The Sulfur-Linked Analogue of O-GlcNAc (S-GlcNAc) Is an Enzymatically Stable and Reasonable Structural Surrogate for O-GlcNAc at the Peptide and Protein Levels

Abstract

Synthetic proteins bearing site-specific posttranslational modifications have revolutionized our understanding of their biological functions in vitro and in vivo. One such modification, O-GlcNAcylation, is the dynamic addition of β-N-acetyl glucosamine to the side chains of serine and threonine residues of proteins, yet our understanding of the site-specific impact of O-GlcNAcylation remains difficult to evaluate in vivo because of the potential for enzymatic removal by endogenous O-GlcNAcase (OGA). Thioglycosides are generally perceived to be enzymatically stable structural mimics of O-GlcNAc; however, in vitro experiments with small-molecule GlcNAc thioglycosides have demonstrated that OGA can hydrolyze these linkages, indicating that S-linked β-N-acetyl glucosamine (S-GlcNAc) on peptides or proteins may not be completely stable. Here, we first develop a robust synthetic route to access an S-GlcNAcylated cysteine building block for peptide and protein synthesis. Using this modified amino acid, we establish that S-GlcNAc is an enzymatically stable surrogate for O-GlcNAcylation in its native protein setting. We also applied nuclear magnetic resonance spectroscopy and computational modeling to find that S-GlcNAc is an good structural mimic of O-GlcNAc. Finally, we demonstrate that site-specific S-GlcNAcylation results in biophysical characteristics that are the same as those of O-GlcNAc within the context of the protein α-synuclein. While this study is limited in focus to two model systems, these data suggest that S-GlcNAc broadly resembles O-GlcNAc and that it is indeed a stable analogue in the context of peptides and proteins.

Total chemical synthesis of glycocin F and analogues: S-glycosylation confers improved antimicrobial activity

“…Indeed, the Pratt group has recently demonstrated the resistance of S -linked GlcNAc to cleavage by human O -GlcNAcase (OGA). 14 By avoiding enzymatic cleavage, the S -linked GlcNAc moieties could remain available for binding at the GccF target receptor, leading to enhanced potency and prolonged bacteriostasis.…”

“… 7 The C -terminal S -glycosidic linkage is of particular interest, as the natural functions of such bonds remain unclear. Further, the improved biochemical stability of S -glycoside linkages 14 compared to their O -linked congeners provides considerable scope for the development of S -glycosylated bacteriocins as both preservatives and therapeutics.…”

“…Our hypothesis is that replacing O -linked sugars with S -linked sugars would not greatly perturb the structural requirements of GccF, thereby preserving the bioactivity but may also result in enhanced enzymatic stability. 14 …”

A Mechanism‐Based Approach to Screening Metagenomic Libraries for Discovery of Unconventional Glycosidases

“…It is well known that normal glycosidases that follow Koshland mechanisms (Figure a) are not effective in hydrolyzing thioglycosides. While in the literature there are examples in which Koshland glycosidases catalyze hydrolysis of aryl thioglycosides that contain thiophenols of low p K a , none of these enzymes have been shown to cleave unactivated thioglycosides corresponding to their natural substrate . However, UGLs follow a distinct mechanism in which the glycosidic bond is cleaved through an elimination mechanism.…”

8-Hydroxyquinoline Glycoconjugates Containing Sulfur at the Sugar Anomeric Position—Synthesis and Preliminary Evaluation of Their Cytotoxicity

“…It was expected that the introduction of a sulfur atom into the anomeric position of the sugar would increase the stability of the obtained glycoconjugates against hydrolytic cleavage. Compared to O -glycosyl derivatives, compounds with the S -glycosidic binding are less susceptible to enzymatic degradation, especially under the action of glycosylhydrolases [ 28 , 29 ]. 1-Thiosugars, due to their enzymatic stability, exhibit great therapeutic potential [ 30 , 31 , 32 ].…”