Monitoring the stability of heparin: NMR evidence for the rearrangement of sulfated iduronate in phosphate buffer.

Heparin, a major anticoagulant drug, comprises a complex mixture of motifs. Heparin is isolated from natural sources while being subjected to a variety of conditions but the detailed effects of these on heparin structure have not been studied in depth. Therefore, the result of exposing heparin to a range of buffered environments, ranging pH values from 7 to 12, and temperatures of 40, 60 and 80 °C were examined. There was no evidence of significant N-desulfation or 6-O-desulfation in glucosamine residues, nor of chain scission, however, stereochemical re-arrangement of α-L-iduronate 2-O-sulfate to α-L-galacturonate residues occurred in 0.1 M phosphate buffer at pH 12/80 °C. The results confirm the relative stability of heparin in environments like those during extraction and purification processes; on the other hand, the sensitivity of heparin to pH 12 in buffered solution at high temperature is highlighted, providing an important insight for heparin manufacturers.

Hydrolytic Degradation of Heparin in Acidic Environments: Nuclear Magnetic Resonance Reveals Details of Selective Desulfation.

Heparin is a complex glycosaminoglycan, derived mainly from pig mucosa, used therapeutically for its anticoagulant activity. Yet, owing largely to the chain complexity, the progressive effects of environmental conditions on heparin structure have not been fully described. A systematic study of the influence of acidic hydrolysis on heparin chain length and substitution has therefore been conducted. Changes in the sulfation pattern, monitored via 2D NMR, revealed initial de-N-sulfation of the molecule (pH 1/ 40 °C) and unexpectedly identified the secondary sulfate of iduronate as more labile than the 6-O-sulfate of glucosamine residues under these conditions (pH 1/ 60 °C). Additionally, the loss of sulfate groups, rather than depolymerization, accounted for most of the reduction in molecular weight. This provides an alternative route to producing partially 2-O-de-sulfated heparin derivatives that avoids using conventional basic conditions and may be of value in the optimization of processes associated with the production of heparin pharmaceuticals.

Thermal degradation and stability of sodium hyaluronate in solid state.

The kinetics and mechanism of depolymerisation of solid sodium hyaluronate at elevated temperatures and various pH have been investigated. Depolymerisation was found to be governed by random chain scission. The activation energy at neutral pH was found to be 127 kJ/mol. The solid polymer was most stable at neutral pH. Results suggest the depolymerisation mechanism in solid- and solution state to be the same. Correlation of log intrinsic viscosity to log weight-average molecular weight was investigated to ensure high quality data for polymer size. Based on more than sixty hyaluronate samples spanning from 0.4 to 2.3 MDa, it was concluded that a second order polynomial regression gives a better fit than the linear regression offered by classical Mark-Houwink-Kuhn-Sakurada description. This finding was supported by literature data and could be expanded to other simple, well behaving linear polymers, such as polystyrene and polyethylene.

Evidence of the presence of 2-O-beta-D-xylopyranosyl-alpha-l-arabinofuranose side chains in barley husk arabinoxylan.

(Glucurono)arabinoxylans were extracted from barley husks and degraded with endo-beta-xylanase or subjected to periodate oxidation. The released oligosaccharide fragments were separated and isolated on Biogel-P2, and their structures were determined by NMR spectroscopy. The oligosaccharides identified consisted of beta-d-(1-->4)-linked xylopyranosyl residues, of which some were substituted at O-3 with alpha-l-arabinofuranosyl groups or at O-2 with 4-O-methylglucuronic acid. In addition to these substituents, a disaccharide side chain, 2-O-beta-d-xylopyranosyl-alpha-l-arabinofuranose, attached at position O-3 of the main chain, was proved to exist in arabinoxylan from barley husks. The compound was fully characterized with NMR, and all (1)H and (13)C NMR signals were assigned. The arabinose to xylose ratio was low (approximately 0.2) and no 2,3-disubstitution existed. No blocks of substituted xylose residues could be observed along the main chain.