Inhibition of selectin-mediated cell adhesion and prevention of acute inflammation by nonanticoagulant sulfated saccharides. Studies with carboxyl-reduced and sulfated heparin and with trestatin a sulfate.

Selectins play a major role in the inflammatory reaction by initiating neutrophil attachment to activated vascular endothelium. Some heparin preparations can interact with L- and P-selectin; however, the determinants required for inhibiting selectin-mediated cell adhesion have not yet been characterized. We now report that carboxyl-reduced and sulfated heparin (prepared by chemical modifications of porcine intestinal mucosal heparin leading to the replacement of carboxylates by O-sulfate groups) and trestatin A sulfate (obtained by sulfation of trestatin A, a non-uronic pseudo-nonasaccharide extracted from Streptomyces dimorphogenes) exhibit strong anti-P-selectin and anti-L-selectin activity while lacking antithrombin-mediated anticoagulant activity. In vitro experiments revealed that both compounds inhibited P-selectin- and L-selectin-mediated cell adhesion under laminar flow conditions. Moreover, carboxyl-reduced and sulfated heparin and trestatin A sulfate were also active in vivo, as assessed by experiments showing 1) that microinfusion of trestatin A sulfate reduced by 96% leukocyte rolling along rat mesenteric postcapillary venules and 2) that both compounds inhibited (by 58-81%) neutrophil migration into thioglycollate-inflamed peritoneum of BALB/c mice. These results indicate that nonanticoagulant sulfated saccharides targeted at P-selectin and L-selectin may have therapeutic potential in inflammatory disorders.

Pivalates in the selective protection and activation of maltose for the synthesis of sulfated 3-deoxy-maltosyl-(1-->4)-alpha, alpha-trehalose.

Pivaloylation of maltose gave, in satisfactory yield, 1,2,6,2',3',4',6'-hepta-O-pivaloyl-beta-maltose which was converted to the 3-deoxygenated analogue in a Barton-McCombie reaction. This compound was used directly in a trimethylsilyl triflate-mediated glycosylation reaction with 2,3,6-tri-O-benzyl-alpha-D-glucopyranosyl 2,3-di-O-benzyl-4,6-O-benzylidene-alpha-D-glucopyranoside to give the corresponding maltosyl-(1-->4)-alpha, alpha-trehalose derivative. After deprotection, the monodeoxygenated tetrasaccharide was sulfated; in the reaction product, one compound fully sulfated at the outer pyranose rings predominated.

New synthetic sulfated oligosaccharides prolong survival of cardiac xenografts by inhibiting release of heparan sulfate from endothelial cells.

Binding of recipient natural antibodies to the endothelium of the graft, complement activation, endothelial cell activation, and microvascular thrombosis are major events in the hyperacute rejection of organ xenografts. The aim of this study was to investigate the effects of two new synthetic sulfated oligosaccharides (A and B) on the survival of discordant cardiac xenografts in the guinea pig-to-rat model. In untreated recipients, hyperacute rejection occurred in 5 min (median; range, 4-6 min) and immunohistological analysis of all the grafts revealed deposition of IgM and C3 along the endothelium. Administration of oligosaccharides A and B prior to revascularization prolonged the survival of xenografts in a dose-dependent manner, up to 113 min (median; range, 42-145 min) and 86 min (median; range, 35-108 min), respectively, when doses of 20 mg/kg were used. There were no bleeding complications. Histological examination of the rejected grafts showed a picture of hyperacute rejection, with no difference in IgM and C3 deposition as compared with the untreated animals. In cell culture experiments, the release of heparan sulfate from guinea pig cardiac endothelial cells induced by rat serum was inhibited by both saccharides in a dose-dependent manner. The results indicate that these new synthetic sulfated oligosaccharides are effective for prolongation of discordant xenograft survival, possibly by interfering with endothelial cell activation. Such substances may be of value in other xenotransplant combinations.

Conformational flexibility in highly sulfated beta-D-glucopyranoside derivatives.

Triggered by findings on heparin-like disaccharides, the conformation of sulfated glucopyranosides was investigated. Sodium (methyl 2,3,4-tri-O-sulfonato-beta-D-glucopyranosid)uronate tetrasodium salt is in a conformational equilibrium, to which a non-chair conformation contributes. The same is true for methyl (methyl 2,3,4-tri-O-sulfonato-beta-D-glucopyranosid)uronate trisodium salt, methyl 2,3,4,6-tetra-O-sulfonato-beta-D-glucopyranoside tetrasodium salt, and octa-O-sulfonato-beta, beta-trehalose octasodium salt, with less obvious non-chair contributions. The effect is charge related. The conformational effect, which does not occur in analogous alpha-D-glucopyranoside derivatives, is discussed in terms of the anomeric effect.

Preparation of 4,6-cyclo-hexopyranoses by palladium-mediated intramolecular cyclodehalogenation.

