Chemoenzymatic synthesis of ultralow and low-molecular weight heparins.

Heparin is a naturally occurring glycosaminoglycan isolated from animal tissues and is medically used as an anticoagulant drug. Adulteration attempts of isolated heparin with chondroitin sulfate in the past resulted in great safety concerns. Also, increasing demands on batch-to-batch homogeneity for better evaluation and control of its pharmacodynamic and pharmacokinetic properties kindled the development of synthetic routes for the production of heparin and its derivatives. The discovery of enzymes involved in glycosaminoglycan biosynthesis and their application in chemoenzymatic synthesis makes it feasible to generate low molecular weight heparins (LMWHs) and ultra-low molecular weight heparins (ULMWHs). Understanding the scope and limitations of these enzymes currently used in the production of synthetic heparins will help to achieve more defined heparins with controlled medicative properties. Here, we summarized the recent advances in the chemoenzymatic synthesis of LMW/ULMW heparins.

Isolation of low-molecular-weight heparin/heparan sulfate from marine sources.

The glycosaminoglycan (heparin and heparan sulfate) are polyanionic sulfated polysaccharides mostly recognized for its anticoagulant activity. In many countries, low-molecular-weight heparins have replaced the unfractionated heparin, owing to its high bioavailability, half-life, and less adverse effect. The low-molecular-weight heparins differ in mode of preparation (chemical or enzymatic synthesis and chromatography fractionations) and as a consequence in molecular weight distribution, chemical structure, and pharmacological activities. Bovine and porcine body parts are at present used for manufacturing of commercial heparins, and the appearance of mad cow disease and Creutzfeldt-Jakob disease in humans has limited the use of bovine heparin. Consequently, marine organisms come across the new resource for the production of low-molecular-weight heparin and heparan sulfate. The importance of this chapter suggests that the low-molecular-weight heparin and heparan sulfate from marine species could be alternative sources for commercial heparin.

De novo synthesis of a narrow size distribution low-molecular-weight heparin.

Heparin, a commonly used anticoagulant drug, is a mixture of highly sulfated polysaccharides with various molecular weights (MWs). The unique sulfation pattern dictates the anticoagulant activity of heparin. Commercial heparins are categorized into three forms according to their average MW: unfractionated heparin (UFH, MWavg 14,000), low-MW heparin (LMWH, MWavg 3500-6500) and the synthetic pentasaccharide (fondaparinux, MW 1508.3). UFH is isolated from porcine intestine while LMWH is derived from UFH by various methods of depolymerization, which generate a wide range of oligosaccharide chain lengths. Different degradation methods result in structurally distinct LMWH products, displaying different pharmacological and pharmacokinetic properties. In this report, we utilized a chemoenzymatic method to synthesize LMWH with the emphasis on controlling the size distribution of the oligosaccharides. A tetrasaccharide primer and a controlled enzyme-based polymerization were employed to build a narrow size oligosaccharide backbone. The oligosaccharide backbones were further modified by a series of sulfation and epimerization steps in order to obtain a full anticoagulation activity. Determination of the anticoagulation activity in vitro and ex vivo indicated that the synthetic LMWH has higher potency than enoxaparin, a commercial LMWH drug in clinical usage.

Antiangiogenic activity of semisynthetic biotechnological heparins: low-molecular-weight-sulfated Escherichia coli K5 polysaccharide derivatives as fibroblast growth factor antagonists.

OBJECTIVE: Low-molecular-weight heparin (LMWH) exerts antitumor activity in clinical trials. The K5 polysaccharide from Escherichia coli has the same structure as the heparin precursor. Chemical and enzymatic modifications of K5 polysaccharide lead to the production of biotechnological heparin-like compounds. We investigated the fibroblast growth factor-2 (FGF2) antagonist and antiangiogenic activity of a series of LMW N,O-sulfated K5 derivatives. METHODS AND RESULTS: Surface plasmon resonance analysis showed that LMW-K5 derivatives bind FGF2, thus inhibiting its interaction with heparin immobilized to a BIAcore sensor chip. Interaction of FGF2 with tyrosine-kinase receptors (FGFRs), heparan sulfate proteoglycans (HSPGs), and alpha(v)beta3 integrin is required for biological response in endothelial cells. Similar to LMWH, LMW-K5 derivatives abrogate the formation of HSPG/FGF2/FGFR ternary complexes by preventing FGF2-mediated attachment of FGFR1-overexpressing cells to HSPG-bearing cells and inhibit FGF2-mediated endothelial cell proliferation. However, LMW-K5 derivatives, but not LMWH, also inhibit FGF2/alpha(v)beta3 integrin interaction and consequent FGF2-mediated endothelial cell sprouting in vitro and angiogenesis in vivo in the chick embryo chorioallantoic membrane. CONCLUSIONS: LMW N,O-sulfated K5 derivatives affect both HSPG/FGF2/FGFR and FGF2/alpha(v)beta3 interactions and are endowed with FGF2 antagonist and antiangiogenic activity. These compounds may provide the basis for the design of novel LMW heparin-like angiostatic compounds.