Glycolipids modulate glycosyl transfer to endogenous protein acceptors

Regulation by gangliosides of glycosylation of endogenous membrane glycoproteins is indicated from in vitro studies in which incorporation of radioactive sugars into endogenous protein acceptors was measured and from in vitro studies where transferase activities of membranes were correlated with ganglioside content during hepatic tumorigenesis. Galactosyl transfer from UDP galactose exhibited a complex response pattern and was stimulated by lactosyl ceramide and the ganglioside N-acetylgalactosaminyl-(N-acetylneuraminyl)-galactosylglucosylceramide (GM2) but was inhibited by higher gangliosides. Except for N-acetylneuraminylgalactosylglucosylceramide (GM3), which had no effect, inhibition was proportional to ganglioside complexity. Inhibition of glycosylation of the exogenous acceptor, ovomucoid, by ganglioside was slight by comparison. While marked structure-linked latency was observed with the high molecular weight exogenous acceptor, no latency was observed for incorporation into endogenous acceptors suggesting that the membranes were permeable to sugar nucleotides. Membrane disruption with detergents lessened rather than enhanced inhibition by gangliosides. Sialyl transfer from CMPsialic acid, on the other hand, was unaffected or stimulated by gangliosides. Stimulation by galactosyl-N-acetylgalactosaminyl-(N-acetylneuraminyl)-galactosylglucosylceramide (GM1) was proportional to concentration and reached 2-fold at 240 micrograms/mg protein. The results suggest that the ganglioside content of membrane may affect glycosylation of membrane glycoproteins.     

Glycoprotein synthesis in lysolecithin-treated cells using sugar nucleotides as glycosyl donors

The 3T3 cells were treated with 50 mu g/ml lysolecithin (LL) followed by the addition of exogenously supplied radiolabelled sugar nucleotides to serve as direct glycosyl donors. These were found to be 1.5 to 3.0 times more active than untreated cells in their glycosyl transferase activities depending on the particular sugar nucleotide used. Mannosyl transferase activity was not inhibited by 2-deoxyglucose or mannose-1-phosphate, indicating that the sugar nucleotide remained intact throughout the assay period. Preincubation of the cells with tunicamycin caused an 85% decrease in mannosyl transfer, which suggested that the normal pathway of glycosylation via lipid intermediates was still operable in the treated cells. Fractionation of control and LL-treated cells after incubation with UDP[3H]galactose revealed that only microsomal and cytosolic proteins from the treated cells were radioactive. Thus, intracellular labelling of permeabilized cells was allowed. About 80% of the radiolabeled product was glycoprotein in nature, based upon its solubilization with pronase.     

Methods of synthesis of glycosyl fluorides

In recent years glycosyl fluorides have been utilized as versatile sugar donors in the synthesis of natural products and carbohydrates. This paper provides an update on the advances made in the preparation of glycosyl fluorides during the last decade (1988-1998).     

Glycoprotein synthesis in lysolecithin-treated cells using sugar nucleotides as glycosyl donors

Summary The 3T3 cells were treated with 50 μg/ml lysolecithin (LL) followed by the addition of exogenously supplied radiolabelled sugar nucleotides to serve as direct glycosyl donors. These were found to be 1.5 to 3.0 times more active than untreated cells in their glycosyl transferase activities depending on the particular sugar nucleotide used. Mannosyl transferase activity was not inhibited by 2-deoxyglucose or mannose-1-phosphate, indicating that the sugar nucleotide remained intact throughouth the assay period. Preincubation of the cells with tunicamycin caused an 85% decrease in mannosyl transfer, which suggested that the normal pathway of glycosylation via lipid intermediates was still operable in the treated cells. Fractionation of control and LL-treated cells after incubation with UDP[³H]galactose revealed that only microsomal and cytosolic proteins from the treated cells were radioactive. Thus, intracellular labelling of permeabilized cells was allowed. About 80% of the radiolabeled product was glycoprotein in nature, based upon its solubilization with pronase. This work was supported by institutional funds granted by Texas Woman's University.     

Trans-N-deoxyribosylase: substrate specificity studies. Purine bases as acceptors.

