Synthesis of oligosaccharide fragments of O-specific Shigella flexneri polysaccharides. II. Synthesis of trisaccharide Glc alpha1----3RHa alpha1----2Rha alpha1----OMe and tetrasaccharide GlcNAc beta1----2(Llc alpha1----3)Rha alpha1----2Rh alpha1----OMe

Methyl glycosides of the title linear trisaccharide and branched tetrasaccharide were synthesized by stepwise glycosylation. These oligosaccharides represent the fragments of O-antigenic polysaccharides of Shigella flexneri serotypes 2b, 3a, 5b, and X.     

Solution conformation of asparagine-linked oligosaccharides: alpha(1-2)-, alpha(1-3)-, beta(1-2)-, and beta(1-4)-linked units

The solution conformation is presented for representatives of each of the major classes of asparaginyl oligosaccharides. In this report the conformation of alpha(1-3)-, alpha(1-2)-, beta(1-2)-, and beta(1-4)-linked units is described. The conformational properties of these glycopeptides were determined by high-resolution 1H nuclear magnetic resonance in conjunction with potential energy calculations. The NMR parameters that were used in this analysis were chemical shifts and nuclear Overhauser enhancements. Potential energy calculations were used to evaluate the preferred conformers available for the different linkages in glycopeptides and to draw conclusions about the behavior in solution of these molecules. It was found that the linkage conformation of the Man alpha 1-3 residues was not affected by substitution either at the 2-position by alpha Man or beta GlcNAc or at the 4-position by beta GlcNAc or by the presence of a bisecting GlcNAc on the adjacent beta Man residue.     

Homonuclear three-dimensional NMR methods for the complete assignment of proton NMR spectra of oligosaccharides--application to Gal beta 1-4(Fuc alpha 1-3)GlcNAc beta 1-3Gal beta 1-4Glc.

Two new homonuclear three-dimensional NMR techniques are described for the simplification of proton resonance assignment in oligosaccharides, namely HOHAHA-COSY and ROESY-COSY. The former technique is of value in the resonance assignment of gluco-configuration monosaccharide residues, whereas the latter is more suited to resonance assignment of galacto-configuration monosaccharide residues. The value of these techniques is illustrated by application to the proton resonance assignment of the pentasaccharide Gal beta 1-4(Fuc alpha 1-3)GlcNAc beta 1-3 Gal beta 1-4Glc, a compound which exhibits a variety of assignment problems due to severe cross-peak overlap in conventional COSY or HOHAHA spectra.     

Primary structure of neutral and acidic oligosaccharide-alditols derived from the jelly coat of the Mexican axolotl. Occurrence of oligosaccharides with fucosyl(alpha 1-3)fucosyl(alpha 1-4)-3-deoxy-D-glycero-D-galacto-nonulosonic acid and galactosyl(alpha 1-4)[fucosyl(alpha 1-2)]galactosyl(beta 1-4)-N-acetylglucosamine sequences.

Six major neutral and acidic oligosaccharide-alditols were prepared from the jelly coat of Mexican axolotl eggs. These compounds were demonstrated to contain 3-deoxy-D-glycero-D-galacto-nonulosonic acid (dNloA) and L-fucose (Fuc). The structures of the six major oligosaccharides were established as follows: [sequence: see text]     

Structural determination of three neutral oligosaccharides in bovine (Holstein-Friesian) colostrum, including the novel trisaccharide; GalNAc alpha 1-3Gal beta 1-4Glc

Two trisaccharides, and a pentasaccharide were obtained from bovine colostrum. Their chemical structures were determined by using methylation and 13C-NMR analyses as follows: GalNac alpha 1-3Gal beta 1-4Glc, Gal alpha-1-3Gal beta 1-4Glc, GaL beta 1-3[Gal beta 1-4GlcNAc beta 1-6]Gal beta 1-4Glc. GalNAc alpha 1-3Gal beta 1-4Glc, which was identified in this study, is a novel oligosaccharide from natural sources. Gal alpha 1-3Gal beta 1-4Glc and Gal beta 1-3[Gal beta 1-4GlcNAc beta 1-6]Gal beta 1-4Glc (lacto-N-novopentaose) have been already found in ovine colostrum, and in horse colostrum and marsupial milk, respectively.     

Isolation and characterization of CNBr peptides of human (alpha 1 (III) )3 collagen and tissue distribution of (alpha 1 (I) )2 alpha 2 and (alpha 1 (III) )3 collagens.

