Changes in the structure and biological property of N—-O sulfate-transferred, N-resulfated heparin.

H Uchiyama,K Nagasawa

doi:10.1016/s0021-9258(20)89564-7

Abstract

The N----O sulfate transfer of heparin has been investigated as an approach to chemical 3-O-sulfation of the D-glucosamine residues in heparin. The pyridinium salt of porcine heparin was heated at 90 degrees C in solid state for 90 min (in vacuo over P2O5) to effect the transfer of the N-sulfate groups to the HO groups in the polysaccharide, followed by N-resulfation. The product (N----O sulfate-transferred, N-resulfated heparin (ST heparin] was depolymerized with HONO to generate a mixture of di- and higher oligosaccharides. The borohydride-reduced oligosaccharides were separated on Bio-Gel P-4 and DEAE-Sephacel. The disaccharide trisulfate fraction (10.4% yield) was found to be a mixture of nearly equal amounts of IdoA(2-SO4)-AManR(3,6-diSO4) and IdoA(2,3-diSO4)-AManR(6-SO4), where IdoA represents L-iduronic acid and AManR represents the alditol formed by reduction of 2,5-anhydro-D-mannose with NaBH4. Chemical and NMR spectroscopic analyses revealed that the N----O sulfate transfer proceeded preferentially at HO-3 positions in both 6-O-sulfo-D-glucosamine and 2-O-sulfo-L-iduronic acid residues. Chromatography on antithrombin III-Sepharose gel indicated that the structural change involved in ST heparin resulted in an obvious increase in the ability to bind antithrombin III. Biological examination also indicated that this structural change resulted in moderate increases in all the activities (blood anti-clotting, anti-Factor IIa, and anti-Factor Xa) and in the strength of intrinsic fluorescence of antithrombin III.

Highlights

Tigated as an approach tcohemical 3-0-sulfation of the D-glucosamine residues in heparin
Thecommercial heparin was purified by the method of R o d h et al [7] to remove contaminatingdermatansulfate.The purified heparin (1g) dissolved in water was passed througha column product (N+O sulfate-transferred, N-resulfatedhep- of Dowex 50W-X2 cation-exchange arin (ST heparin)) was depolymerized with HONO to resin at room temperature
The borohydride-reducedoligosaccharides were sepa- mg) as awhitepowder.Bovine antithrombin 111 was preparedas rated on Bio-Gel P-4 and DEAE-Sephacel

Summary

EXPERIMENTAL PROCEDURES

Tigated as an approach tcohemical 3-0-sulfation of the D-glucosamine residues in heparin. Identification of a pentasaccharide as the shortest sequence Time Course of N-0 Sulfate Transfer of Heparin-Aliquots of able to activate antithrombi1n11. This uniqueoligosaccharide contains several sulfate groups, including a critically important 3-0-sulfate group in the central D-glucosamine unit of the oligosaccharide [1, 2]. Transfer anSdubsequent N-Resulfationof Heparinsalt of heparin (286 mg), which had been dried over investigated the intramolecularN-0 sulfate transferof hep- P,O, in uacuo a t room temperature, was placed into a Pyrex glass arinasanapproachto chemical 3-0-sulfation of theD- tube (2.5 X 15 cm) and heated at 90 “C for 90 min

ST heparin

ST Heparin

RESULTS AND DISCUSSION

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