Straightforward Entry to S‑Glycosylated Fmoc-Amino Acids and Their Application to Solid Phase Synthesis of Glycopeptides and Glycopeptidomimetics Daniela Comegna, † Ivan de Paola, † Michele Saviano, ‡ Annarita Del Gatto, † and Laura Zaccaro* ,† † Institute of Biostructures and Bioimaging, National Research Council, Napoli, Italy ‡ Institute of Crystallography, National Research Council, Bari, Italy * S Supporting Information ABSTRACT: Streamlined access to S-glycosylated Fmoc- amino acids was developed. The process provides diverse glycosylated modified amino acids in high yield and stereo- selectivity taking advantage of the in situ generation of a glycosylthiolate obtained from carbohydrate acetates in a few steps. Mild basic conditions make the conjugation reaction compatible with Fmoc-iodo-amino acids. To validate the strategy the glycosylated building blocks were used for SPPS and the unprecedented incorporation of a long thio- oligosaccharide to the peptide chain was demonstrated. D rug discovery based on peptides and proteins is a widely explored area in biomedical research that very often relies on the “peptidomimetic approach”. As a matter of fact, introduction of modified amino acids or higher homologues thereof results in an expanded molecular diversity that can be exploited as a very effective tool in medicinal chemistry. 1 Indeed, peptidomimetics based on β- and γ-amino acids feature enhanced in vivo stability, but can also be of valuable interest for the investigation of new highly ordered architectures. 2,3 Over the past years, the critical role played by the protein glycosylation in biology has been widely unveiled, as witnessed by the involvement of glycoproteins in a wide set of events such as cell adhesion and proliferation, trafficking, cell -cell recognition, inflammation, virulence and host immune re- sponse. 4 Glycosylation also plays a pivotal role in protein folding and proteolytic stability, and it is widely investigated for tuning the biological activity of non-naturally glycosylated peptides and proteins. 5 In this regard, synthetic access to homogeneous natural or modified glycosylated peptides is a relevant topic in organic chemistry and different synthetic conjugation methods have been developed, 6 providing naturally occurring O- and N-linked, and uncommon C- and S-linked, glycopeptides. In particular, the thioglycoside bond is well-known to be chemically and enzymatically more stable than the O- linked counterpart, and it is well tolerated in biological systems because of its isosteric mimicry. 7 To date, several methods for the assembly of S- glycosylated peptides have been reported, 8 but very few of them are compatible with use of Fmoc-protected amino acids. 9 As to the saccharide moiety of the reported glycosylated Fmoc-amino acids and peptides, the known synthetic strategies entail preliminary generation and purification of a glycosylthiol, 9b,c,10 a glycosylthioacetate, 9a or a glycosylthiomethylating agent 11 via the corresponding glycosyl bromides. Access to these 1-thiosugar derivatives is a tedious, time-consuming process and requires harsh acidic conditions for the generation of the 1-bromo intermediate, which can be an especially demanding issue when applying the method to longer oligosaccharides. In fact, a postsynthetic strategy of trisaccharide S-conjugation with a peptide was already described, 12 but saccharide sequences longer than two residues were never thio-anchored to Fmoc-amino acids prior to their solid-phase incorporation into a growing chain. On this basis, we directed our effort toward the implementa- tion of an operationally simple synthetic approach to S- glycosylated Fmoc-protected amino acids, which is endowed with both the compatible direct use of commercial Fmoc-amino acids and the applicability to carbohydrates more complex than mono- or disaccharides. To this aim, we have suitably adapted a straightforward strategy recently described for the synthesis of thioglycosides, entailing the conversion of per-O-acetylated carbohydrates into a glycosyl thiolate intermediate which is entrapped in situ with a suitable electrophile. 13 The synthetic sequence is advantageous because the sequential generation of the two requisite intermediates (namely, a glycosyl iodide and a glycosylthiouronium) takes very short reactions, and no purification of any saccharide intermediate is needed. First, we have obtained iodo β- and γ-amino acids starting from Fmoc-L-aspartic and Fmoc-L-glutammic acids 4- and 5-tert-butyl esters respectively (a-b, Scheme 1) and iodo α-amino acids starting from corresponding 1-tert-butyl esters (c-d, Scheme 1), one of which belongs to the D-series (e, Scheme 1). 9c The free Received: December 19, 2014 Letter pubs.acs.org/OrgLett © XXXX American Chemical Society A DOI: 10.1021/ol503664t Org. Lett. XXXX, XXX, XXX-XXX