Patent ID: 7781488
Filing Date: 2010-08-24
Classification: C07K

Abstract:
1. A method for preparing an oligopeptide or polypeptide product, comprising: (a) ligating in a first reaction, first and second oligopeptides each having an N-terminus and a C-terminus, wherein said first oligopeptide comprises a C-terminus having a thioester group, and an N-terminus that is substantially unreactive with said thioester group, wherein said second oligopeptide comprises an N-terminus having an unprotected cysteine, an unprotected 1,2-amino thiol, or an unprotected 1,3-amino thiol, and a C-terminus having an inactive thioester precursor, and wherein said ligating forms a first oligopeptide ligation product comprising a C-terminus having an inactive thioester precursor, and an N-terminus that is substantially unreactive with said thioester group; (b) ligating in a second reaction, third and fourth oligopeptides each having an N-terminus and a C-terminus, wherein said third oligopeptide comprises an N-terminus having a protected cysteine, a protected 1,2-amino thiol, or a protected 1,3-amino thiol, and a C-terminus comprising a thioester, wherein said fourth oligopeptide comprises an N-terminus having an unprotected cysteine, an unprotected 1,2-amino thiol, or an unprotected 1,3-amino thiol, and a C-terminus that is substantially unreactive with said unprotected cysteine, said unprotected 1,2-amino thiol, or said unprotected 1,3-amino thiol, and wherein said ligating forms a second oligopeptide ligation product comprising an N-terminus having a protected cysteine, a protected 1,2-amino thiol, or a protected 1,3-amino thiol, and a C-terminus that is substantially unreactive with said unprotected cysteine, said unprotected 1,2-amino thiol, or said unprotected 1,3-amino thiol; (c) transforming said inactive thioester precursor of said first oligopeptide ligation product of step (a) into a thioester to form a third oligopeptide ligation product having a C-terminus comprising a thioester, and an N-terminus that is substantially unreactive with said thioester group; (d) deprotecting said N-terminus of said second oligopeptide ligation product of step (b) to form a fourth ligation product comprising an N-terminus having an unprotected cysteine, an unprotected 1,2-amino thiol, or an unprotected 1,3-amino thiol, and a C-terminus that is substantially unreactive with said unprotected cysteine, said unprotected 1,2-amino thiol, or said unprotected 1,3-amino thiol; and (e) ligating said third oligopeptide ligation product of step (c) to said fourth oligopeptide ligation product of step (d) to form a fifth oligopeptide ligation product comprising an N-terminus that is substantially unreactive with an oligopeptide bearing a C-terminal thioester group, and a C-terminus that is substantially unreactive with an oligopeptide bearing an unprotected N-terminal cysteine, an unprotected 1,2-amino thiol, or an unprotected 1,3-amino thiol, wherein: said inactive thioester precursor at said C-terminus is defined by the formula: wherein: Xaa m is an integer from 2 to 200; n is an integer from 1 to 10; R is an amino acid side chain; R′ is a member selected from the group consisting of a sulfur protecting group and —SR″; R″ is a member selected from the group consisting of C Y is an electron withdrawing group selected from the group consisting of R each R p is an integer from 1 to 2; R each R when two R