Biomedical subjects
Publications and source records attributed to M Kiso.
Novel analogs of the nonreducing-sugar subunit of bacterial lipid A, which were composed only of 3-hydroxytetradecanoic acid and its homologs, were synthesized. These analogs exhibited significant mitogenic activity.
Structural variations among gangliosides significantly influence their immunosuppressive activity. By total chemical synthesis, a sialic acid-containing glycoconjugate, 2-(tetradecylhexadecyl)-O-(5-acetamido-3,5-dideoxy-D-glycero-alpha -D-galacto-2-nonulopyranosylonic acid)-(2-->3)-O-beta-D-galactopyrannosyl-(1-->4)-beta-D- glucopyrannoside was synthesized. This glycoconjugate has the same carbohydrate structure as does GM3 ganglioside and a branched alkane in place of ceramide. It markedly inhibits the tetanus toxoid-induced human lymphoproliferative response in vitro (ID90 < 7 microM) and is five-fold more active than d18:1-C18:0 GM3 ganglioside, to which it is structurally related. This glycoconjugate is also a potent inhibitor of the murine alloimmune response in vivo: 10 nmol of the molecule injected subcutaneously together with an allogeneic cell challenge markedly inhibits the cellular immune response in the draining popliteal lymph node. In fact, the effect is quantitatively similar to that of systemically administered cyclosporin A, a well-studied immunosuppressive agent.
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Sialidase L releases 2,7-anhydro-NeuAc from sialoglycoconjugates (Li, Y.-T., Nakagawa, H., Ross, S. A., Hansson, G., and Li, S.-C. (1990) J. Biol. Chem. 265, 21629-21633). This enzyme has been purified more than 10,000-fold from Macrobdella leech. The final preparation gives a single protein band on SDS-polyacrylamide gel electrophoresis with the molecular mass of 84 kDa. The pI is determined to be 6.0 using isoelectric focusing. With 4-methylumbelliferyl-alpha-NeuAc as substrate, the pH optimum is between pH 5.5-7.0. Unlike regular sialidases, sialidase L is not inhibited by 2-deoxy-2,3-dehydro-NeuAc. Two of the seven tryptic peptides derived from sialidase L contain the consensus repeat S-X-D-X-G-X-T-W that has been found in the regular sialidases. Among various sialoglycoconjugates tested, sialidase L cleaves only the NeuAc alpha 2-->3Gal linkage. NeuAc alpha 2-->6Gal, NeuAc alpha 2-->6GalNAc, NeuAc alpha 2-->6GlcNAc, NeuAc alpha 2-->8-NeuAc, and NeuAc alpha 2-->9NeuAc linkages are not hydrolyzed. At pH 7.0, sialidase L and Clostridial sialidase release 46 and 92% of sialic acid, respectively, from bovine fetuin, indicating that sialidase L selectively cleaves NeuAc alpha 2-->3Gal linkages in fetuin. Sialidase L is the first sialidase found to exhibit a strict specificity toward the hydrolysis of the NeuAc alpha 2-->3Gal linkage, and it should become useful for the selective cleavage of NeuAc alpha 2-->3Gal linkages in sialoglycoconjugates without destroying other sialosyl linkages.
