Synthesis of 3-O-Sulfated Oligosaccharides to Understand the Relationship between Structures and Functions of Heparan Sulfate

Abstract

The sulfation at the 3-OH position of glucosamine is an important modification in forming structural domains for heparan sulfate to enable its biological functions. Seven 3-O-sulfotransferase isoforms in the human genome are involved in the biosynthesis of 3-O-sulfated heparan sulfate. As a rare modification present in heparan sulfate, the availability of 3-O-sulfated oligosaccharides is very limited. Here, we report the use of a chemoenzymatic synthetic approach to synthesize six 3-O-sulfated oligosaccharides, including three hexasaccharides and three octasaccharides. The synthesis was achieved by rearranging the enzymatic modification sequence to accommodate the substrate specificity of 3-O-sulfotransferase 3. We studied the impact of 3-O-sulfation on the conformation of the pyranose ring of 2-O-sulfated iduronic acid using NMR, and on the correlation between ring conformation and anticoagulant activity. We identified a novel octasaccharide that interacts with antithrombin and displays anti factor Xa activity. Interestingly, the octasaccharide displays a faster clearance rate than fondaparinux, an FDA-approved pentasaccharide drug, in a rat model, making this octasaccharide a potential short-acting anticoagulant drug candidate that could reduce bleeding risk. Having access to a set of critically important 3-O-sulfated oligosaccharides offers the potential to develop new heparan sulfate-based therapeutics.

Fig 1. Structures and synthetic schemes for different HS oligosaccharides

Panel A shows the structures of 11 oligosaccharides used in this study. The synthesis of Compound 1 to 6 was accomplished for the first time. Subsequently, these compounds were subjected to purity and structural analysis as described in the text. The synthesis of Compound 7* to 11* was reported in previous publications ^, . Panel B shows the schemes for the syntheses of compound 1 to 6. Both starting materials, NS2S 6-mer substrate and NS2S 8-mer substrate, were synthesized in a previous publication . 6-OSTs represent 6-O-sulfotransferase isoform 1 and isoform 3. Panel C depicts the structures of three conformers of an IdoA2S residue.

Fig 2. Purity and structural analysis compound 6

Panel A shows the structure of the compound 6. Panel B shows the chromatogram of DEAE-HPLC analysis. Panel C shows the ESI-MS spectrum of compound 6. The molecular ions carrying 5, 6, and 7 negative charges are indicated.

Fig 3. Biochemical and biological evaluation of synthesized oligosaccharides

Panel A shows the in vitro inhibition curve of the activity of FXa. The data is represented as the mean ± S.D. (n =3). Panel B shows the representative data for the ITC analysis of compound 5. Panel C shows the anti-FXa effects of compound 5, compound 11* and fondaparinux in a rat model. The Xa activity measurements at different time points are presented as mean ± S.D. (n =4). Compared with the control group (treated with saline), statistical analysis was conducted on each experimental group using a two-tailed Student’s t-test, where * represents a p-value of < 0.05. Panel D shows the blood concentrations of compound 5, 11* and fondaparinux at different time points in the animals after injection. The concentrations of compound 5, compound 11* and fondaparinux were obtained based on the anti-Xa activity, which was converted into the concentration of drugs by standard curves as shown in Supplementary Fig S29. The data are presented as mean ± S.D. (n= 2).

Fig 4. Molecular Dynamic (MD) analysis of the interaction between AT and fondaparinux

Panel A shows the structure of AT-binding pentasaccharide. The critical sulfo groups for AT-binding affinity and the IdoA2S residue (residue b) are highlighted in blue. The ²S_O conformation at residue b position is required for binding to AT. The structural requirement for residue d is promiscuous. Panel B depicts the interaction energy (ΔΔG) of the five MD simulations of AT complexed with fondaparinux and its derivatives. The graphical representation is expressed as mean ± SD (*p ≤ 0.0001 compared to fondaparinux). Panel C presents an image from three simulations of AT in which residue d of fondaparinux is either GlcA (purple), IdoA2S-¹C₄ (orange), or IdoA2S-²S_O (green). The simulations were aligned on AT (grey), and the carboxyl groups of residue d are depicted at multiple time points during the simulations (10 frames each from the latter 150 ns).