Chemoenzymatic synthesis of heparan sulfate and heparin oligosaccharides and NMR analysis: paving the way to a diverse library for glycobiologists

Abstract

Heparan sulfate (HS) is a member of the glycosaminoglycans (GAG) family that plays essential roles in biological processes from animal sources. Heparin, a highly sulfated form of HS, is widely used as anticoagulant drug worldwide. The high diversity and complexity of HS and heparin represent a roadblock for structural characterization and biological activity studies. Access to structurally defined oligosaccharides is critical for the successful development of HS and heparin structure-activity relationships. In this study, a library of 66 HS and heparin oligosaccharides covering different sulfation patterns and sizes was prepared through an efficient method of chemoenzymatic synthesis. A systematic nuclear magnetic resonance spectroscopy study was firstly undertaken for every oligosaccharide in the library. In addition to the availability of different oligosaccharides, this work also provides spectroscopic data helpful for characterizing more complicated polysaccharide structures providing a safeguard to ensure the quality of the drug heparin. This HS/heparin library will be useful for activity screening and facilitate future structure-activity relationship studies.

Scheme 1. Route for the chemoenzymatic synthesis of seven key intermediates. Reagents and conditions: (a) pmHS2, GlcNAc; (b) pmHS2, GlcA; (c) pmHS2, GlcNTFA; (d) de-N-trifluoroacetylation, NST, PAPS; (e) C₅-epimerase; (f) 2-OST, PAPS. PmHS2, Pasteurella multocida heparosan synthase-2; NST, N-sulfotransferase; 2-OST, 2-O-sulfotransferase; PAPS, 3′-phosphoadenosine-5′-phosphosulfate. Each intermediate was purified to minimum 97% purity by HPLC with a TSKgel DNA-NPR column (ESI).

Fig. 1. HS and heparin oligosaccharides 1–66 were synthesized using a chemoenzymatic approach. The azide tag is N-(6-azidohexanamidyl) p-aminophenyl (pNA-N₃) and the chemical structure is presented in Fig. 2A.

Fig. 2. Structures and synthetic schemes for different HS and heparin oligosaccharides. Panel A shows the structure of the compounds 28, 46, 65 and 66. Panel B shows the scheme for the synthesis of GlcNS3S6S containing compounds and panel C for the GlcA2S containing compounds. Reagents and conditions: (a) pmHS2, GlcNTFA; (b) deacetylation, NST, PAPS; (c) pmHS2, GlcA; (d) 6-OST, PAPS; (e) 3-OST-1, PAPS; (f) 2-OST, PAPS.

Fig. 3. Purity and structure analysis of hexasaccharide 14. Panel A shows the chemical structure of hexasaccharide 14. Panel B shows the chromatogram of TSKgel DNA-NPR HPLC analysis. Panel C shows the ESI-MS spectrum of hexasaccharide 14. The molecular ions carrying 4, 5 and 6 negative charges are indicated.

Fig. 4. NMR characterization of hexasaccharide 14 (structure see Fig. 3A). Panel A, B, C show the 1D ¹H NMR, 2D ¹H–¹H COSY and ¹H–¹³C HSQC spectra, respectively. Peaks corresponding to the anomeric protons of these compounds can be clearly identified.