Rational Design of Anticoagulant Drugs Using Oligosaccharide Chemistry

For a long time, heparin and low molecular weight heparins have been the drugs of choice for the management of thrombosis. Discovery of the antithrombin binding domain in heparin, a critical element in the anticoagulant activity of this polysaccharide, allowed a rational approach based on medicinal carbohydrate chemistry in the design of new anticoagulants. The fully synthetic pentasaccharide fondaparinux that selectively targets blood coagulation factor Xa was first to be developed as a drug. Fondaparinux was followed by various heparin mimicking oligosaccharides prepared with a view to replace polydisperse heparin and low molecular weight heparins by structurally-defined anticoagulants with no unwanted side-effects

Stereoselective protecting group free synthesis of d,l-gulose ethyl glycoside via multicomponent enyne cross metathesis—hetero Diels–Alder reaction

An efficient and stereoselective synthesis of d,l-gulose was described. The key step of the synthetic route is represented by a multicomponent enyne cross metathesis—hetero Diels–Alder reaction which allows the formation of the pyran ring from cheap and commercially available substrates in a single synthetic step. The synthesis of d,l-gulose was accomplished without the use of protecting groups making this approach highly desirable also in terms of atom economy

Elucidating glycosaminoglycan–protein–protein interactions using carbohydrate microarray and computational approaches

Glycosaminoglycan polysaccharides play critical roles in many cellular processes, ranging from viral invasion and angiogenesis to spinal cord injury. Their diverse biological activities are derived from an ability to regulate a remarkable number of proteins. However, few methods exist for the rapid identification of glycosaminoglycan–protein interactions and for studying the potential of glycosaminoglycans to assemble multimeric protein complexes. Here, we report a multidisciplinary approach that combines new carbohydrate microarray and computational modeling methodologies to elucidate glycosaminoglycan–protein interactions. The approach was validated through the study of known protein partners for heparan and chondroitin sulfate, including fibroblast growth factor 2 (FGF2) and its receptor FGFR1, the malarial protein VAR2CSA, and tumor necrosis factor-α (TNF-α). We also applied the approach to identify previously undescribed interactions between a specific sulfated epitope on chondroitin sulfate, CS-E, and the neurotrophins, a critical family of growth factors involved in the development, maintenance, and survival of the vertebrate nervous system. Our studies show for the first time that CS is capable of assembling multimeric signaling complexes and modulating neurotrophin signaling pathways. In addition, we identify a contiguous CS-E-binding site by computational modeling that suggests a potential mechanism to explain how CS may promote neurotrophin-tyrosine receptor kinase (Trk) complex formation and neurotrophin signaling. Together, our combined microarray and computational modeling methodologies provide a general, facile means to identify new glycosaminoglycan–protein–protein interactions, as well as a molecular-level understanding of those complexes

Synthesis of carbohydrate capped silicon nanoparticles and their reduced cytotoxicity, in vivo toxicity, and cellular uptake

The development of smart targeted nanoparticles (NPs) that can identify and deliver drugs at a sustained rate directly to cancer cells may provide better efficacy and lower toxicity for treating primary and advanced metastatic tumors. Obtaining knowledge of the diseases at the molecular level can facilitate the identification of biological targets. In particular, carbohydrate‐mediated molecular recognitions using nano‐vehicles are likely to increasingly affect cancer treatment methods, opening a new area in biomedical applications. Here, silicon NPs (SiNPs) capped with carbohydrates including galactose, glucose, mannose, and lactose are successfully synthesized from amine terminated SiNPs. The MTT [3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide] analysis shows an extensive reduction in toxicity of SiNPs by functionalizing with carbohydrate moiety both in vitro and in vivo. Cellular uptake is investigated with flow cytometry and confocal fluorescence microscope. The results show the carbohydrate capped SiNPs can be internalized in the cells within 24 h of incubation, and can be taken up more readily by cancer cells than noncancerous cells. Moreover, these results reinforce the use of carbohydrates for the internalization of a variety of similar compounds into cancer cells

Comparative analysis and validation of the malachite green assay for the high throughput biochemical characterization of terpene synthases

Terpenes are the largest group of natural products with important and diverse biological roles, while of tremendous economic value as fragrances, flavours and pharmaceutical agents. Class-I terpene synthases (TPSs), the dominant type of TPS enzymes, catalyze the conversion of prenyl diphosphates to often structurally diverse bioactive terpene hydrocarbons, and inorganic pyrophosphate (PPi). To measure their kinetic properties, current bio-analytical methods typically rely on the direct detection of hydrocarbon products by radioactivity measurements or gas chromatography–mass spectrometry (GC–MS).  In this study we employed an established, rapid colorimetric assay, the pyrophosphate/malachite green assay (MG), as an alternative means for the biochemical characterization of class I TPSs activity.  •We describe the adaptation of the MG assay for turnover and catalytic efficiency measurements of TPSs. •We validate the method by direct comparison with established assays. The agreement of kcat/KM among methods makes this adaptation optimal for rapid evaluation of TPSs. •We demonstrate the application of the MG assay for the high-throughput screening of TPS gene libraries

Homogeneous low-molecular-weight heparins with reversible anticoagulant activity

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

Characterization of anticoagulant heparinoids by immunoprofiling

Heparinoids are used in the clinic as anticoagulants. A specific pentasaccharide in heparinoids activates antithrombin III, resulting in inactivation of factor Xa and–when additional saccharides are present–inactivation of factor IIa. Structural and functional analysis of the heterogeneous heparinoids generally requires advanced equipment, is time consuming, and needs (extensive) sample preparation. In this study, a novel and fast method for the characterization of heparinoids is introduced based on reactivity with nine unique anti-heparin antibodies. Eight heparinoids were biochemically analyzed by electrophoresis and their reactivity with domain-specific anti-heparin antibodies was established by ELISA. Each heparinoid displayed a distinct immunoprofile matching its structural characteristics. The immunoprofile could also be linked to biological characteristics, such as the anti-Xa/anti-IIa ratio, which was reflected by reactivity of the heparinoids with antibodies HS4C3 (indicative for 3-O-sulfates) and HS4E4 (indicative for domains allowing anti-factor IIa activity). In addition, the immunoprofile could be indicative for heparinoid-induced side-effects, such as heparin-induced thrombocytopenia, as illustrated by reactivity with antibody NS4F5, which defines a very high sulfated domain. In conclusion, immunoprofiling provides a novel, fast, and simple methodology for the characterization of heparinoids, and allows high-throughput screening of (new) heparinoids for defined structural and biological characteristics