An efficient method has been developed for the salicylaldehyde ester-mediated ligation of unprotected peptides at serine (Ser) or threonine (Thr) residues. The utility of this peptide ligation approach has been demonstrated through the convergent syntheses of two therapeutic peptides--ovine-corticoliberin and Forteo--and the human erythrocyte acylphosphatase protein (∼11 kDa). The requisite peptide salicylaldehyde ester precursor is prepared in an epimerization-free manner via Fmoc-solid-phase peptide synthesis.andmark advances in the field of synthetic protein chemistry have enabled the preparation of complex, homogeneous proteins (1-3), including those that carry specific posttranslational modifications (PTMs) (4-11). Of particular significance, Danishefsky and coworkers have obtained glycosylated human folliclestimulating hormone (7) and erythropoietin (11), solely through chemical synthesis. In general, protein synthesis consists of two key phases: (i) solid-phase peptide synthesis (SPPS) allows for the generation of moderately sized peptide segments (up to ∼30 amino acids) (12); and (ii) chemical ligation serves to chemoselectively join these synthetic peptide fragments (13-16). In the 1970s, Kemp and coworkers conceptually devised a promising peptide ligation strategy, involving prior capture followed by acyl transfer, which has laid the foundation for the development of chemical ligation in the convergent peptide synthesis (17)(18)(19)(20)(21)(22). A milestone advance in the field was the discovery, by Kent and coworkers, of native chemical ligation (NCL) (13), in which a C-terminal peptide thioester and an N-terminal cysteine-containing peptide--both in side-chain unprotected forms--are selectively coupled to generate a natural peptidic linkage (Xaa-Cys) at the site of ligation. Because the Kent NCL approach exploits the unique nucleophilicity of the cysteine thiol group, the relatively low abundance of cysteine residues in natural proteins can pose a significant challenge to NCL-based protein synthesis efforts. In an effort to expand the scope of NCL, a number of research groups have developed variants, wherein β-or γ-thiol containing amino acids are temporarily installed on the peptide N terminus, thus promoting NCL-like ligation. Subsequent desulfurization serves to restore the natural amino acid at the site of ligation (23)(24)(25)(26). Whereas this NCLdesulfurization strategy has been widely used in protein synthesis, a menu of complementary thiol-independent ligation approaches (27-34) may offer new opportunities for convergent protein synthesis. Nevertheless, these thiol-independent ligations require the installation of unique reaction functionalities, often tediously, on both sides of the N-terminal peptide segment and the C-terminal peptide segment to induce a chemoselective coupling reaction.Along these lines, our laboratory has been pursuing the development of methods for ligation at N-terminal serine and threonine residues to generate natural Xaa-Ser/Thr linkages directly, using natural serin...

A total synthesis of daptomycin, the first natural product antibiotic launched in a generation, was achieved. This convergent synthesis relies on an efficient macrocyclization via a serine ligation to assemble the 31-membered cyclic depsipeptide. The difficult esterification by the nonproteinogenic amino acid kynurenine was accomplished via the esterification of a threonine residue by a suitably protected Trp ester, followed by ozonolysis. This synthesis provides a foundation and framework to prepare varied analogues of daptomycin to establish its structure-activity profile.

A serine/threonine-based chemoselective ligation method is described. It uses an O-salicylaldehyde ester at the C-terminus, reacting with N-terminal serine or threonine to realize peptide ligations. The utility of the O-salicylaldehyde ester enables the rapid coupling reaction and the production of an N,O-benzylidene acetal intermediate, which is readily converted into natural peptidic linkages (Xaa-Ser/Thr) at the ligation site.

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.