[Alpha 1(III)]3 collagen was solubilized by pepsin digestion of normal human placental membranes and was purified by differential salt precipitation and carboxymethylcellulose chromatography. This collagen was digested with CNBr, and the resultant nine peptides were isolated and characterized. The chains are cross-linked by cysteinyl residues in the COOH-terminal peptide. Isolation of peptides derived from CNBr digestion of insoluble tissues was used as an assay for the presence of [alpha 1(I)]2alpha 2 and [alpha 1(III)]3 collagens. Both types are present in human skin, intestine, liver, spleen, kidney, lung, aorta, umbilical cord, placental membranes, and myocardium. Bone and tendon contain [alpha 1(I)]2alpha 2 collagen but, unlike the other tissues, lack [alpha 1(III)]3 collagen. Both [alpha 1(I)]2alpha 2 and[alpha 1(III)]3 collagens are present in scars of human skin, myocardium, tendon, and liver and of rabbit skin. The degree of hydroxylation of proline was 4 to 5% lower in the same peptides in skin, bone, and tendon than in the other tissues. The degree of hydroxylation of lysine in the same peptides derived from different tissues varied more widely.     

Synthesis of allyl 6-O-(3-deoxy-alpha- and -beta-D-manno-oct-2- ulopyranosylonic acid)-(1----6)-2-deoxy-2-[(3R)-3-hydroxytetradecanamido]- beta-D-glucopyranoside 4-phosphate and of the copolymer of the alpha anomer with acrylamide.

The title disaccharides were synthesized by mercuric cyanide-catalyzed condensation of methyl (4,5,7,8-tetra-O-acetyl-3-deoxy-alpha-D-manno-oct-2-ulopyranosyl bromide)onate and allyl 2-[(3R)-3-acetoxy-tetradecanamido]-3-O-benzyl-2-deoxy-beta-D- glucopyranoside, followed by phosphorylation of the alcoholic function of the amino sugar. The phosphorylated anomers were separated by chromatography and deprotected by conventional methods. Polymeric material was obtained by copolymerisation, catalyzed by peroxosulphate and N,N,N',N'-tetramethylethylenediamine, of the alpha anomer with acrylamide; it contained ca. one disaccharide unit for 18 acrylamide residues.     

Synthesis of O-alpha-L-fucopyranosyl-(1----3)-O-beta-D-galactopyranosyl-(1----4)-2- acetamido-2-deoxy-D-glucopyranose (N-acetyl-3'-O-alpha-L-fucopyranosyllactosamine)

Methyl 2-O-benzyl-beta-D-galactopyranoside (6) was obtained in five, good yielding steps from methyl beta-D-galactopyranoside (1). Treatment of 1 with tert-butylchlorodiphenylsilane in N,N-dimethylformamide in the presence of imidazole afforded a 6-(tert-butyldiphenylsilyl) ether, which was converted into its 3,4-O-isopropylidene derivative (3). Benzylation of 3 with benzyl bromide-silver oxide in N,N-dimethylformamide, and subsequent cleavage of its acetal and ether groups then afforded 6. On similar benzylation, followed by the same sequence of deprotection, benzyl 2-acetamido-3,6-di-O-benzyl-4-O-[6-O-(tert-butyldiphenylsilyl)-3,4 -O- isopropylidene-beta-D-galactopyranosyl]-2-deoxy-alpha-D-glucopyranoside gave the 2-O-benzyl derivative (10). Compound 10 was converted into its 4,6-O-benzylidene acetal (11). Glycosylation (catalyzed by halide-ion) of 11 with 2,3,4-tri-O-benzyl-alpha-L-fucopyranosyl bromide afforded the fully protected trisaccharide derivative (13). Cleavage of the benzylidene and then the benzyl groups of 13 furnished the title trisaccharide (16). The structure of 16 was established by 13C-n.m.r. spectroscopy.     

Enzymatic in vitro synthesis of radiolabeled pentasaccharides GlcNAc beta 1-3(Gal beta 1-4GlcNAc beta 1-6)Gal beta 1-4GlcNAc/Glc and the isomeric Gal beta 1-4GlcNAc beta 1-3(GlcNAc beta 1-6)Gal beta 1-4GlcNAc/Glc.

