Mapping of Metabolic Glycan Labeling Tolerance Based on SERS-Imaging of N-Substituted Unnatural Sugar Analogs in the Hexosamine Biosynthetic Pathway.

Unnatural forms of sialic acid can be introduced to mammalian cell surfaces by the metabolic transformation of glycan precursors of N-acetylhexosamine (ManNAc and GlcNAc). Previous studies have shown that mannosamine analogs with N-acyl groups, whether unacetylated or acetylated, containing up to five carbon atoms, and N-alkyl glucosamine derivatives (GlcNPhAlk) can be recognized by the biosynthetic machinery and converted into sialoglycoconjugates on the cell surface. These structural changes to cell surface glycans can be used to investigate carbohydrate-dependent cellular phenomena pertinent to advanced diagnostics by tracking the changes in cell surface glycans. Herein, we report the tolerance levels of the hexosamine biosynthetic pathway (HBP) based on structural alterations of the N-acyl, N-alkyl, and N-acyloxy substituents of mannosamine and glucosamine. A series of analogs has been synthesized with N-acyl, N-alkyl, and N-acyloxy groups of hexosamines (GlcNPhAlk, ManNPhAlk, GlcNAcPhAlk, ManNAcPhAlk, GlcNAcOPhAlk, and ManNAcOPhAlk) and examined their metabolic conversion to cell surface glycans using the metabolic glycan labeling coupled surface-enhanced Raman scattering (MGL-SERS) mapping technique. We observed that ManNAcPhAlk imposed the highest glycan labeling efficiency-based SERS-mapped images in the cell-surface milieu, followed by ManNPhAlk and GlcNPhAlk, while GlcNAcOPhAlk, ManNAcOPhAlk, and GlcNAcPhAlk are subsequently less efficiently metabolized by the biosynthetic machinery. Furthermore, these findings have been validated through far western blot analysis using azide-PEG3-biotin, which consistently complemented the outcome of the MGL-SERS technique. Subsequently, inhibitor studies using N-glycan and O-glycan inhibitors with MGL-SERS techniques revealed that all these glycan analogs are mostly labeling N-glycans, which was further substantiated by the same far-western blot analysis. Finally, azido naphthalimide fluorophore was utilized to complement the alkyne-tag of the highest labeling precursor ManNAcPhAlk, enabling click chemistry to generate a triazole product with an intense fluorescence and reconfirmed mapped images on the cell surface. Collectively, the key structural requirements for substrate recognition within the HBP provide a clear understanding of its metabolic tolerance to design novel chemical tools for glycoengineering.