A series of purine bases and analogues were tested as substrates for trans-N-deoxyribosylase (EC 2.4.2.6). It was observed that the pyrimidine ring and its substituents on positions 1, 2 and 6, are of minor importance. On the other hand only a few modifications are tolerated on the imidazole moiety, as follows. 1. A tautomeric proton must be present on the imidazole ring. The "usual" shift is between position 9 and 7. 2. The position of the tautomeric proton governs the site of substitution. 3. For steric reasons no substituent is allowed on position 8.     

Significant rate accelerated synthesis of glycosyl azides and glycosyl 1,2,3-triazole conjugates

An efficient and significantly rapid access of a series of glycosyl azides and glycosyl 1,2,3-triazole conjugates is reported using modified one-pot reaction conditions. In both cases yields were excellent and single diastereomers were obtained. Figure Rapid preparation of 4-substituted glycosyl 1,2,3-triazole conjugates from glycosyl bromides. CDRI communication no. 7340.     

Oligosaccharide synthesis by dextransucrase: new unconventional acceptors

The acceptor reactions of dextransucrase offer the potential for a targeted synthesis of a wide range of di-, tri- and higher oligosaccharides by the transfer of a glucosyl group from sucrose to the acceptor. We here report on results which show that the synthetic potential of this enzyme is not restricted to 'normal' saccharides. Additionally functionalized saccharides, such as alditols, aldosuloses, sugar acids, alkyl saccharides, and glycals, and rather unconventional saccharides, such as fructose dianhydride, may also act as acceptors. Some of these acceptors even turned out to be relatively efficient: alpha-D-glucopyranosyl-(1-->5)-D-arabinonic acid, alpha-D-glucopyranosyl-(1-->4)-D-glucitol, alpha-D-glucopyranosyl-(1-->6)-D-glucitol, alpha-D-glucopyranosyl-(1-->6)-D-mannitol, alpha-D-fructofuranosyl-beta-D-fructofuranosyl-(1,2':2,3')-dianhydride, 1,5-anhydro-2-deoxy-D-arabino-hex-1-enitol ('D-glucal'), and may therefore be of interest for future applications of the dextransucrase acceptor reaction.     

Stereoselective synthesis of glycosyl amides by traceless Staudinger ligation of unprotected glycosyl azides

The stereoconservative Staudinger ligation of unprotected alpha- and beta-glucosyl azides with diphenylphosphanyl-phenyl esters to afford alpha- and beta-glucosyl amides is described.     

Use of a phenyl 1-selenogalactofuranoside as a glycosyl donor for the synthesis of galactofuranosyl-containing disaccharides.

The use of acetylated phenyl 1-seleno-beta-D-galactofuranoside as a glycosyl donor for the synthesis of protected D-Galf-beta-(1-->3)-alpha-D-Manp as its methyl or ethylthio glycoside has been demonstrated. Activation of the selenoglycoside over a thioglycoside acceptor by NIS/TfOH is extremely selective and gives the ethylthio disaccharide in 91% yield. The parent disaccharide is found as a terminal and branched unit in the lipopeptidophosphoglycan oligosaccharides of the protozoan Trypanosoma cruzi, the causative agent of Chagas' disease.     

Synthesis of glycosyl phosphoramidates: novel isosteric analogues of glycosyl phosphates.

Several newer isosteric analogues of glycosyl phosphates, namely of glycosyl phosphoramidates, were synthesized in good yields using Staudinger reaction of their corresponding azides with trimethyl phosphite followed by de-O-acetylation. The structure and conformation of the fully protected analogue synthesized, namely 2,3,4,6-tetra-O-acetyl-beta-D-glucopyranosyl bismethoxyphosphoramidate, was established by X-ray crystallography.     

