Silver Oxide Promoted Synthesis of Alpha O‑GalNAc Containing Glyco-Amino Acids: Synthesis of Core 2 Containing Glyco-Amino Acids for Solid Phase Synthesis of Glycopeptides Ousman Boye, ∥ Lisa Nicholson, ∥ Anna Marstall, ∥ Brooke Van Engen, ∥ Marika Van Slageren, ∥ Noah Mulder, ∥ Mostafa Ali Eldeen, Abe Hall, Anjaneyulu Putta, Sandeep K. Misra, Joshua S. Sharp, and Hailiang Joshua Zhu* Cite This: https://doi.org/10.1021/acs.joc.4c01572 Read Online ACCESS Metrics & More Article Recommendations *sı Supporting Information ABSTRACT: O-GalNAc glycans on glycoproteins with eight different core structures sharing a common α-glycosidic linkage (O-GalNAc-α- Ser/Thr) are critical in various physiological and pathological processes. Among the eight O-GalNAc glycan cores, core 2 characterized by a GlcNAcβ1−6(Galβ1−3)GalNAc structural motif plays a significant role in regulating diverse biological processes, such as immune response modulation, adhesive properties of selectins, and gastrointestinal tract protection. However, the large-quantity synthesis of core 2 containing glyco-amino acids for downstream solid-phase peptide synthesis is challenging. In this work, we successfully employed a silver oxide for coupling a 2-azido-galactosyl chloride donor with two acceptors, Fmoc-Ser/Thr-O t Bu, respectively, for the large-scale syn- thesis of the two important intermediates, α-GalN 3 −Fmoc-Ser/Thr- O t Bu, which can be further utilized for the large-scale synthesis of core 2 containing glyco-amino acids. The two intermediates, α- GalN 3 −Fmoc-Ser/Thr-O t Bu, were utilized for synthesizing core 2 containing Fmoc-Ser/Thr-COOH. The synthesis of core 2 containing Fmoc-Ser-COOH was achieved on a 1.95 g scale, while the synthesis of core 2 containing Fmoc-Thr-COOH was achieved on a 0.38 g scale. Additionally, the synthesis of the 2-azido-galactosyl chloride donor was optimized into a three-step process with only one column chromatography purification. Finally, core 2 containing Fmoc-Ser/Thr-COOH were applied for the synthesis of glycosylated CCR1 and CCR5 N-terminal peptides. ■ INTRODUCTION O-linked glycosylation is a widely existing glycoform on human cell surface. 1 Alpha-linked N-acetylgalactosamine (GalNAc) on serine/threonine is one of the major types of O-linked glycosylation, such as T/Tn antigens. O-GalNAc glycans can be derived into branched sugars, rendering eight core structures of O-GalNAc glycans which are commonly found in biological systems. 2 Among the eight core structures, the core 2 structure was found in human glycoproteins with critical biological functions. 3 For example, core 2 containing glycans are commonly required for certain selectin-mediated inter- actions between endothelial cells and leukocytes. 4 In order to study the detailed functions of core 2 containing glycans, synthetic core 2 containing glycans are needed to provide structure-defined derivatives of core 2 glycans, which can provide structural information on how the sugars are involved in their biological functions. It is a challenging task to synthesize glycopeptides and glycoproteins with core 2 containing glycans. One of the major challenges is the large quantity needed of core 2 containing glyco-amino acids because solid-phase synthesis of core 2 containing glycopeptides/glycoproteins needs core 2 contain- ing glyco-amino acids in large quantities in general. The formation of the α-glycosidic linkage and the introduction of the azido group have been the two major limiting points for the synthesis of core 2 containing glyco-amino acids on a large scale. In the past decades, core 2 containing glyco-amino acids and their derivatives have been synthesized utilizing Schmidt glycosylation or thioglycoside donors for introducing the critical α-glycosidic linkage (O-GalNAc-α-Ser/Thr). 5 To our knowledge, most of the up-to-date strategies mainly utilize thioglycoside donors for introducing the α-glycosidic linkage, while recent work by Xiao et al. utilized GalN 3 N-phenyl Received: June 21, 2024 Revised: November 20, 2024 Accepted: November 28, 2024 Article pubs.acs.org/joc © XXXX American Chemical Society A https://doi.org/10.1021/acs.joc.4c01572 J. Org. Chem. XXXX, XXX, XXX−XXX Downloaded via UNIV OF MISSISSIPPI on December 17, 2024 at 12:57:57 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.