REVERSIBLE HEPARIN MOLECULES AND METHODS OF MAKING AND USING THE SAME

Methods and systems for synthesizing heparin compounds are provided . The chemoenzymatic synthesis of structurally homogeneous low molecular weight heparins that have a reversible anticoagulant activity is provided . Also disclosed are heparin compounds having anticoagulant activity , including a binding affinity to antithrombin and an anti - Xa activity , but no detectable anti - lla activity . Additionally , provided are synthetic , low - molecular weight heparin com pounds with reversible anticoagulant activity , where the anticoagulant activity is reversible by protamine

Chemoenzymatic synthesis of heparan sulfate and heparin oligosaccharides and NMR analysis: Paving the way to a diverse library for glycobiologists

Heparin and heparan sulfate are sulfated carbohydrates that display a wide range of biological functions. A chemoenzymatic method is becoming a promising approach to synthesize heparin-like oligosaccharides with high efficiency

Negative ion fast-atom bombardment tandem mass spectrometry to determine sulfate and linkage position in glycosaminoglycan-derived disaccharides

AbstractNegative ion fast-atom bombardment tandem mass spectrometry has been used in the analysis of monosulfated disaccharides. These commercially obtained disaccharides have been enzymatically prepared from glycosaminoglycans using polysaccharide lyases. Three disaccharides from chondroitin sulfate and dermatan sulfate and two disaccharides from heparan sulfate and chemically derivatized heparin were analyzed. All five disaccharides were isomeric, with differences in sulfate position and linkage position. The full-scan mass spectra are useful in differentiating isomers when the sulfate group resides on different saccharide units. This structural information was obtained from fragment ions produced through cleavage at the glycosidic linkage. the full-scan mass spectra of each monosulfated disaccharide also produced intense molecular anions having long lifetimes. Collisional activation of these resulted in tandem mass spectra rich in significant product ions. Some of these fragment ions were formed through ring cleavage and were useful in the determination of both sulfate and linkage position

Chloroquine reduces arylsulphatase B activity and increases chondroitin-4-sulphate: implications for mechanisms of action and resistance

<p>Abstract</p> <p>Background</p> <p>The receptors for adhesion of <it>Plasmodium falciparum</it>-infected red blood cells (RBC) in the placenta have been identified as chondroitin-4-sulphate (C4S) proteoglycans, and the more sulphate-rich chondroitin oligosaccharides have been reported to inhibit adhesion. Since the anti-malarial drug chloroquine accumulates in lysosomes and alters normal lysosomal processes, the effects of chloroquine on the lysosomal enzyme arylsulphatase B (ASB, N-acetylgalactosamine-4-sulphatase), which removes 4-sulphate groups from chondroitin-4-sulphate, were addressed. The underlying hypothesis derived from the recognized impairment of attachment of parasite-infected erythrocytes in the placenta, when chondroitin-4-sulphation was increased. If chloroquine reduced ASB activity, leading to increased chondroitin-4-sulphation, it was hypothesized that the anti-malarial mechanism of chloroquine might derive, at least in part, from suppression of ASB.</p> <p>Methods</p> <p>Experimental methods involved cell culture of human placental, bronchial epithelial, and cerebrovascular cells, and the <it>in vitro </it>exposure of the cells to chloroquine at increasing concentrations and durations. Measurements of arylsulphatase B enzymatic activity, total sulphated glycosaminoglycans (sGAG), and chondroitin-4-sulphate (C4S) were performed using <it>in vitro </it>assays, following exposure to chloroquine and in untreated cell preparations. Fluorescent immunostaining of ASB was performed to determine the effect of chloroquine on cellular ASB content and localization. Mass spectrometry and high performance liquid chromatography were performed to document and to quantify the changes in chondroitin disaccharides following chloroquine exposure.</p> <p>Results</p> <p>In the human placental, bronchial epithelial, and cerebrovascular cells, exposure to increasing concentrations of chloroquine was associated with reduced ASB activity and with increased concentrations of sGAG, largely attributable to increased C4S. The study data demonstrated: 1) decline in ASB activity following chloroquine exposure; 2) inverse correlation between ASB activity and C4S content; 3) increased content of chondroitin-4-sulphate disaccharides following chloroquine exposure; and 4) decline in extent of chloroquine-induced ASB reduction with lower baseline ASB activity. Confocal microscopy demonstrated the presence of ASB along the cell periphery, indicating extra-lysosomal localization.</p> <p>Conclusions</p> <p>The study data indicate that the therapeutic mechanism of chloroquine action may be attributable, at least in part, to reduction of ASB activity, leading to increased chondroitin-4-sulphation in human placental, bronchial epithelial, and cerebrovascular cells. In vivo, increased chondroitin-4-sulphation may reduce the attachment of <it>P. falciparum</it>-infected erythrocytes to human cells. Extra-lysosomal localization of ASB and reduced impact of chloroquine when baseline ASB activity is less suggest possible mechanisms of resistance to the effects of chloroquine.</p