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Analogues of sialyl-lactotetraosylceramide, sialyl-neolactotetraosylceramide, and sialyl Lewis X ganglioside, in which the N-acetylneuraminic acid residue is replaced by a 3-deoxy-D-glycero-D-galacto-2-nonulopyranosylonic acid (KDN) unit, have been synthesized. Methyl O-(methyl 4,5,7,8,9-penta-O-acetyl-3-deoxy-D-glycero-alpha-D-galacto-2- nonulopyranosylonate)-(2-->3)-2,4,6-tri-O-benzoyl-1-thio-beta-D- galactopyranoside (4) was prepared from 2-(trimethylsilyl)ethyl O-(methyl 4,5,7,8,9-penta-O-acetyl-3-deoxy-D-glycero-alpha-D-galacto-2- nonulopyranosylonate)-(2-->3)-6-O-benzoyl-beta-D-galactopyranoside , via O-benzoylation, replacement of the 2-(trimethylsilyl)ethyl group by acetyl, and introduction of the methylthio group with trimethyl(methylthio)silane. Glycosylation of 2-(trimethylsilyl)ethyl O-(2-acetamido-4,6-O-benzylidene-2-deoxy-beta-D-glucopyranosyl)-(1-->3)- O- (2,4,6-tri-O-benzyl-beta-D-galactopyranosyl)-(1-->4)-2,3,6-tri-O-benzyl- beta- D-glucopyranoside (5) or of 2-(trimethylsilyl)ethyl O-(2-acetamido-3,6-di-O-benzyl-2-deoxy-beta-D-glucopyranosyl)-(1-->3)-O- (2,4,6-tri-O-benzyl-beta-D-galactopyranosyl)-(1-->4)-2,3,6-tri-O-benzyl- beta- D-glucopyranoside, prepared from 5 via O-benzylation and reductive opening of the benzylidene acetal ring, with 4 as a donor gave the corresponding pentasaccharides 9 and 13 in good yields. In the same way, 4 was reacted with 2-(trimethylsilyl)ethyl O-(2,3,4-tri-O-benzyl-alpha-L-fucopyranosyl)-(1-->3)-O-(2-acetamido-6- O-benzyl-2-deoxy-beta-D-glucopyranosyl)-(1-->3)-O-(2,4,6-tri-O-benzyl-be ta- D-galactopyranosyl)-(1-->4)-2,3,6-tri-O-benzyl-beta-D-glucopyranoside to yield the hexasaccharide 17. These three oligosaccharides 9, 13, and 17 were converted via reductive removal of the benzyl groups and benzylidene group, O-acetylation, removal of the 2-(trimethylsilyl)ethyl group, and subsequent reaction with trichloroacetonitrile, into the corresponding trichloroacetimidates 12, 16, and 20, respectively. Glycosylation of (2S,3R,4E)-2-azido-3-O-benzoyl-4-octadecene-1,3-diol with 12, 16, and 20 in the presence of boron trifluoride etherate afforded the expected beta-glycosides, which were transformed via selective reduction of the azido group, coupling with octadecanoic acid, O-deacylation, and de-esterification, into the target gangliosides in high yields.
Several neutral and sialylated pyridylamino (PA) oligosaccharides were separated on an amine-bearing silica column, PALPAK Type N. Neutral PA-oligosaccharides were fractionated according to the number of sugar residues by amine adsorption. Sialylated PA-oligosaccharides were separated by ion-exchange chromatography. An amine-bearing column was eluted with a mobile phase consisting of acetonitrile and water containing acetic acid titrated to pH 7.3 with triethylamine (TEAA buffer). A mixture of neutral and sialylated PA-oligosaccharides was separated by double-mode HPLC with a solvent program of decreasing acetonitrile concentration (70 to 51%) with a constant TEAA buffer concentration (0.03 M), and then an increasing TEAA buffer concentration (0.03 to 0.49 M) with a constant acetonitrile concentration (51%). This HPLC technique was applied to the comparative oligosaccharide pattern analysis of human Asn-linked oligosaccharides of normal and pathological IgG by hydrazinolysis. The result indicated clearly that oligosaccharides of IgG myeloma proteins have different core structures and ratios of sialic acid than those of IgG normal proteins.
Methyl thioglycoside derivatives of 2-, 3-, and 4-deoxy-L-fucopyranose have been prepared as glycosyl donors for the synthesis of sialyl Le(x) ganglioside analogues containing modified alpha-L-fucopyranose residues. Reductive dethioacylation of 2-(trimethylsilyl)ethyl 3,4-di-O-benzoyl-2-O-(phenoxy)thiocarbonyl-beta-L-fucopyranoside, prepared from L-fucose in eight steps, gave the 2-deoxy compound, which was transformed via selective removal of the 2-(trimethylsilyl)ethyl group, subsequent acetylation, and displacement of the 1-acetoxy group by a methylthio group, into methyl 3,4-di-O-benzoyl-2,6-dideoxy-1-thio- alpha,beta-L-lyxo-hexopyranoside (11). 2-(Trimethylsilyl)ethyl 2,4-di-O-benzoyl-3-O-(phenoxy)thiocarbonyl-beta- L-fucopyranoside, prepared from the unsubstituted glycoside in four steps, and 2-(trimethylsilyl)ethyl 2,3-di-O-benzoyl-4-O-(phenoxy)thiocarbonyl-beta-L-fucopyranoside, similarly prepared in two steps, were transformed via reduction of the (phenoxy)thiocarbonyloxy group, selective removal of the 2-(trimethylsilyl)ethyl group, O-acetylation, displacement of the 1-acetoxy group by a methylthio group as described for 11, and finally replacement of the benzoyl groups by benzyl groups, into the analogous, protected methyl 3- and 4-deoxy-1-thio-beta-L-fucopyranosides.
