Preparation of Low Molecular Weight Heparin from a Remodeled Bovine Intestinal Heparin.

Bovine intestinal heparins are structurally distinct from porcine intestinal heparins and exhibit lower specific anticoagulant activity (units/mg). The reduced content of N-sulfo, 3-O-sulfo glucosamine, the central and critical residue in heparin's antithrombin III binding site, is responsible for bovine intestinal heparin's reduced activity. Previous studies demonstrate that treatment of bovine intestinal heparin with 3-O-sulfotransferase in the presence of 3'-phosphoadenosine-5'-phosphosulfate afforded remodeled bovine heparin with an enhanced activity reaching the United States Pharmacopeia's requirements. Starting from this remodeled bovine intestinal heparin, we report the preparation of a bovine intestinal low molecular weight heparin having the same structural properties and anti-factor IIa and anti-factor Xa activities of Enoxaparin. Moreover, this bovine intestinal heparin-derived "Enoxaparin" showed comparable platelet factor-4 binding affinity, suggesting that it should exhibit similarly low levels of heparin induced thrombocytopeneia, HIT.

Advances in the preparation and synthesis of heparin and related products.

Heparin is a naturally occurring glycosaminoglycan from livestock, principally porcine intestine, and is clinically used as an anticoagulant drug. A limitation to heparin production is that it depends on a single animal species and potential problems have been associated with animal-derived heparin. The contamination crisis in 2008 led to a search for new animal sources and the investigation of non-animal sources of heparin. Over the past 5 years, new animal sources, chemical, and chemoenzymatic methods have been introduced to prepare heparin-based drugs. In this review, we describe advances in the preparation and synthesis of heparin and related products.

N-glycolyl chondroitin synthesis using metabolically engineered E. coli.

N-glycolyl chondroitin (Gc-CN) is a metabolite of N-glycolylneuraminic acid (Neu5Gc), a sialic acid that is commonly found in mammals, but not humans. Humans can incorporate exogenous Neu5Gc into their tissues from eating red meat. Neu5Gc cannot be biosynthesized by humans due to an evolutionary mutation and has been implicated in causing inflammation causing human diseases, such as cancer. The study Neu5Gc is important in evolutionary biology and the development of potential cancer biomarkers. Unfortunately, there are several limitations to detecting Neu5Gc. The elimination of Neu5Gc involves a degradative pathway leading to the incorporation of N-glycolyl groups into glycosaminoglycans (GAGs), such as Gc-CN. Gc-CN has been found in humans and in animals including mice, lamb and chimpanzees. Here, we present the biosynthesis of Gc-CN in bacteria by feeding chemically synthesized N-glycolylglucosamine to Escherichia coli. A metabolically engineered strain of E. coli K4, fed with glucose supplemented with GlcNGc, converted it to N-glycolylgalactosamine (GalNGc) that could then be utilized as a substrate in the chondroitin biosynthetic pathway. The final product, Gc-CN was converted to disaccharides using chondroitin lyase ABC and analyzed by liquid chromatography-tandem mass spectrometry with multiple reaction monitoring detection. This analysis showed the incorporation of GalNGc into the backbone of the chondroitin oligosaccharide.

Glycosylation in room temperature ionic liquid using unprotected and unactivated donors.

Glycosylation in room temperature ionic liquid is demonstrated using unprotected and unactivated donors. Modest yields of simple benzyl glycosides and disaccharides of glucose, mannose and N-acetylgalactosamine were obtained in 1-ethyl-3-methylimidazolium benzoate with Amberlite IR-120 (H(+)) resin or p-toluenesulfonic acid as promoters.

Synthesis of double C-glycoside analogue of sTn.

A sTn double C-glycoside, sTn analogue 2, was synthesized using samarium chemistry developed in our laboratory. Complications in the oxidation reaction affording aldehyde acceptor were overcome by double protection of amide and the use of a room-temperature ionic liquid as solvent. Studies are underway to conjugate the sTn double C-glycoside hapten 2 to KLH carrier protein for biological evaluation as a vaccine.

Solid-phase chemoenzymatic synthesis of C-sialosides.

The chemoenzymatic regioselective acylation of Neu5Ac followed by SmI2-mediated C-glycosylation on a solid support is described for five C-glycosides. This method should facilitate the construction of combinatorial libraries of inhibitors of neuraminidase activity and hemagglutinin interaction as potential antiviral agents.