Chemoenzymatic Synthesis of Homogeneous Heparan Sulfate and Chondroitin Sulfate Chimeras

Abstract

Heparan sulfate (HS) and chondroitin sulfate (CS) are two structurally distinct natural polysaccharides. Here, we report the synthesis of a library of seven structurally homogeneous HS and CS chimeric dodecasaccharides (12-mers). The synthesis was accomplished using six HS biosynthetic enzymes and four CS biosynthetic enzymes. The chimeras contain a CS domain on the reducing end and a HS domain on the nonreducing end. The synthesized chimeras display anticoagulant activity as measured by both in vitro and ex vivo experiments. Furthermore, the anticoagulant activity of H/C 12-mer 5 is reversible by protamine, a U.S. Food and Drug Administration-approved polypeptide to neutralize anticoagulant drug heparin. Our findings demonstrate the synthesis of unnatural HS-CS chimeric oligosaccharides using natural biosynthetic enzymes, offering a new class of glycan molecules for biological research.

Figure 1.

Reactivities of oligosaccharide substrates to 6-OST-3 and CS 6-OST modifications. (a) HPLC chromatograms of NS 6-mer with or without 6-OST-3 modification (right) and NS 6-mer with or without CS 6-OST modification. The reaction catalyzed by 6-OST-3 is shown on top of the panel. (b) HPLC chromatograms of CS 7-mer with or without 6-OST-3 modification (left) and CS 7-mer with or without CS 6-OST modification. The reaction catalyzed by CS 6-OST is shown on top of the panel. (c) HPLC chromatograms of CS 7-mer-4S with or without CS 6-OST modification (left) and CS 7-mer-4S with or without CS 6-OST modification. The reaction catalyzed by CS 6-OST is shown on top of the panel. (d) MW of the products after the sulfotransferase modification. The analysis was carried out by LC–MS. The keys for the shorthand symbols are shown at the bottom of the figure.

Figure 2.

Chemical structures of the HS-CS chimeras synthesized in the present study. The reducing end of each chimera contains a CS heptasaccharide domain, which is boxed for emphasis.

Figure 3.

Schemes for the chemoenzymatic synthesis of chimeric H/C 12-mers. (a) Shows the synthesis CS/HS backbone. KfoC was used to construct CS 7-mer, in which the linkage GlcA and GalNAc is β1 → 3, and the linkage between GalNAc and GlcA is β1 → 4. The CS 7-mer was further elongated to the H/C 12-mer intermediate. H/C 12-mer 1 was obtained by converting GlcNTFA to GlcNS. The glycosidic linkages in the HS domain of H/C 12-mer 1 are either →4)GlcA(β1 → 4)GlcNAc(α1 → and → 4)GlcNAc(α1 → 4)GlcA(β1→. (b) Synthesis of H/C 12-mer 6 and H/C 12-mer 7 was initiated from H/C 12-mer 1 using CS-6-OST and CS 4-OST, respectively. (c) Synthesis of H/C 12-mer 2, 3, 4, and 5. The synthesis of H/C 12-mer 2 was initiated from H/C 12-mer 1 using four HS biosynthetic enzymes. H/C 12-mer 2 was converted to H/C 12-mer 3, 4, and 5 with CS-6OST, CS-4OST, and GalNAc-4S-6OST, respectively. The abbreviations for enzymes are shown in Supplementary Table S1.

Figure 4.

Determination of the anticoagulant activity of H/C 12-mers in vitro and ex vivo. (a) 12-mer products were assessed for anti-FXa activity in a standard chromogenic assay. The values of IC₅₀ are showed in legenda. (b) Neutralization of the anticoagulant activity of H/C 12-mers by protamine sulfate. H/C 12-mer 4 and 5 were selected for this assay. Dekaparin and Fondaparinux were used as positive and negative controls, respectively. (c) Anti-FXa effects of H/C 12-mer 5 after subcutaneous injection into 8-week-old C57BL/6 J male mice. The FXa activity measurements at different time points are presented as mean ± SD (n = 3). (d) Plasma concentrations of H/C 12-mer 5 at different time points in the animals after injection. The concentration of H/C 12-mer 5 was obtained based on the standard curve of the anti-FXa activity plotted against the concentration of the drug.