Since the pioneering days of Bruce Merrifield in solid phase peptide synthesis, in the early 60’s, the field of peptide synthesis has considerably evolved; from tBOC to FMOC chemistry. The peptide chemist is now able to obtain fairly long peptides (upto 150 residues in length) by straight chemical synthesis. However, not every peptide is amenable to be assembled by direct chemical synthesis. The idea of being able to ligate native peptides arose from the work of Stephen Kent in 1994 and James Tam and his colleagues in 1995.
The concept lies on being able to use unique functionalities at both the carboxy and amino end of two fully deprotected peptides (native peptides). In the case of Kent’s method, he makes use of an N-terminal Cys that functions as the inside thioester capture, in effect forming a new thioester via transesterification; the covalent S-acyl intermediate goes through S- to N- acyl migration forming a peptide bond via five membered ring intermediate.
Tam’s method was coined chemoselective capture activation ligation via an azothiolidine intermediate. In any event all ligation methods are able to bring the reactive sites of the two segments in close proximity, resulting in intramolecular acyl transfer to establish the peptide bond at the ligation site; that is to say the design brings close to each other the only two possible reactive sites among the 2 peptide fragments, a very ingenious design indeed.
Most ligation procedures take place under aqueous conditions at pH 7-8, in the presence of 6-7M guanidinium chloride; in order to prevent, N,S-bisacylated product and disulfide formation, reducing conditions are recommended (thiophenol addition). In summary we have described briefly the potential benefits of native chemical ligation; whereas a number of long peptides/proteins, such us IL8 has been accomplished it must be said, that considerable effort and expertise may be needed to master the methodologies for its efficient use.
An excellent review of chemical ligation is given below:
- J.P.Tam, Q.Yu, Z.Miao, Biopolymers 1999, 51, 311