Azra Pervin

Separation of negatively charged carbohydrates by capillary electrophoresis

Capillary electrophoresis (CE) has recently emerged as a highly promising technique consuming an extremely small amount of sample and capable of the rapid, high-resolution separation, characterization, and quantitation of analytes. CE has been used for the separation of biopolymers, including acidic carbohydrates. Since CE is basically an analytical method for ions, acidic carbohydrates that give anions in weakly acid, neutral, or alkaline media are often the direct objects of this method. The scope of this review is limited to the use of CE for the analysis of carbohydrates containing carboxylate, sulfate, and phosphate groups as well as neutral carbohydrates that have been derivatized to incorporate strongly acidic functionality, such as sulfonate groups.

NMR solution conformation of heparin-derived tetrasaccharide.

The solution conformation of the homogeneous, heparin-derived tetrasaccharide delta UA2S(1-->4)-alpha-D-GlcNpS6S(1-->4)-alpha-L-IdoAp2S (1-->4)-alpha-D-GlcNpS6S (residues A, B, C and D respectively, where IdoA is iduronic acid) has been investigated by using 1H- and 13C-NMR. Ring conformations have been defined by J-coupling constants and inter-proton nuclear Overhauser effects (NOEs), and the orientation of one ring with respect to the other has been defined by inter-ring NOEs. NOE-based conformational modelling has been done by using the iterative relaxation matrix approach (IRMA), restrained molecular dynamics simulations and energy minimization to refine structures and to distinguish between minor structural differences and equilibria between various ring forms. Both glucosamine residues B and D are in the 4C1 chair conformation. The 6-O-sulphate group is oriented in the gauche-trans configuration in the D ring, whereas in the B ring the gauche-gauche rotomer predominates. Uronate (A) and iduronate (C) residues are mostly represented by 1H2 and 2S0 twisted boat forms, respectively, with small deviations in expected coupling constants and NOEs suggesting minor contributions from other A and C ring conformations.

LOW MOLECULAR WEIGHT DERMATAN SULFATE AS AN ANTITHROMBOTIC AGENT STRUCTURE-ACTIVITY RELATIONSHIP STUDIES

A structure-activity relationship of low molecular weight dermatan sulfate was undertaken to understand better this new non-heparin, glycosaminoglycan-based antithrombotic agent. A dermatan sulfate prepared from bovine intestinal mucosa [average molecular weight (MWavg) 25,000], and currently in clinical trials as an antithrombotic agent, was used in this study. Dermatan sulfate was partially depolymerized using hydrogen peroxide and copper(II) as catalyst to MWavg 5600 to obtain a low molecular weight dermatan sulfate. This low molecular weight dermatan sulfate was then fractionated by gel permeation chromatography to obtain four subfractions having MWavg 7800, 5500, 4200 and 1950. The dermatan sulfate, low molecular weight dermatan sulfate and its subfractions showed substantially different optical rotations. The 1H-NMR spectroscopic analysis of dermatan sulfate samples showed some differences including increased content of GalpNAc4S6S residues and improved resolution in ring resonances for low molecular weight dermatan sulfate fractions, primarily the result of reduced molecular weight and lowered heterogeneity. Saccharide compositional analysis relied on chondroitin ABC lyase treatment followed by capillary electrophoresis. Polyacrylamide gel-based oligosaccharide mapping was also performed by treating dermatan sulfate samples with chondroitin B, AC and ABC lysases. These analyses showed increased amounts of sulfation as the MWavg decreased. In vitro bioassay showed maximum anti-Xa activity in the 4.2 kDa fraction and maximum heparin cofactor II-mediated anti-IIa activity in the 5.5 kDa fraction. The in vivo antithrombotic activity of these fractions was measured using a modified Wessler stasis thrombosis model. The 4.2 kDa fraction showed greater antithrombotic activity than the other low molecular weight dermatan sulfate fractions, dermatan sulfate, and low molecular weight dermatan sulfate. This enhanced activity may result from several structural features of the 4.2 kDa fraction including: a high content of 4,6- and 2,4-disulfated disaccharide sequences; the requirement of specific chain length; a change in the ratio of iduronic to glucuronic acid; and the presence of chondroitin ABC lyase resistant material.

Comparison of the activity of two chondroitin AC lyases on dermatan sulfate

Chondroitin sulfates are widely distributed in human and animal tissues and are the main constituents of cartilage’. The two major isomeric chondroitin sulfates, A (ChS-A) and C (ChS-C) are isolated as glycosaminoglycan from the proteoglycans present in tissues’. Structural investigations have shown that ChS-A and ChS-C both are co-polymers of D-glucuronic acid and sulfated 2-acetamido-2-deoxy-pgalactose. ChS-A primarily contains 4-sulfated 2-acetamido-2-deoxy-D-galactose residues, while ChS-C primarily contains 6-sulfated 2-acetamido-2-deoxy-o-galactose residues. Dermatan sulfate (DS) is a related glycosaminoglycan, often called chondroitin sulfate B (ChS-B), composed of 4-sulfated 2-acetamido-2-deoxy-ogalactose residues. DS differs from ChS-A and ChS-C since its primary uranic acid residue is L-iduronic acid instead of p-glucuronic acid. Despite these structural differences ChS-A, ChS-C, and DS also contain the minor uranic acid C-S epimer (L-iduronic acid in ChS-A and ChS-C and p-glucuronic acid in DS) in their structures. The chondroitin lyases depolymerize the ChS-A, ChS-C, and DS by an elimination mechanism into oligosaccharides containing a A,,,-unsaturated uranic acid residue at the nonreducing end3T4 (Fig. 1). This residue exhibits an absorbance maximum at 232 nm permitting the detection of the oligosaccharide products of the chondroitin lyases using UV spectroscopy. The chondroitin lyases include ABC, AC, B, and C lyases and are microbial enzymes produced by the using ChS or DS as an inducer3*4. Two different chondroitin AC lyases are commercially available. Chondroitin AC lyase I (AC Flavo) is prepared from Fluvobacterium heparinum and chondroitin AC lyase II (AC Arthro) is prepared from Arthrobacter aurescens. F. heparinum also contains a chondroitin B lyase while A. aurescens does not3. Other bacteria also reportedly produce chondroitin AC lyase including: