A concise methodology for the stereoselective synthesis of O- glycosylated amino acid building blocks: Complete ¹H NMR assignments and their application in solid-phase glycopeptide synthesis

A facile strategy for the stereoselective synthesis of suitably protected O-glycosylated amino acid building blocks, namely, N(α)-Fmoc-Ser- [Ac₄-β-D-Gal-(1-3)-Ac₂-α or β-D-GalN₃]-OPfp and N(α)-Fmoc-Thr-[Ac₄- β-D-Gal-(1-3)-Ac₂-α or β-D-GalN₃]-OPfp is described. What is new and novel in this report is that Koenigs-Knorr type glycosylation of an aglycon serine/threonine derivative (i.e. N(α)Fmoc-Ser-OPfp or N(α)-Fmoc-Thr-OPfp) with protected β-D-Gal(1-3)-D-GalN₃ synthon mediated by silver salts resulted in only α- and/or β-isomers in excellent yields under two different reaction conditions. The subtle differences in stereoselectivity were demonstrated clearly when glycosylation was carried out using only AgClO₄ at -40°C which afforded α-isomer in a quantitative yield (α:β = 5:1). On the other hand, the β-isomer was formed exclusively when the reaction was performed in the presence of Ag₂CO₃/AgClO₄ at room temperature. A complete assignment of ¹H resonances to individual sugar ring protons and the characteristic anomeric α-1H and β-1H in Ac₄Galβ(1- 3)Ac₂GalN₃ α and/or β linked to Ser/Thr building blocks was accomplished unequivocally by two-dimensional double-quantum filtered correlated spectroscopy and nuclear Overhauser enhancement and exchange spectroscopy NMR experiments. An unambiguous structural characterization and documentation of chemical shifts, including the coupling constants for all the protons of the aforementioned α- and β-isomers of the O-glycosylated amino acid building blocks carrying protected β-D-Gal(1-3)-D-GalN₃, could serve as a template in elucidating the three-dimensional structure of glycoproteins. The synthetic utility of the building blocks and versatility of the strategy was exemplified in the construction of human salivary mucin (MUC7)-derived, O- linked glycopeptides with varied degrees of glycosylation by solid-phase Fmoc chemistry. Fmoc/tert-butyl-based protecting groups were used for the peptidic moieties in conjunction with acetyl sugar protection. The transformation of the 2-azido group into the acetamido derivative was carried out with thioacetic acid on the polymer-bound glycopeptides before the cleavage step. After cleaving the glycopeptide from the resin, the acetyl groups used for sugar OH-protection were removed with sodium methoxide in methanol. Finally, the glycopeptides were purified by reversed-phase high-performance liquid chromatography and their integrity was confirmed by proton NMR as well as by mass spectral analysis. Secondary structure analysis by circular dichroism of both the glycosylated and nonglycosylated peptides revealed that carbohydrates did not exert any profound structural effect on the peptide backbone conformation.