Four radiolabeled pentasaccharides, GlcNAc beta 1-3(Gal beta 1-4GlcNAc beta 1-6)Gal beta 1-4GlcNAc, Gal beta 1-4GlcNAc beta 1-3(GlcNAc beta 1-6)Gal beta 1-4GlcNAc, GlcNAc beta 1-3(Gal beta 1-4GlcNAc beta 1-6)Gal beta 1-4Glc, and Gal beta 1-4GlcNAc beta 1-3(GlcNAc beta 1-6)Gal beta 1-4Glc, were prepared in virtually pure form. They were obtained by partial enzymic beta 1,4-galactosylations of the appropriate tetrasaccharide acceptors or by partial enzymic degalactosylations of the appropriate hexasaccharides, followed by paper chromatographic separations. All four pentasaccharides contain two nonidentical distal branches, making them valuable primers for enzymatic in vitro synthesis of larger oligo(N-acetyllactosaminoglycans).     

The crystal structures of Man(alpha1-3)Man(alpha1-O)Me and Man(alpha1-6)Man(alpha1-O)Me in complex with concanavalin A.

The crystal structures of concanavalin A in complex with Man(alpha1-6)Man(alpha1-O)Me and Man(alpha1-3)Man(alpha1-O)Me were determined at resolutions of 2.0 and 2.8 A, respectively. In both structures, the O-1-linked mannose binds in the conserved monosaccharide-binding site. The O-3-linked mannose of Man(alpha1-3)Man(alpha1-O)Me binds in the hydrophobic subsite formed by Tyr-12, Tyr-100, and Leu-99. The shielding of a hydrophobic surface is consistent with the associated large heat capacity change. The O-6-linked mannose of Man(alpha1-6)Man(alpha1-O)Me binds in the same subsite formed by Tyr-12 and Asp-16 as the reducing mannose of the highly specific trimannose Man(alpha1-3)[Man(alpha1-6)]Man(alpha1-O)Me. However, it is much less tightly bound. Its O-2 hydroxyl makes no hydrogen bond with the conserved water 1. Water 1 is present in all the sugar-containing concanavalin A structures and increases the complementarity between the protein-binding surface and the sugar, but is not necessarily a hydrogen-bonding partner. A water analysis of the carbohydrate-binding site revealed a conserved water molecule replacing O-4 on the alpha1-3-linked arm of the trimannose. No such water is found for the reducing or O-6-linked mannose. Our data indicate that the central mannose of Man(alpha1-3)[Man(alpha1-6)]Man(alpha1-O)Me primarily functions as a hinge between the two outer subsites.     

Three-dimensional structure of a glycosphingolipid having a novel carbohydrate linkage, Gal beta 1-4(Fuc alpha 1-3)Glc beta 1-3Gal beta, determined by theoretical calculations

The novel glycosphingolipid, SEGLx (Gal beta 1-4(Fuc alpha 1-3)Glc beta 1-3Gal beta Cer), which was identified by us (Kawakami Y, et al. (1993) J Biochem 114: 677-83), shows a characteristic spectrum on 1H-NMR analysis, in which the anomeric proton resonances of a reducing end galactose and a glucose are split. To elucidate the structural characteristics of SEGLx, we determined its three-dimensional (3D) structure by means of computer simulation, involving such techniques as molecular mechanics (MM2), the semiempirical molecular orbital method (AM1), molecular dynamics (Amber), and computer 3D modelling. With the hypothesis that all OH group(s) of a ceramide participate in intramolecular hydrogen bonds, two kinds of stable conformers, horizontal and right-angled ones, were formed, depending on the ceramide species. The present findings suggest that the chemical species of both the long chain base and fatty acid moieties, mainly the occurrence of OH group(s), affect the chemical shifts of the anomeric proton resonances not only of the reducing terminal galactose but also the penultimate glucose through the formation of intramolecular hydrogen bonds. Computer simulation through theoretical calculation and 3D modelling was shown to be the best means of confirming the results obtained by experimental analysis.     

Synthesis of (5alpha)-17-azaandrostan-3-ols and (5alpha)-17-aza-D-homoandrostan-3-ols and their N-acylated derivatives.

Two groups of N-acylated D-azasteroids (4 and 5) were synthesized to explore structure-activity relationships for steroid modulation of GABA(A) receptor function. The target compounds were prepared conveniently from (5alpha)-3-hydroxyandrostan-17-ones (6 and 7) via the intermediate (5alpha)-17-aza-D-homoandrostan-3-ols (14 and 15) or (5alpha)-17-azaandrostan-3-ols (18 and 19) precursors in high overall yields. A Beckmann rearrangement and a Hofmann rearrangement were employed as two key steps in the synthetic sequences.     

