Electrophoresis for the analysis of heparin purity and quality

The adulteration of raw heparin with oversulfated chondroitin sulfate (OSCS) in 2007-2008 produced a global crisis resulting in extensive revisions to the pharmacopeia monographs and prompting the FDA to recommend the development of additional methods for the analysis of heparin purity. As a consequence, a wide variety of innovative analytical approaches have been developed for the quality assurance and purity of unfractionated and low-molecular-weight heparins. This review discusses recent developments in electrophoresis techniques available for the sensitive separation, detection, and partial structural characterization of heparin contaminants. In particular, this review summarizes recent publications on heparin quality and related impurity analysis using electrophoretic separations such as capillary electrophoresis (CE) of intact polysaccharides and hexosamines derived from their acidic hydrolysis, and polyacrylamide gel electrophoresis (PAGE) for the separation of heparin samples without and in the presence of its relatively specific depolymerization process with nitrous acid treatment.     

Polysaccharide lyases

Polysaccharide lyases (or eliminases) are a class of enzymes (EC 4.2.2.-) that act to cleave certain activated glycosidic linkages present in acidic polysaccharides. These enzymes act through an eliminase mechanism, rather than through hydrolysis, resulting in unsaturated oligosaccharide products. Acidic polysaccharides are ubiquitous and so are the lyases that degrade them. This review article examines lyases that act on acidic polysaccharides of plant, animal, and microbial origin. These lyases are predominantly of microbial origin and come from a wide variety of both pathogenic and nonpathogenic bacteria and fungi. The lyases discussed include alginate lyase (EC 4.2.2.3), pectin lyase (EC 4.2.2.10), pectate lyase (EC 4.2.2.2), oligogalacturonide lyase (EC 4.2.2.6), exopolygalacturonate lyase (EC 4.2.2.9), chondroitin lyases (EC 4.2.2.4 and EC 4.2.2.5), hyaluronate lyase (EC 4.2.2.1), heparin lyase (EC 4.2.2.7), heparan lyase (EC 4.2.2.8), and other unclassified lyases. This review examines the sources, regulation, purification, and properties of these polysaccharide lyases.     

An immobilized microbial heparinase for blood deheparinization

A new medical application of an immobilized microbial enzyme is described. Extracorporeal devices require systemic heparin administration to prevent thrombus formation; however, the use of heparin often leads to serious hemorrhagic complica tions. Heparinase isolated from Flavobacterium has been immobilized and used in a fluidized bed reactor to eliminate heparin from blood passing through an extracorporeal circuit both in vitro and in vivo. This paper discusses the stepwise de velopment of this heparinase reactor including: (1) improvements in the fermentation resulting in an inexpensive large-scale source of heparinase without the addition of the previously required inducer, heparin; (2) the use of batch processes to adapt previous purification schemes to large-scale heparinase production and the subsequent purifica tion of heparinase to a single SDS-PAGE banding protein; (3) the immobilization of heparinase with a 91% activity recovery and good stability, (4) the design and suc cessful testing of a fluidized bed reactor containing immobilized heparinase in the re moval of clinically used quantities of heparin from both human blood in vitro and ca nine blood in vivo; and (5) the initiation of animal studies focusing on the toxicology of heparinase-derived heparin degradation products and the short and long term effects of exposure to these products and to heparinase.     

Carbohydrate analysis of glycoproteins A review

Many of the products prepared by biotechnological approaches, including recombinant genetic engineering, cell tissue culture, and monoclonal technologies, are glycoproteins. As little as five years ago, glycosylation was believed to play no significant role in the function of glycoproteins. Recent large scale testing of glycoprotein-based pharmaceuticals has indicated that both the extent and type of glycosylation can play a central role in glycoprotein activity. Although methods for compositional and sequence analysis of proteins and nucleic acids are generally available, similar methods have yet to be developed for carbohydrate oligomers and polymers. This review focuses on new, developing methods for the analysis and sequencing of the carbohydrate portion of glycoproteins. Included are: (1) the release of oligosaccharides and hydrolysis of carbohydrate chains using enzymatic and chemical methods; (2) fractionation by LPLC, electrophoresis, HPLC, and lectin affinity chromatography; (3) detection through the preparation of derivatives or by new electrochemical methods; (4) analysis by spectroscopic methods, including MS and high-field NMR; and (5) their sequencing through the use of multiple, well-integrated techniques. The ultimate goal of the analytical approaches discussed is to firmly establish structure and, thus, permit the study of structure-function relationships and eventually to allow the intelligent application of carbohydrate remodeling techniques in the preparation of new glycoproteins.     

