Kinetics of interaction of some α- and β-D-monosaccharides with concanavalin A

The rates of formation and dissociation of concanavalin A with some 4-methylumbelliferyl and p-nitrophenyl derivatives of α- and β-D-mannopyranosides and glucopyranosides were measured by fluorescence and spectral stopped-flow methods. All process examined were uniphasic. The second-order formation rate constants varied only from 6.8 · 10⁴ to 12.8 · 10⁴ M^?. s^?1, whereas the first-order dissociation rate constants ranged from 4.1. to 220 s^?1, all at ph 5.0, I = 0.3 M, and 25°C. Dissociation rates thus controlled the value of binding constant. The effect of temperature on these reactions was examined, from which enthalpies and entropies of activation and of reaction could be calculated. The effects of pH at 25°C on the reaction rates of 4-methylumbelliferyl α-D-mannopyranoside and 4-methylumbelliferyl α-D-glucopyranoside with concanavalin A were examined. The value of the binding constant K_ap (derived from the kinetics) at any pH could be related to the intrinsic binding constant K by the expression K_ap = K_aK(K_a + [H⁺])^?1. The values of K_a, the ionization constant of the protein segment responsive to sugar binding, were 3 · 10^?4 M and 1 · 10^?4 M for 4-methylumbelliferyl α-D-mannopyranoside and 4-methylumbelliferyl α-D-glucopyranoside, respectively. The binding constant of p-nitrophenyl α-D-mannopyranoside is surprisingly much less sensitive to a pH change from 5.0 to 2.7. Ionic strength had little effect on the binding characteristics of 4-methylumbelliferyl α-D-mannopyranoside to concanavalin A at pH 5.2 and 25°C.     

Synthesis of methyl 3-O- and 2-O-carbamoyl-α-D-mannopyranosides and carbamoyl-group migration between them

Methyl 3-O- and 2-O-carbamoyl-α-D-mannopyranosides, (2 and 3), were synthesized from methyl α-D-mannopyranoside via ammonolysis of a cyclic carbonate or a p-nitrophenoxycarbonate, as shown in Charts 1 and 2. Carbamoyl-group migration between the C-2 and C-3 hydroxyl groups, in methyl α-D-mannopyranoside under alkaline conditions, was also studied.     

Syntheses of methyl ethers of methyl α-D-mannopyranoside,and hydrogen bonding in diastereoisomers of methyl 4-O-(1-ethoxyethyl)-α-D-mannopyranoside

The synthesis of the 4-methyl, the 2,4-dimethyl, and the 2,3,6-trimethyl ethers of methyl α-D-mannopyranoside has been accomplished by the use of selective, benzoyl protecting groups, the 1-ethoxyethyl protecting group, and methylation with diazomethane. Considerable differences were noted in the i.r.- and n.m.r.-spectroscopic and t.l.c. properties of the diastereoisomers of methyl 4-O-[1-ethoxyethyl]-α-D-mannopyranoside. A structure, analogous to that of trans-decalin, maintained by intramolecular hydrogen-bonding is proposed for these compounds. The differences in physical properties of the two diastereoisomers are interpreted on the basis that the (R) isomer has an axially attached methyl group, and, therefore, the ring involved cannot be so stable as that of the (S) isomer.     

Mannose residues on phagocytes as receptors for the attachment of Escherichia coli and Salmonella typhi.

The attachment of Escherichia coli and Salmonella typhi to mouse peritoneal macrophages was inhibited by D-mannose, methyl α-D-mannopyranoside and yeast mannan, but not by any other sugar tested. D-Mannose and its derivatives also inhibited the attachment of E. coli to human polymorphonuclear leucocytes. Mannan inhibited phagocytosis when preincubated with E. coli, but not when preincubated with leucocytes. Attachment of opsonized bacteria to leucocytes was not inhibited by D-mannose or methyl α-D-mannopyranoside nor by any other sugar tested. Our results suggest that the surface of phagocytes, like that of epithelial cells, contains D-mannose residues which serve for the attachment of certain Gram negative bacteria.     

An assay for iduronate sulfatase (Hunter corrective factor)

Acetylation of benzyl α-D-mannopyranoside with acetic anhydride-sodium acetate at room temperature gave crystalline benzyl 2,3,6-tri-O-acetyl-α-D-manno-pyranoside (25%) and benzyl 2,3,4,6-tetra-O-acetyl-α-D-mannopyranoside (≈65%). Similar esterification of benzyl β-D-glucopyranoside yielded the crystalline benzyl 2,4,6-triacetate (66%), whereas the corresponding galactopyranoside gave the crystalline 3,4,6-, 2,3,6-, and 2,4,6-triacetates (3, 25, and 9%. respectively). The structures of these compounds were established by methylation with diazomethane-boron trifluoride etherate and were confirmed by n.m.r. studies.     

Synthesis of methyl 4,6-O-methylene-D-glycopyranosides

Methyl 4,6-O-methylene-D-glycopyranosides having the α-D-altro, α- and β-D-gluco, α-D-manno, and α-D-galacto configurations were prepared in 3.4 to 27.4% yields by condensing formaldehyde from 1,3,5-trioxane with the methyl glucosides in anhydrous 1,4-dioxane at 95° with boron trifluoride as the catalyst. A crystalline methyl 2,3:4,6-di-O-methylene-α-D-mannopyranoside was also isolated. Crystalline methyl 4,6-O-methylene 2,3-di-O-p-tolylsulfonyl-α-D-galacto- and α-D-glucopyranosides were prepared in 78 and 54.4% yields. N.m.r. coupling constants of the 2,3-di-O-acetyl derivatives of the 4,6-O-methylene glycosides were used to establish the Cl(D) conformation for each derivative.     

Biochemical Characterization of C-type Lectin in the Diamondback Moth,Plutella xylostella (Yponomeutidae: Lepidoptera)

Lectins due to their affinity to carbohydrate moiety are involved in diverse functions like cell attachment in embryogenesis, organogenesis and cellular trafficking as well as nonself recognition in immune responses. Agglutinating activity was detectable in Plutella xylostella (Yponomeutidae: Lepidoptera) against 14 different species including bacterial and yeast cells, among which the whole body homogenate of P. xylostella agglutinated Providencia vermicola, Flavobacterium sp., and Saccharomyces cerevisiae with high titers. On analysis of physico-chemical properties, this putative agglutinating factor (s) was specifically dependent on the presence of Ca⁺⁺ for its activity and was reversibly sensitive to EDTA. The agglutinating activity was stable at pH 6–8, but was heat-labile. The agglutinating factor (s) was proteinaceous in nature as it was completely precipitable by ammonium sulphate. Its carbohydrate binding activity was demonstrated by inhibition assay, which revealed that methyl α-D-mannopyranoside inhibited agglutination against P. vermicola. In contrast, P. xylostella parasitized by an endoparasitoid wasp, Cotesia plutellae (Braconidae: Hymenoptera), also showed the agglutination properties with somewhat higher activity than the nonparasitized. Carbohydrate inhibition assay with methyl α-D-mannopyranoside was detectable at one-fold higher concentration in the homogenate of the parasitized larvae, suggesting that the agglutinating factor (s) is inducible or due to de novo parasitism-specific synthesis. These results suggest the presence of calcium-dependent lectin in P. xylostella and an alteration in the agglutinating property by C. plutellae parasitization.     

Agglutination of an Arbovirus by Concanavalin A

MANY enveloped viruses contain carbohydrates as components of glycoproteins^1–5 or glycolipid⁶. We have found (unpublished results) that the envelope of Semliki Forest virus (SFV), a group A arbovirus, contains a glycoprotein in which the principal sugars are mannose, galactose and N-acetylglucos-amine and also a glucose-containing glycolipid. It was therefore possible that the virus would react with concanavalin A, the phytohaemagglutinin from jackbean, which specifically binds sugar residues with the α-D-mannopyranoside, α-D-glucopyranoside and α-D-glucosaminyl residues, at branched or terminal non-reducing ends of polysaccharides⁷.     