Sialyl Lex ganglioside analogs containing 2-, 3-, and 4-deoxyfucose in the place of L-fucose have been synthesized. Glycosylation of 2-(trimethylsilyl)ethyl O-(2-acetamido-4,6-O-benzylidene-2-deoxy-beta-D-glucopyranosyl)-(1-->3)- 2,4,6-triO-benzyl-beta-D-galactopyranoside with the methyl 1-thioglycoside derivatives of the respective deoxyfucoses, using dimethyl(methylthio)sulfonium triflate (DMTST) as a promoter, gave the corresponding three protected 2-(trimethylsilyl)ethyl dideoxy-alpha-L-heoxpyranosyl-(1-->3)- O-2(acetamido-2-deoxy-beta-glucopyranosyl)-(1-->3)-beta-D-galactop yranosides. These were transformed by reductive ring-opening of their benzylidene acetal groups into the glycosyl acceptors 6, 8, and 10. Dimethyl(methylthio)sulfonium triflate promoted glycosylation of 6, 8, and 10 with methyl O-(methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-alpha- D-galacto-2-nonulopyranosylonate)-(2-->3)-2,4,6-tri-O-benzoyl-1-th io-beta-D-galactopyranoside afforded the desired pentasaccharides, which were converted via reductive removal of their benzyl groups, O-acetylation, selective removal of the 2-(trimethylsilyl)ethyl group, and reaction with trichloroacetonitrile, into the corresponding alpha-trichloroacetimidates 14, 18, and 22. Glycosylation of (2S,3R,4E)-2-azido-3-O-benzoyl-4-octadecene-1,3-diol with 14, 18, and 22 in the presence of boron trifluoride etherate afforded the expected beta-glycosides, which were transformed in good yields, via selective reduction of the azido group, coupling with octadecanoic acid, O-deacylation, and deesterification, into the target compounds.
Lipid A is the active principle of lipopolysaccharide (LPS). Synthetic lipid A analogues with monosaccharide backbones, GLA-60, GLA-69 and GLA-58, which exhibit potent, weak and scarce agonistic activities of LPS, respectively, induced tolerance against LPS lethality in galactosamine-(GalN)-sensitized mice while none of them were pyrogenic in rabbits. The tolerance-inducing mechanisms were investigated focusing on the regulation of tumor-necrosis-factor-alpha(TNF-alpha)-mediated lethal pathway of LPS. Induction of serum TNF-alpha in LPS-challenged mice was suppressed by prior administration of these analogues as well as LPS. Prior treatment of murine macrophages with the substances suppressed LPS-stimulated TNF-alpha production in the culture supernatant and TNF-alpha mRNA expression in the cells as well. Lethal toxicity of TNF-alpha in GalN-sensitized mice was effectively suppressed by prior treatment with LPS, GLA-60 and GLA-69 but not by GLA-58. This protective effect was suggested to be mediated by endogenous TNF-alpha, which was induced by prior treatment with the effective substances, because either neutralization of endogenously induced TNF-alpha activity with an antibody or deletion of its induction by using LPS-resistant C3H/HeJ mice reduced the protective effect, and a detectable amount of TNF-alpha was produced by stimulating macrophages with the effective substances but not with GLA-58. These results indicated that multiple regulation steps (one is prior to and the others are following TNF-alpha production) are participating in the tolerance induction by LPS and some lipid A analogues and that GLA-58 is a characteristic compound which induces the tolerance by only blocking the step prior to TNF-alpha production.
In earlier studies of oligosaccharide probes (neoglycolipids) generated from an ovarian cystadenoma glycoprotein, one of the components that strongly supported binding of the endothelial adhesion molecule, E-selectin, was identified as an equimolar mixture of tetrasaccharides of blood group Le(a) and Le(x) type sulfated at position 3 of the outer galactose (C.-T. Yuen, A. M. Lawson, W. Chai, M. Larkin, M. S. Stoll, A. C. Stuart, F. X. Sullivan, T. J. Ahern, and T. Feizi (1992) Biochemistry 31, 9126-9131). In the present studies, the individual sulfated Le(a) and sulfated Le(x) oligosaccharides synthesized chemically have been investigated, first, for their ability to support E-selectin binding when converted into neoglycolipids, and second, for their ability to inhibit E-selectin binding to immobilized lipid-linked sialyl-Le(a), sialyl-Le(x), or sulfated Le(a) pentasaccharides; their activities have been compared with those of the sialyl-Le(a) and sialyl-Le(x) analogues. From these studies, the sulfated Le(a) tetra- and pentasaccharides emerge as the most potent E-selectin ligands so far. In particular, the inhibitory activity of the sulfated Le(a) pentasaccharide is substantially greater than that of the sialyl-Le(x) trisaccharide, which is currently the most widely used inhibitor of E-selectin binding: 45-, 35-, or 15-fold greater depending on whether adhesion is to sialyl-Le(a), sulfated Le(a), or sialyl-Le(x) pentasaccharides, respectively. These findings have an important bearing on design of new generations of inhibitors of E-selectin binding as antiinflammatory compounds.