Structural analysis of trisialylated biantennary glycans isolated from mouse serum transferrin. Characterization of the sequence Neu5Gc(alpha 2-3)Gal(beta 1-3)[Neu5Gc(alpha 2-6)]GlcNAc(beta 1-2)Man

Five variants of mouse serum transferrin (mTf, designated mTf-I to mTf-V) with respect to carbohydrate composition have been isolated by DEAE-cellulose chromatography in the following relative percentages: mTf-I: 0.55; mTf-II: 0.79; mTf-III: 71.80; mTf-VI: 21. 90 and mTf-V: 4.96. The primary structures of the major glycans from mTf-III and mTf-IV were determined by methylation analysis and 1H-nuclear magnetic resonance (NMR) spectroscopy. All glycans possessed a common trimannosyl-N,N'-diacetylchitobiose core. From the glycovariant mTf-III two isomers of a conventional biantennary N-acetyllactosamine type were isolated, in which two N-glycolylneuraminic acid (Neu5Gc) residues are linked to galactose either by a (alpha 2-6) or (alpha 2-3) linkage. A subpopulation of this glycovariant contains a fucose residue (alpha 1-6)-linked to GlcNAc-1. The structure of the major glycan found in variant mTf-IV contained an additional Neu5Gc and possessed the following new type of linkage: Neu5Gc(alpha 2-3)Gal(beta 1-3)[Neu5Gc(alpha 2-6)]GlcNAc(beta 1-2 )Man(alpha 1-3). In addition to this glycan, a minor compound contained the same antennae linked to Man(alpha 1-6). In fraction mTf-V, which was found to be very heterogeneous by (1)H NMR analysis, carbohydrate composition and methylation analysis suggested the presence of tri'-antennary glycans sialylated by Neu5Gc alpha-2,6- and alpha-2, 3-linked to the terminal galactose residues. In summary, mTf glycans differed from those of other analyzed mammalian transferrins by the presence of Neu5Gc and by a Neu5Gc(alpha 2-6)GlcNAc linkage in trisialylated biantennary structures, reflecting in mouse liver, a high activity of CMP-Neu5Ac hydroxylase and (alpha 2-6)GlcNAc sialyltransferase.     

Synthesis and proaggregatory activity of 1-O-(2-methyloctadecyl)-2-O-acetyl-rac-glycero-3-phosphocholine.

Racemic 1-O-(2-methyloctadecyl)-2-O-acetyl-glycero-3-phosphocholine, a branched chain PAF species, was prepared by chemical synthesis and investigated for biological activity on human blood platelets in vitro. The synthesis started from 2-O-benzylglycerol and 2-methyloctadecyl-1-methyl sulfonate and was accomplished in five reaction steps. A comparison with 'octadecyl-rich' PAF showed that the PAF species described here exerts a 22-fold weaker proaggregatory activity. Based on [3H]PAF-binding studies, an obstruction of PAF-binding or the signal transduction by the branched alkyl chain in C-1 position of the glycerol backbone is suggested.     

Characterization of glycosphingolipids from Schistosoma mansoni eggs carrying Fuc(alpha1-3)GalNAc-, GalNAc(beta1-4)[Fuc(alpha1-3)]GlcNAc- and Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc- (Lewis X) terminal structures.

The carbohydrate moieties of glycosphingolipids from eggs of the human parasite, Schistosoma mansoni, were enzymatically released, labelled with 2-aminopyridine (PA), fractionated and analysed by linkage analysis, partial hydrolysis, enzymatic cleavage, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and nano-electrospray ionization mass spectrometry. Apart from large, highly fucosylated structures with five to seven HexNAc residues, we found short, oligofucosylated species containing three to four HexNAc residues. Their structures have been determined as Fuc(alpha1-3)GalNAc(beta1-4)[ +/- Fuc (alpha1-3)]GlcNAc(beta1-3)GalNAc(beta1-4)Glc-PA, GalNAc(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-3)GlcNAc(beta1-3)GalNAc(beta1-4) Glc-PA, Fuc(alpha1-3)GalNAc(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-4) GlcNAc(beta1-3)GalNAc(beta1-4)Glc-PA, and Fuc(alpha1-3) GalNAc(beta1-4)[ +/- Fuc(alpha1-2) +/- Fuc(alpha1-2)Fuc(alpha1-3)]Glc NAc(beta1-3)GlcNAc(beta1-3)GalNAc(beta1-4)Glc-PA. The last structure exhibits a trifucosyl sidechain previously identified on the cercarial glycocalyx. These structures stress the importance of 3-fucosylated GalNAc as a terminal epitope in schistosome glycoconjugates. To what degree these glycans contribute to the pronounced antigenicity of S. mansoni egg glycolipids remains to be determined. In addition, we have identified the compounds GlcNAc(beta1-3)GalNAc(beta1-4)Glc-PA, Gal(beta1-4)[Fuc(alpha1-3)]GlcNAc(beta1-3) GalNAc (beta1-4)Glc-PA, the latter of which is a Lewis X-pentasaccharide identical to that present on cercarial glycolipids, as well as Gal(beta1-3)GalNAc(1-4)Gal(1-4)Glc-PA, which corresponds to asialogangliotetraosylceramide and is most probably derived from the mammalian host.     