Interactions of an ovalbumin glycopeptide with concanavalin A.

The interaction of a highly purified glycopeptide isolated from ovalbumin with Concanavalin A has been investigated by measuring solvent proton relaxation rates over a wide range of magnetic fields. We find that binding of the glycopeptide to Mn-Ca-Concanavalin A uniformly reduces the solvent proton relaxation rates in the same manner as that of simple saccharides such as methyl α-D-mannopyranoside, but that the magnitude of the reduction is not as great. Furthermore, we observe that the glycopeptide is capable of precipitating the lectin, and that the precipitation reaction can be readily reversed by addition of methyl α-D-mannopyranoside. The latter results indicate that the branched chain glycopeptide appears to be bivalent with respect to binding by the lectin.     

Slow mono- and biphasic Ca2+-binding kinetics to Ni2+-concanavalin A

Biphasic kinetics of Ca²⁺ binding to Ni²⁺-concanavalin A were studied at pH 5.2 and 16 °C in the min-time range, using 4-methylumbelliferyl α-D-mannopyranoside as an indicator. The results contrast with the monophasic Ca²⁺-induced decrease of Tyr absorption followed in the same time range. Both kinetic phenomena can be consistent with two protomeric forms of dimeric Ni²⁺-concanavalin A in solution.     

Optimizing lectin-carbohydrate interactions: improved binding of divalent α-mannosylated ligands towards Concanavalin A

The synthesis and binding properties to Jack bean phytohaemagglutinin in (Concanavalin A, Con A) of a new family of divalent α-D-mannopyranoside ligands are described. The synthesis of these ligands is based on the coupling of commercially available diamines to p-isothiocyanatophenyl 2,3,4,6 tetra-O-acetyl-α-D-mannopyranoside (4). The resulting dimers 6, 15 to 22 and 30 were tested for their relative inhibitory potency by solid-phase enzyme-linked lectin assays (ELLA) using methyl α-D-mannopyranoside as standard. Divalent mannosylated ligand 35 bearing a non-aromatic aglycon was also tested for comparison purposes. Concentrations necessary for 50% inhibition (IC50s) of binding of yeast mannan to Jack bean phytohaemagglutinin (Con A) were determined. The inhibitions showed dimers to be approximately 10- to 90-fold more potent than methyl α-D-mannopyranoside. Variations in the intra-mannosyl distance proved to be an important factor for optimum binding. This revised version was published online in November 2006 with corrections to the Cover Date.     

Concanavalin A binds of purified prolyl hydroxylase and partially inhibits its enzymic activity.

Prolyl hydroxylase [(EC 1.14.11.2; prolyl-glycyl peptide, 2-oxoglutarate dioxygenase (4-hydroxylating)] was electrophoresed on polyacrylamide gels and the enzyme in the gels was shown to bind [acetyl-³H]concanavalin A. The enzyme-lectin complex was dissociated by treating the gel with methyl α-D-mannopyranoside, a sugar known to inhibit binding of concanavalin A to glycoproteins. Furthermore, prolyl hydroxylase activity was partially inhibited by concanavalin A when the enzyme was assayed in the absence of bovine serum albumin, a protein which enhances enzymic activity. The inhibition of enzyme activity was prevented by sugars known to react with concanavalin A.     

Towards a stable noeuromycin analog with a D-manno configuration: synthesis and glycosidase inhibition of D-manno-like tri- and tetrahydroxylated azepanes

Noeuromycin is a highly potent albeit unstable glycosidase inhibitor due to its hemiaminal function. While stable D-gluco-like analogs have been reported, no data are available for D-manno-like structures. A series of tri- and tetrahydroxylated seven-membered iminosugars displaying either a D-manno-or a L-gulo-like configuration, were synthesized from methyl α-D-mannopyranoside using a reductive amination-mediated ring expansion as the key step. Screening towards a range of commercial glycosidases demonstrated their potency as competitive glycosidase inhibitors while cellular assay showed selective albeit weak glycoprotein processing mannosidase inactivation.     

Syntheses of methyl ethers of methyl α-D-mannopyranoside, and hydrogen bonding in diastereoisomers of methyl 4-O-(1-ethoxyethyl)-α-D-mannopyranoside

The synthesis of the 4-methyl, the 2,4-dimethyl, and the 2,3,6-trimethyl ethers of methyl α-D-mannopyranoside has been accomplished by the use of selective, benzoyl protecting groups, the 1-ethoxyethyl protecting group, and methylation with diazomethane. Considerable differences were noted in the i.r.- and n.m.r.-spectroscopic and t.l.c. properties of the diastereoisomers of methyl 4-O-[1-ethoxyethyl]-α-D-mannopyranoside. A structure, analogous to that of trans-decalin, maintained by intramolecular hydrogen-bonding is proposed for these compounds. The differences in physical properties of the two diastereoisomers are interpreted on the basis that the (R) isomer has an axially attached methyl group, and, therefore, the ring involved cannot be so stable as that of the (S) isomer.     

The α-glucosidases of Dictyostelium discoideum: I. Identification and characterization

We have identified four isozymes of α-glucosidase in the cellular slime mold, Dictyostelium discoideum. The isozymes can be distinguished by their physical and enzymatic properties. α-Glucosidase-1, α-glucosidase-2, and α-gluocosidase-3 are all present in vegetative cells, while α-glucosidase-4 is present only after the cells have proceeded through aggregation. Three of the four enzymes, α-glucosidase-1, α-glucosidase-3, and α-glucosidase-4, have acidic pH optima of 3.5, 2.2, and 4.0, respectively. In contrast, α-glucosidase-2 has a neutral pH optimum, 7.25. α-Glucosidase-1, α-glucosidase-2, and α-glucosidase-3 are distinguishable by electrophoresis in native polyacrylamide gels. α-Glucosidase-4 comigrates with α-glucosidase-2 on native gels but they can be resolved by isoelectric focusing. The isozymes also differ with respect to affinity for the substrates p-nitrophenyl-α-d-glucoside and 4-methyl-umbelliferyl-α-d-glucopyranoside and the relative maximal rates of hydrolysis of these substrates. α-Glucosidases-1, -2, and -4 have apparent K_m's in the millimolar range while the apparent K_m of α-glucosidase-3 for p-nitrophenyl-α-d-glucoside is much higher. This may suggest that isozyme 3 is an endoglycosidase or may have greater affinity for other sugar substrates. α-Glucosidase-1 is the major isozyme in vegetative cells.     

The synthesis of some deoxygenated analogues of early intermediates in the biosynthesis of glycosylphosphatidylinositol (GPI) membrane anchors

Syntheses are described of 2-azido-4,6-di-O-benzyl-2,3-dideoxy-d-ribo-hexopyranosyl fluoride, 6-O-acetyl-2-azido-3-O-benzyl-2,4-dideoxy-d-xylo-hexopyranosyl fluoride and 2-azido-3,4-di-O-benzyl-2,6-dideoxy-d-glucopyranosyl fluoride. These glycosyl donors were coupled with the acceptor 1d-2,3,4,5-tetra-O-benzyl-1-O-(4-methoxybenzyl)-myo-inositol and the α-coupled products were transformed into α-d-3dGlcpN-PI, α-d-4dGlcpN-PI and α-d-6dGlcpN-PI by way of the H-phosphonate route. Brief mention is made of the biological evaluation of these deoxy-sugar analogues and their N-acetylated forms as candidate substrate/inhibitors of the N-deacetylase and α-(1→4)-d-mannosyltransferase activities present in trypanosomal and HeLa (human) cell-free system.     