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A systematic synthesis of the N-methyl-1-deoxynojirimycin-containing oligosaccharides related to the Lewis x, Lewis a, sialyl-Lewis x and sialyl-Lewis a antigens has been achieved. The couplings of the suitably protected 1-deoxynojirimycin derivative 10 with methyl-1-thioglycosides (glycosyl donors) of L-fucose (11), D-galactose (15) and alpha-sialyl-(2-->3)-D-galactose (27) were carried out by using dimethyl(methylthio)sulfonium triflate (DMTST) or N-iodosuccinimide/trifluoromethanesulfonic acid (NIS/TfOH) as the glycosyl promoter. The resulting di- and tri-saccharides were each converted, by further cross glycosylations with 11, 15 or 27, to the desired tri- and tetra-saccharides 3-6 that inhibit the recognition between sialyl-Lewis x and selectins, a family of leukocyte cell adhesion molecules.
Recognition specificity for sialylsugar chains by the hemagglutinin of influenza B viruses isolated in different years from 1940 through 1990 (B/Lee/40, B/Setagaya/3/56, B/Tokyo/7/66, B/Kagoshima/1/68, B/Gifu/2/73, B/Kanagawa/3/76, B/Ibaraki/2/85, B/Yamagata/16/88, and B/Bangkok/163/90) was studied using 13 gangliosides. Reactivity of the viruses' hemagglutinin binding to gangliosides was determined by using thin-layer chromatography/virus-binding assay, and also by measuring virus binding to erythrocytes modified by incubation with gangliosides in terms of the absorbance of hemoglobin released from the infected cells. Eight strains preferentially recognized a novel ganglioside, carrying lacto-series type I and II sugar chains with the Neu5Ac alpha 2-6Gal linkage. It was found that B/Gifu/2/73 strain binds to lacto-series gangliosides containing Neu5Ac alpha 2-6Gal and Neu5Ac alpha 2-3Gal linkages. Other gangliosides studied, including GM4, GM3(alpha 2-3), GM3(alpha 2-6), GM2, GM1a, GD3, GD1a, GD1b, and GT1b, were poor receptors.
We have investigated the inhibitory mechanism of the initial arrest of L5178Y-ML25 lymphoma cells in a target organ (liver) by using recombinant fibronectin fragments with cell- and/or heparin-binding domains (C-274, H-271 or the fusion fragment CH-271). Pretreatment of hepatic sinusoidal endothelial (HSE) cell monolayers with lymphoma cells or their conditioned medium for 4 to 6 h resulted in the enhancement of lymphoma cell adhesion to HSE cell monolayer. The increased tumor adhesiveness was completely abolished by preincubation of the conditioned medium with anti interleukin-1 beta monoclonal antibody (mAb). Synthetic sialyl Le(x) (SLe(x)) as a ligand for endothelial cell leukocyte adhesion molecule-1 (ELAM-1) adhesion receptor and anti ELAM-1 mAb blocked the conditioned medium-induced enhancement of tumor-endothelial cell interaction, while pretreatment of the activated HSE cell monolayer with anti vascular cell adhesion molecule-1 (VCAM-1) mAb did not affect the enhanced tumor cell adhesion. These results indicate that tumor cell interaction with the stimulated HSE cells is mediated by ELAM-1 molecules on HSE cells. However, the expression of SLe(x) and SLe(a) on the tumor surface was not observed by flow cytometric analysis. ELAM-1-mediated enhancement of tumor cell adhesion to HSE monolayer was also inhibited in a concentration-dependent manner by CH-271 fusion polypeptide or the sulfated chitin derivative sulfated carboxymethyl-chitin, which can bind to the heparin-binding domain of CH-271. In addition, CH-271 inhibited not only tumor-endothelium interaction but also tumor cell invasion into reconstituted basement membrane Matrigel in vitro.