Abstract
Serine/Threonine ligation (STL) has emerged as an alternative tool for protein chemical synthesis, bioconjugations as well as macrocyclization of peptides of various sizes. Owning to the high abundance of Ser/Thr residues in natural peptides and proteins, STL is expected to find a wide range of applications in chemical biology research. Herein, we have fully investigated the compatibility of the STL strategy for X-Ser/Thr ligation sites, where X is any of the 20 naturally occurring amino acids. Our studies have shown that 17 amino acids are suitable for ligation, while Asp, Glu, and Lys are not compatible. Among the working 17 C-terminal amino acids, the retarded reaction resulted from the bulky β-branched amino acid (Thr, Val, and Ile) is not seen under the current ligation condition. We have also investigated the chemoselectivity involving the amino group of the internal lysine which may compete with the N-terminal Ser/Thr for reaction with the C-terminal salicylaldehyde (SAL) ester aldehyde group. The result suggested that the free internal amino group does not adversely slow down the ligation rate.
Highlights
Chemical methods enabling merger of two fully side chain unprotected peptide segments at the termini, termed peptide ligation, provide access to complex, long peptides and even proteins beyond the limit of solid phase peptide synthesis (SPPS) (Kent, 2009)
In pursuing the development of the easy-handling chemoselective peptide ligation, we have recently introduced a serine/threonine ligation (STL) strategy, enabling the peptide ligation of side chain unprotected peptide segments with the generation of natural peptidic bond (Xaa-Ser/Thr) at the ligation site (Li et al, 2010)
The leftover of K2 was much less than K0, which excluded that possibility that more K2 was accidently added than K0 peptide to the reaction to cause the more K2 ligated product
Summary
Chemical methods enabling merger of two fully side chain unprotected peptide segments at the termini, termed peptide ligation, provide access to complex, long peptides and even proteins beyond the limit of solid phase peptide synthesis (SPPS) (Kent, 2009). Ever since the introduction of native chemical ligation (NCL) (Dawson et al, 1994; Tam et al, 1995; Dawson and Kent, 2000), protein chemical synthesis has become reachable and provided a general strategy for the flexible and precise incorporation of natural or unnatural elements into a protein molecule. The realization of NCL lies in the super nucleophilicity of the Nterminal cysteine to mediate a chemoselective peptide ligation, thereby enabling merger of two side chain unprotected peptide segments with the generation of the natural Xaa-Cys at the ligation site. Examples include the use of ß-mercaptophenylalanine (Crich and Banerjee, 2006), γ-mercaptovaline/penicillamine (Chen et al, 2008; Haase et al, 2008), ß-mercaptoleucine (Harpaz et al, 2010; Tan et al, 2010), γ-mercaptothreonine (Chen et al, 2010), δ-mercaptolysine
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