A CONCISE METHODOLOGY FOR THE STEREOSELECTIVE SYNTHESIS OF O-GLYCOSYLATED AMINO-ACID BUILDING-BLOCKS - COMPLETE H-1-NMR ASSIGNMENTS AND THEIR APPLICATION IN SOLID-PHASE GLYCOPEPTIDE SYNTHESIS

A facile strategy for the stereoselective synthesis of suitably protec ted O-glycosylated amino acid building blocks, namely, N-alpha-Fmoc-Se r-[Ac-4-beta-D-Gal-(1-3)-Ac-2-alpha or beta-D-GalN(3)]-OPfp and N-alph a-Fmoc-Thr-[Ac-4-beta-D-Gal-(1-3)-Ac-2-alpha or beta-D-GalN(3)]-OPfp i s described. What is new and novel in this report is that Koenigs-Knor r type glycosylation of an aglycon serine/threonine derivative (i.e. N -alpha-Fmoc-Ser-OPfp or N-alpha-Fmoc-Thr-OPfp) with protected beta-D-G al(1-3)-D-GalN(3) synthon mediated by silver salts resulted in only al pha- and/or beta-isomers in excellent yields under two different react ion conditions. The subtle differences in stereoselectivity were demon strated clearly when glycosylation was carried out using only AgClO4 a t -40 degrees C which afforded alpha-isomer in a quantitative yield (a lpha:beta = 5:1). On the other hand, the beta-isomer was formed exclus ively when the reaction was performed in the presence of Ag2CO3/AgClO4 at room temperature. A complete assignment of H-1 resonances to indiv idual sugar ring protons and the characteristic anomeric alpha-1H and beta-1H in Ac(4)Gal beta(1-3)Ac(2)GalN(3) alpha and/or beta linked to Ser/Thr building blocks was accomplished unequivocally by two-dimensio nal double-quantum filtered correlated spectroscopy and nuclear Overha user enhancement and exchange spectroscopy NMR experiments. An unambig uous structural characterization and documentation of chemical shifts, including the coupling constants for all the protons of the aforement ioned alpha- and beta-isomers of the O-glycosylated amino acid buildin g blocks carrying protected beta-D-Gal(1-3)-D-GalN(3,) could serve as a template in elucidating the three-dimensional structure of glycoprot eins. The synthetic utility of the building blocks and versatility of the strategy was exemplified in the construction of human salivary muc in (MUC7)-derived, O-linked glycopeptides with varied degrees of glyco sylation by solid-phase Fmoc chemistry. Fmoc/tert-butyl-based protecti ng groups were used for the peptidic moieties in conjunction with acet yl sugar protection. The transformation of the 2-azido group into the acetamido derivative was carried out with thioacetic acid on the polym er-bound glycopeptides before the cleavage step. After cleaving the gl ycopeptide from the resin, the acetyl groups used for sugar OH-protect ion were removed with sodium methoxide in methanol. Finally, the glyco peptides were purified by reversed-phase high-performance liquid chrom atography and their integrity was confirmed by proton NMR as well as b y mass spectral analysis. Secondary structure analysis by circular dic hroism of both the glycosylated and nonglycosylated peptides revealed that carbohydrates did not exert any profound structural effect on the peptide backbone conformation.