Synthesis of the 6-methyl and 3,6- and 4,6-dimethyl ethers of methyl 2-acetamido-2-deoxy-α-D-mannopyranoside

The 6-mono- (6) and 4,6- (16) and 3,6-di-methyl (25) ethers of methyl 2-acetamido-2-deoxy-α-D-mannopyranoside have been synthesized from 6-O-trityl, 4,6-O-benzylidene, and 3-O-methyl derivatives, respectively, by way of O-benzoyl and of O-allyl derivatives. The yields were respectively 37 and 43% for 6, 34 and 50% for 16, and 14 and 25% for 25. These ethers are used as standard compounds for the structure elucidation, by methylation, of polymers containing 2-amino-2-deoxy-D-mannose.     

A conformational change in concanavalin A detected by a calorimetric study of the binding of methyl α-D-mannopyranoside

The binding of methyl α-D-mannopyranoside to the lectin concanavalin A was studied by means of calorimetry. An apparent enthalpy of binding was also calculated from the variation of the equilibrium constant with temperature (van't Hoff ΔH). The ΔH measured directly was ?30 to ?38 kJ/mole indicating that the binding is driven by the ΔH change. In contrast, the van't Hoff ΔH was substantially smaller, about zero at pH 5.2. The difference in the ΔH measured directly and the van't Hoff ΔH implies that the conformation of concanavalin A undergoes a temperature dependent change at both pH's but most predominantly at pH 5.2. The existence of this conformational change was verified by difference absorption spectroscopy.     

Synthesis of 4-O-β-D-mannopyranosyl-L-rhamnopyranose

The title disaccharide (16) has been synthesized in 50% overall yield by way of condensation of 4,6-di-O-acetyl-2,3-O-carbonyl-α-D-mannopyranosyl bromide 5 with methyl 2,3-O-isopropylidene-α-L-rhamnopyranoside (1) in chloroform solution, in the presence of silver oxide. The disaccharide was characterized as the crystalline isopropyl alcoholate of methyl 4-O-β-D-mannopyranosyl-α-L-rhamnopyranoside (11) and as 1,2,3-tri-O acetyl-4-O- (2,3,4,6-tetra-O-acetyl-β-D-mannopyranosyl)-α-L-rhamnopyranose (15). Methyl β-D-mannopyranoside isopropyl alcoholate 7 was readily obtained in 85% yield via the reaction of bromide 5 with methanol.Reduction of 2,3-di-O-methyl-L-rhamnose with sodium borohydride, followed by acetylation, may result in the formation of an appreciable proportion of a boric ester, namely 1,5-di-O-acetyl-4-deoxy-2,3-di-O-methyl-L-rhamnitol-4-yl dimethyl borate, depending on the procedure used.     

Synthesis of the 3- and 4-methyl, 3,4-dimethyl, and 3,4,6-trimethyl ethers of methyl 2-acetamido-2-deoxy-alpha-D-mannopyranoside

The methyl ethers of 2-amino-2-deoxy-D-mannose are reference compounds in studies, by the methylation procedure, of the chemical structure of polysaccharides containing 2-amino-2-deoxy-D-mannose and 2-amino-2-deoxy-D-mannuronic acid residues. Methylation of methyl 2-acetamido-2-deoxy-α-D-mannopyranoside (1) gave the 3,4,6-trimethyl ether. Methylation of the 6-trityl ether of 1, followed by detritylation, gave the 3,4-dimethyl ether of 1. Methylation of the 4,6-O-benzylidene derivative (6) of 1, followed by removal of the benzylidene group, gave the 3-methyl ether of 1. Benzoylation of 6, followed by removal of the benzylidene group and monobenzoylation, gave the 3,6-dibenzoate of 1, which was methylated, and the product saponified, to give the 4-methyl ether of 1; the latter compound was also obtained by a similar route via the 3-O-acetyl-6-O-benzoyl derivative.