Low-molecular-weight heparins (LMWHs) are carbohydrate-based anticoagulants clinically used to treat thrombotic disorders, but impurities, structural heterogeneity or functional irreversibility can limit treatment options. We report a series of synthetic LMWHs prepared by cost-effective chemoenzymatic methods. The high activity of one defined synthetic LMWH against human factor Xa (FXa) was reversible in vitro and in vivo using protamine, demonstrating that synthetically accessible constructs can have a critical role in the next generation of LMWHs.

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.

The L-iduronic acid (IdoA) residue is a critically important structural component in heparan sulphate polysaccharide for the biological functions. The pyranose ring of IdoA is present in 1C4-chair, 2SO-skew boat, and less frequently, in 4C1-chair conformations. Here, we analyzed the conformation of IdoA residue in eight hexasaccharides by NMR. The data demonstrate a correlation between the conformation of IdoA and sulphations in the surrounding saccharide residues. For the 2-O-sulpho IdoA residue, a high degree of sulphation on neighboring residues drives ring dynamics towards the 2SO-skew boat conformer. In contrast, the nonsulphated IdoA residue is pushed towards the 1C4-chair conformer when the neighboring residues are highly sulphated. Our data suggest that the conformation of IdoA is regulated by the sulphation pattern of nearby saccharides that is genetically controlled by the heparan sulphate biosynthetic pathway.

Background: Sulfotransferases with distinct specificities act in sequence in the heparan sulfate biosynthetic pathway. Results: The crystal structure of 2-O-sulfotransferase with bound substrate reveals its requirements for substrate recognition. Conclusion: The 2-O-sulfotransferase recognizes N-sulfate but excludes 6-O-sulfate on substrates. Significance: The results advance the understanding of cellular control for the biosynthesis of heparan sulfate.

Three new diterpenoids, 2-O-lactoylborapetoside B (1), 6'-O-lactoylborapetoside B (2), and tinocrispol A (3), and nine known diterpenoids (4-12) were isolated from an EtOH extract of Tinospora crispa vines. Their structures were elucidated by spectroscopic analyses. The C-6 glucosyloxy group in borapetoside C (6) was revised to be α-oriented. The in vivo hypoglycemic activities of the major components, borapetosides A-C (4-6), were examined. Intraperitoneal injection of 4 and 6 (5 mg/kg) showed significant lowering of plasma glucose levels in normal and streptozotocin-induced type 1 diabetic mice. Borapetoside C increased glucose utilization in peripheral tissues and reduced hepatic gluconeogenesis, accounting for the hypoglycemic effect.