alpha(1)-Adrenoceptor agonists: the identification of novel alpha(1A )subtype selective 2'-heteroaryl-2-(phenoxymethyl)imidazolines

Novel 2'-heteroaryl-2-(phenoxymethyl)imidazolines have been identified as potent agonists of the cloned human alpha(1)-adrenoceptors in vitro. The nature of the 2'-heteroaryl group can have significant effects on the potency, efficacy, and subtype selectivity in this series. alpha(1A) Subtype selective agonists have been identified.     

Purification and substrate specificity of UDP-D-xylose:beta-D-glucoside alpha-1,3-D-xylosyltransferase involved in the biosynthesis of the Xyl alpha1-3Xyl alpha1-3Glc beta1-O-Ser on epidermal growth factor-like domains

A unique O-glycan structure, Xylalpha1-3Xylalpha1-3Glcbeta1-O-Ser is found on the consensus sequence C-X-S-X-P-C (X denotes any amino acid) in epidermal growth factor (EGF)-like domains of plasma proteins such as clotting factor VII and IX. One of the enzymes involved in the biosynthesis of this trisaccharide, UDP-d-xylose:beta-d-glucoside 1,3-d-xylosyltransferase has been identified in HepG2 cells (Omichi, K., Aoki, K., Minamida, S., and Hase, S. Eur. J. Biochem. 245, 143-146 [1997]). Here, we report that this enzyme activity can be detected in bovine liver and that the enzyme has been purified from the microsomal fraction. The enzyme was purified 6200-fold in terms of specific activity and ran as a single band on native-PAGE and isoelectric focusing gel electrophoresis. The best acceptor substrate of those tested was the EGF-like domain of bovine factor IX carrying beta-glucoside at Ser53. The Km value for this substrate was 34 muM. Comparison of initial velocity with various acceptor substrates shows that this xylosyltransferase recognizes not only the glucose moiety to which xylose is transferred but also the tertiary structure of the EGF-like domain. With regard to the donor substrate, the enzyme does not recognize UDP-d-glucose but does recognize UDP-d-xylose.     

Synthesis and evaluation of N-[(1S,2S)-3-(4-chlorophenyl)-2-(3-cyanophenyl)-1-methylpropyl]-2-methyl-2-aminopropanamide as human cannabinoid-1 receptor (CB1R) inverse agonists

Obesity is a chronic medical condition that is affecting large population throughout the world. CB1 as a target for treatment of obesity has been under intensive studies. Taranabant was discovered and then developed by Merck as the 1st generation CB1R inverse agonist. Reported here is part of our effort on the 2nd generation of CB1R inverse agonist from the acyclic amide scaffold. We replaced the oxygen linker in taranabant with nitrogen and prepared a series of amino heterocyclic analogs through a divergent synthesis. Although in general, the amine linker gave reduced binding affinity, potent and selective CB1R inverse agonist was identified from the amino heterocycle series. Molecular modeling was applied to study the binding of the amino heterocycle series at CB1 binding site. The in vitro metabolism of representative members was studied and only trace glucuronidation was found. Thus, it suggests that the right hand side of the molecule may not be the appropriate site for glucuronidation.     

Synthesis and antitumor activity of 1-mesityl-3-(2-naphthoylmethano)-1H-imidazolium bromide

An imidazolium salt, 1-mesityl-3-(2-naphthoylmethano)-1H-imidazolium bromide (MNIB), has been investigated for its antitumor properties. In vitro studies demonstrate that MNIB is active against K562, SMMC-7721, EJ, AGZY, HEP-2, A549, HepG2, and Raji tumor cells, and can induce the G1 phase cell cycle arrest and apoptosis in K562 cells. Moreover, administration of MNIB significantly inhibited tumor growth in human non-small lung tumor (A549) xenografts.