4,6-Cyclo-4,6-dideoxy-hexapyronoses were obtained by palladium-mediated intramolecular cyclodehalogenation. Thus, methyl 2,3-di-O-acetyl-4,6-dideoxy-4,6-diiodo-beta-D-galactopyranoside (3) afforded methyl 2,3-di-O-acetyl-4,5-cyclo-4,6-dideoxy-beta-D-galactopyranoside (5) in 56% yield upon treatment with hydrogen in the presence of palladium-on-charcoal and diethylamine. The structure of 5 was proven by MS, NMR including NOE measurements, and by independent conversion of 4 to 5 by zinc-mediated Wurtz synthesis. Similarly, methyl 2,3-di-O-acetyl-4,6-cyclo-4,6-dideoxy-alpha-D-galactopyranoside (6) and O-(2,3,-di-O-acetyl-4,6-cyclo-4,6-dideoxy-alpha-D-galactopyranosyl)-(1-- >4)-1,2,3,6-tetra-O-acetyl-beta-D-glucopyranose (17) were obtained along with the respective 4,6-dideoxy analogues. Also methyl 2,3-di-O-acetyl-4,6-dideoxy-4,6-diiodo-beta-D-glucopyranoside (19) gave galacto-configured 5 stereoselectively.

All-alpha-D-linked tetra- and penta-saccharide substructures of Trestatin A by block syntheses with triflic anhydride as promoter.

The perbenzylated maltosyl and maltotriosyl fluorides 6 and 16 were treated with 2,3,2',3',6'-penta-O-benzyl-4,6-O-benzylidene-alpha,alpha-trehalose (7) using triflic anhydride as a promoter to give all-alpha-D-linked tetra- and penta-saccharides which were finally deblocked to the free oligosaccharides 4-O-alpha-maltosyl-9 and 4-O-alpha-maltotriosyl-alpha,alpha-trehaloses 18. The 1H NMR spectra of some of the compounds were fully analyzed by 1D TOCSY and ROESY experiments.

Heparin, carboxyl-reduced sulfated heparin, and Trestatin A sulfate. Antiproliferative and anticoagulant activities.

Human smooth muscle cells were used to investigate the antiproliferative activities of sulfated carbohydrates. The antiproliferative potencies of coarse heparin fractions prepared by ultrafiltration increased with the mean molecular-weight, whereas the anticoagulant activities of a high-molecular-weight fraction had submaximal values. Furthermore, the dependence of antiproliferative activity on sulfate content is discussed. Carboxyl-reduction of heparin abolished both antiproliferative and anticoagulant activities. Sulfation of this compound yielded CRS-heparin with restored antiproliferative potency but devoid of antithrombin III-mediated anticoagulant activity. Sulfation of the pseudo-nonasaccharide, Trestatin A, yielded a compound having the highest antiproliferative activity, so far observed for a low-molecular-weight compound, and having only weak anticoagulant properties.

Synthesis of the trisaccharide moiety of gangliotriosylceramide (asialo GM2).

The synthesis of the trisaccharide methyl glycoside beta-D-GalNAc-(1----4)-beta-D-Gal-(1----4)-beta-D-Glc-OMe, which corresponds to the carbohydrate portion of gangliotriosylceramide (asialo GM2), was accomplished by the reaction of 4-O-acetyl-3,6-di-O-benzoyl-2-deoxy-2-phthalimido-D-galactopyranosyl bromide (18) with a benzylated derivative of methyl 4-O-beta-D-galactopyranosyl-beta-D-glucopyranoside. Comparative studies with a 6'-benzyl ether and a 6'-benzoate revealed that the substituent at O-6' is crucial to the outcome of glycosylations at O-4', the ether derivative being much the more reactive. tert-Butyl 4-O-acetyl-3,6-di-O-benzoyl-2-deoxy-2-phthalimido-D-galactopyranoside, which was readily converted into the corresponding bromide 18, was obtained from the gluco derivative via a single-step, crown ether-assisted epimerization.

Artificial carbohydrate antigens: a block synthesis of a linear, tetrasaccharide repeating-unit of the Shigella flexneri variant Y polysaccharide.

Glycosylation of methyl 2,4-di-O-benzoyl-alpha-L-rhamnopyranoside with 2,3,4-tri-O-acetyl-alpha-L-rhamnopyranosyl bromide gave methyl 2,4-di-O-benzoyl-3-O-(2,3,4-tri-O-acetyl-alpha-L-rhamnopyranosyl) -alpha-L-rhamnopyranoside (4) in 93% yield. Conversion of 4 into the corresponding glycosyl bromide was accomplished with dibromomethyl methyl ether. Under Koenigs-Knorr conditions, this bromide reacted with 8-(methoxycarbonyl)octyl 2-O-(2-acetamido-4,6-O-benzylidene-2-deoxy-beta-D-glycopyranosyl)- 3,4-di-O- benzyl-alpha-L-rhamnopyranoside, to provide the protected tetrasaccharide in 91% yield. Removal of blocking groups gave 8-(methoxycarbonyl)octyl O-alpha-L-rhamnopyranosyl-(1---- 3)-O-alpha-L-rhamnopyranosyl-(1---- 3)-O-2-acetamido-2-deoxy-beta-D-glucopyranosyl-(1----2)-alpha-L- rhamnopyranoside. Together with previously synthesized tetrasaccharides of the Shigella flexneri Y O-antigen, this oligosaccharide has been used to study the conformation of O-antigens and to assist in the selection of S. flexneri, variant Y, specific monoclonal antibodies.