Electron-Induced Dissociation of Glycosaminoglycan Tetrasaccharides

Electron detachment dissociation (EDD) Fourier transform mass spectrometry has recently been shown to be a powerful tool for examining the structural features of sulfated glycosaminoglycans (GAGs). The characteristics of GAG fragmentation by EDD include abundant cross-ring fragmentation primarily on hexuronic acid residues, cleavage of all glycosidic bonds, and the formation of even- and odd-electron product ions. GAG dissociation by EDD has been proposed to occur through the formation of an excited species that can undergo direct decomposition or ejects an electron and then undergoes dissociation. In this work, we perform electron-induced dissociation (EID) on singly charged GAGs to identify products that form via direct decomposition by eliminating the pathway of electron detachment. EID of GAG tetrasaccharides produces cleavage of all glycosidic bonds and abundant cross-ring fragmentation primarily on hexuronic acid residues, producing fragmentation similar to EDD of the same molecules, but distinctly different from the products of infrared multiphoton dissociation or collisionally activated decomposition. These results suggest that observed abundant fragmentation of hexuronic acid residues occurs as a result of their increased lability when they undergo electronic excitation. EID fragmentation of GAG tetrasaccharides results in both even- and odd-electron products. EID of heparan sulfate tetrasaccharide epimers produces identical fragmentation, in contrast to EDD, in which the epimers can be distinguished by their fragment ions. These data suggest that for EDD, electron detachment plays a significant role in distinguishing glucuronic acid from iduronic acid

Neutralizing the anticoagulant activity of ultra-low-molecular-weight heparins using N -acetylglucosamine 6-sulfatase

Heparin has been the most commonly used anticoagulant drug for nearly a century. The drug heparin is generally categorized into three forms according to its molecular weight (MW), unfractionated (UF, average MW 13,000), low molecular weight (LMW, average MW 5,000), and ultra-low molecular weight heparin (ULMWH, average MW 2,000). Overdose of anticoagulant heparin can lead to very dangerous bleeding in patients. Protamine sulfate can be administered as an antidote to reverse heparin’s anticoagulant effect. There is not an effective antidote for ULMWH. In the current study, we examine human N-acetylglucosamine 6-sulfatase (NG6S), expressed in Chinese hamster ovary cells as a reversal agent for ULMWH. NG6S removes a single 6-O-sulfo group at the non-reducing end of the ULMWH Arixtra® (fondaparinux) effectively removing its ability to bind to antithrombin and preventing its inhibition of coagulation factor Xa. These results pave the way to develop human NG6S as an antidote for neutralizing the anticoagulant activity of ULMWHs

Preparation and application of a ‘clickable’ acceptor for enzymatic synthesis of heparin oligosaccharides

A “clickable” disaccharide was prepared by treating the aldehyde precursor with hydroxylamine, followed by the catalytic hydrogenation and diazotransfer reaction. This disaccharide was successfully applied to the elongation of the backbone construction of ultralow molecular weight (ULMW) heparins using two bacterial glycosyl transferases, N-acetyl glucosaminyl transferase from Escherichia coli K5 (KfiA) and heparosan synthase-2 (pmHS2) from Pasteurella multocida

Tandem MS can distinguish hyaluronic acid from N-acetylheparosan

Isobaric oligosaccharides enzymatically prepared from hyaluronic acid (HA) and N-acetylheparosan (NAH), were distinguished using tandem mass spectrometry. The only difference between the two series of oligosaccharides was the linkage pattern (in HA 1→3 and in NAH 1→4) between glucuronic acid and N-acetylglucosamine residues. Tandem mass spectrometry afforded spectra, in which glycosidic cleavage fragment ions were observed for both HA and NAH oligosaccharides. Cross-ring cleavage ions 0,2An and 0,2An-h (n is even number) were observed only in GlcNAc residues of NAH oligosaccharides. One exception was an 0,2A2 ion fragment observed for the disaccharide from HA. These cross-ring cleavage fragment ions are useful to definitively distinguish HA and NAH oligosaccharides

Chemoenzymatic synthesis of unmodified heparin oligosaccharides: cleavage of p-nitrophenyl glucuronide by alkaline and Smith degradation

A heparin oligosaccharide having a completely natural structure was successfully synthesized through a chemoenzymatic approach using an unnatural glycosyl acceptor, p -nitrophenyl glucuronide (GlcA- p NP)