the peptide chain is the recombinant protein comprising a tag for purification.
10. The process according to claim 2 , wherein R 1 is a —O—C 6 aryl group.
11. The process according to claim 2 , wherein R 2 is a halogen atom, or a substituted or unsubstituted —S—C 6-10 aryl group.
12. The process according to claim 2 , wherein Y is an NH group.
13. The process according to claim 2 , wherein R 3 is an acetyl group.
14. The process according to claim 2 , wherein the thiol in the step (c) is thiol represented by the following formula (III):
R 4 —SH (Formula III)
wherein R 4 is any one selected from a substituted or unsubstituted benzyl group, a substituted or unsubstituted aryl group, and a substituted or unsubstituted alkyl group.
15. The process according to claim 2 , wherein the peptide chain is a recombinant protein.