Acetylated methyl glucopyranuronate trichloroacetimidate as a glycosyl donor for efficient synthesis of disaccharides

Allyl (methyl 2,3,4-tri-O-acetyl-beta-D-glucopyranosyl uronate)-(1-->3)-4,6-O-benzylidene-2-deoxy-2-phthalimido-beta-D-glucopyranoside (4) and benzyl (methyl 2,3,4-tri-O-acetyl-beta-D-glucopyranosyl uronate)-(1-->3)-4,6-O-benzylidene-2-deoxy-2-phthalimido-beta-D-glucopyranoside (5) have been efficiently synthesized by coupling allyl 4,6-O-benzylidene-2-deoxy-2-phthalimido-beta-D-glucopyranoside (2) or benzyl 4,6-O-benzylidene-2-deoxy-2-phthalimido-beta-D-glucopyranoside (3) with methyl (2,3,4-tri-O-acetyl-1-O-trichloroacetimidoyl)-alpha-D-glucopyranuronate (1), respectively, using trimethylsilyl triflate as promoter.     

Toward the automated solid-phase synthesis of oligoglucosamines: systematic evaluation of glycosyl phosphate and glycosyl trichloroacetimidate building blocks

Glucosamines are common components of many biologically important oligosaccharides. Reported is a systematic evaluation of glucosamine phosphates and trichloroacetimidates as glycosylating agents for the efficient construction of beta-(1 --> 6) glucosamine linkages. A set of differentially protected glucosamine donors incorporating a host of amine protecting groups, including 2-phthaloyl, benzyloxycarbonyl (Z), trichloroetheoxycarbonyl (Troc) and trichloroacetyl (TCA) protective groups, were prepared. Donors were initially evaluated for reactivity and protecting group compatibility in a solution-phase study with a model 6-hydroxyl galactose acceptor. Based on these results, glucosamine donor 10 was selected for the solution-phase synthesis of a beta-(1 --> 6)-glucosamine pentasaccharide. Finally, building block 10 proved well suited for use in the automated solid-phase synthesis of a repeating unit trisaccharide. An assessment of glucosamine phosphate donors as potential glycosylating agents for a variety of glucosamine linkages is also discussed.     

Study of the stereoselectivity of 2-azido-2-deoxygalactosyl donors: relationship to the steric factors of glycosyl acceptors

The stereoselectivity of a 2-azido-2-deoxygalactosyl (GalN₃) donor is found to strongly depend on the nature of the acceptors in glycosylation reactions. The order of the acceptor, the stereochemistry, and the configuration of the monosaccharide all affect the stereochemistry outcome. More reactive acceptors are observed to favor β-products, while less reactive acceptors afford more α-products.     

Design,synthesis and biological evaluation of azithromycin glycosyl derivatives as potential antibacterial agents

A series of 11,12-cyclic carbonate azithromycin-4″-O-carbamoyl glycosyl derivatives were designed, synthesized, and evaluated as antibacterial agents to search for target compounds with excellent activity. The results of preliminary antibacterial tests against eight strains in vitro revealed that all of the title compounds exhibited improved activities with broad spectrum compared with the parent compound. The glycosylated side chains may be the pharmacophores responsible for the improved activity.     

New developments in the synthesis of phosphopolyprenols and their glycosyl esters.

Efficient methods were developed in our group in recent years for chemical synthesis of polyprenyl phosphates, polyprenyl monophosphate sugars, and polyprenyl diphosphate sugars, which were known to serve as important intermediates in biosynthesis of complex carbohydrates. A simple procedure was developed involving the phosphorylation of aliphatic alcohols with tetra-n-butylammonium dihydrogen phosphate and trichloroacetonitrile. Monophosphates of various natural and modified dolichols and polyprenols, as well as the derivatives of retinol, cholesterol, and nonacosanol, were prepared in high yields. First syntheses of dolichyl thiophosphate and dolichyl hydrogen phosphonate were developed, and these derivatives were of interest as analogs of dolichyl phosphate. Polyprenyl monophosphate sugars, including derivatives of alpha- and beta-anomers of D-glucopyranose, D-galactopyranose, D-mannopyranose, and 2-acetamido-2-deoxy-D-glucopyranose, were obtained smoothly from moraprenyl trichloroacetimidate and acylated glycosyl phosphates after deprotection. A method for the synthesis of polyprenyl diphosphate sugars from polyprenyl phosphoroimidazolidate and unprotected glycosyl phosphates was shown to be applicable for a wide range of the monosaccharide derivatives including hexoses, deoxyhexoses, 2-acetamido-2-deoxyhexoses, and uronic acids. A series of the oligosaccharide derivatives was also prepared by this method.