Polysaccharide lyases

Applied Biochemistry and Biotechnology - Polysaccharide lyases (or eliminases) are a class of enzymes (EC 4.2.2.-) that act to cleave certain activated glycosidic linkages present in acidic...     

Total synthesis of quercetin 3-sophorotrioside

5,7-dihydroxy-3-[beta-D-glucopyranosyl-(1-->2)-beta-D-glucopyranosyl-(1-->2)-beta-D-glucopyranosyl]-2-(3,4-dihydroxyphenyl)-4H-1-benzopyran-4-one (quercetin 3-sophorotrioside), a flavonol triglycoside, isolated from Pisum sativum shoots and showing protective effects on liver injury induced by chemicals, was synthesized for the first time. The target compound was successfully synthesized in eight linear steps and in 39% overall yield through a combination of phase-transfer-catalyzed (PTC) quercetin C-3 glycosylation and silver triflate (AgOTf) promoted carbohydrate chain elongation using both sugar bromide and trichloroacetimidate donors.     

Synthesis of the Bis-Tetrahydropyrano[2,3-b:2′,3′-e] Piperazine Ring System: A New Tricyclic Heterocycle from D-Glucosamine

ABSTRACT

2-Amino-3,4,6-tri-O-benzyl-2-deoxy-D-glucopyranose was transformed into its bis(tetrahydropyranyl)piperazine dimer (4) by reaction with 1,1′-thionyldiimidazole or 1,1′-sulfonyldiimidazole. This dimeric form of glucosamine is the first representative of this previously unknown heterocyclic ring system.     

Regioselective Synthesis of L-Idopyranuronic Acid Derivatives: Intermolecular Aglycon Transfer of Dithioacetal Under Standard Glycosylation Conditions

Abstract

C-1-thioacetalization of L-idofuranurono-6,3-lactone followed by regioselective p-methoxybenzylidenation at C-2 and C-4 gave the hydroxylactones 4 and 19, which were protected at C-5 with TBDMS. Lactone ring opening with methylamine followed by regioselective reductive cleavage of the 1,3-dioxane furnished acceptors 9 and 24. Intermolecular ethyl and phenyl thio group transfers were observed during the attempted preparation of disaccharide 35 through coupling reactions of either 9 or 24 with trichloroacetimidate donor 33, leading to the formation of thioglycoside of donor 33 and the thioglycoside of acceptors 9 or 24 in the furanose form. This intermolecular aglycon transfer was investigated under various glycosylation conditions. Finally, the free 4-hydroxyl groups in acceptors 9 and 24 were acetylated. Desilylation at C-5 followed by ring closure with mercuric salts afforded, in both cases, the IdopA donor and/or acceptor precursor 16.     

Synthesis of Floridoside

Floridoside (2-O-glycerol-α-D-galactopyranoside) is a natural glycerol glycoside found in red algae and is believed to play important roles in carbon storage, transport, and assimilation and in the regulation of osmotic balance. We describe here a rapid, high-yield, and high-stereoselectivity synthesis of floridoside in which the key step involves the 1,2-cis O-glycosylation of 1,3-dibenzylglycerol with ethyl 2,3,4,6-tetra-O-benzyl-1-thio-β-D-galactopyranoside using iodonium dicollidine perchlorate (IDCP) or N-iodosuccinimide/trimethylsilyl triflate (NIS/TMSOTf) as promoters.