Realizing serine/threonine ligation: scope and limitations and mechanistic implication thereof

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.

Serine/threonine ligation for the chemical synthesis of proteins

“…Interestingly, the ligation at the bulky b branched amino acid Thr behaved similarly to that at the least hindered amino acid Gly. Accordingly, we have classified the C-terminal amino acids into three groups: fast reaction with Ala, Gly, Ser, Glu, Thr, Phe and Cys(StBu), modest reaction with Val, Leu, Ile, Asn Phe, Met and Tyr, and slow reaction with Arg, Trp and Pro [26 ]. The same trend was observed in serine/threonine ligation-mediated peptide cyclization.…”

“…Next, we studied the effect of an unprotected lysine on the serine/threonine ligation [26 ]. The first step of Ser/Thr ligation is the imine formation between the amino group of the N-terminal serine or threonine residue and the aldehyde group of the C-terminal SAL ester; thus it is likely that the internal Lys would compete for the imine formation, thereby slowing down or inhibiting the ligation, even though it could not form the productive oxazoline intermediate.…”

Chemical and Biological Tools for the Preparation of Modified Histone Proteins

“…The ability to carry out desulfurization, when combined with non-native 1,2-aminothiol derivatives of amino acids, has allowed the expansion of ligation sites to many more residues, reviewed in [101]. For example, penicillamine is a β-thiol derivative of valine that is commercially available in Fmoc- and Boc-protected forms, including as the thiazolidine derivative required for complex ligation schemes discussed in section 4.4.…”

Use of Serine/Threonine Ligation for the Total Chemical Synthesis of HMGA1a Protein with Site‐Specific Lysine Acetylations

“…[13][14][15] Expectedly, STL can provide wide synthetic design possibilities for protein synthesis, owing to the significant abundance of serine and threonine in natural proteins (12.7 %). [16] The good compatibility of STL with variable Cterminal residues (for the peptide salicylaldehyde ester fragment), [17] the ready availability of requisite peptide salicylaldehyde (SAL) esters via both Fmoc-SPPS [14] and Boc-SPPS [18] , and mild reaction conditions render STL an useful tool for construction of proteins bearing PTMs. To achieve more complex protein targets in convergent manner, both the C-to-N [14,19] and N-to-C [20] sequential STL have been developed, relying on the temporal protection of the N-terminus of the middle fragment and semicarbazone as the SAL ester surrogate respectively.…”