A concise methodology for the stereoselective synthesis of O‐glycosylated amino acid building blocks: complete ¹HNMR assignments and their application in solid‐phase glycopeptide synthesis

Abstract

A facile strategy for the stereoselective synthesis of suitably protected O‐glycosylated amino acid building blocks, namely, Nα‐Fmoc‐Ser‐[Ac4‐β‐d‐Gal‐(1‐3)‐Ac2α or β‐d‐GalN3]‐OPfp and Nα‐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. Nα‐Fmoc‐Ser‐OPfp or Nα‐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 Ag2CO3AgClO4 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)Ac2GalN3α 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 a‐ and p‐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

Thermodynamic Switch in Binding of Adhesion/Growth Regulatory Human Galectin-3 to Tumor-Associated TF Antigen (CD176) and MUC1 Glycopeptides

“…The purity of the Thr building block 7 was confirmed by RP-HPLC and MALDI-MS (see Figure S1 of the Supporting Information ). The NMR spectra are in agreement with the previously published data 21 , 34 and confirmed the anomeric purity of 7 (see Figures S4 and S5 of the Supporting Information ).…”

“…Compounds 1–6 were prepared according to the protocol described previously. , Compounds 9–12 were synthesized according to the method described by Shao et al…”

“…Compounds 1−6 were prepared according to the protocol described previously. 20,21 Compounds 9−12 were synthesized according to the method described by Shao et al 22 Synthesis of N α -(Fluoren-9-ylmethoxycarbonyl)-3-O-[2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl-(1→3)-2-azido-4,6-di-O-acetyl-2-deoxy-α-D-galactopyranosyl]-L-threonine Pentafluorophenyl Ester (7). Based on Scheme 2.…”