Synthesis of undecaprenyl pyrophosphate-linked glycans as donor substrates for bacterial protein N-glycosylation

Synthesis of undecaprenyl pyrophosphate (Und-PP)-linked glycans is described. Bacterial ([E]₃,[Z]₇)-undecaprenol was synthesized from trans-geranylgeranyl sulfone and isoprenoid building blocks, which was converted to undecaprenyl phosphate (Und-P). It was coupled with glycosyl phosphates to afford Und-PP-linked glycans, including core trisaccharide of Campylobacter jejuni N-glycan. Our synthetic method for Und-PP-linked glycan would provide various substrates as a useful tool for systematic analysis of bacterial protein N-glycosylation.     

Efficient synthesis of kaempferol 3,7-O-bisglycosides via successive glycosylation with glycosyl ortho-alkynylbenzoates and trifluoroacetimidates

Selective glycosylation of the 3-OH of 5,4′-di-O-acetyl-kaempferol was achieved with glycosyl ortho-alkynylbenzoates as donors under the catalysis of Ph₃PAuNTf₂, and subsequent glycosylation of the remaining 7-OH with glycosyl trifluoroacetimidates under the catalysis of BF₃·OEt₂, after global deprotection, afforded the kaempferol 3,7-O-bisglycosides conveniently.     

Efficient synthesis of core 2 class glycosyl amino acids by one-pot glycosylation approach

We describe the one-pot synthesis of core 2 class branched oligosaccharides initiated by chemo-selective glycosylation of silyl ether. Glycosylation of 6-O-silyl-4-benzyl-2-azido-thiogalactoside with glycosyl fluoride provided selectively 6-glycosylated thioglycoside without both O-glycosylation at the 3 position and S-glycosylation. Subsequent coupling of galactosyl fluoride and amino acids afforded the protected branched oligosaccharides in good yields.     

Characterization of the N-linked glycosylation site of recombinant pectate lyase

Recombinant pectate lyase from Aspergillus niger was overexpressed in Aspergillus nidulans. The two recombinant proteins produced differed in molecular mass by 1200 Da, which suggested that the larger molecular weight protein was glycosylated. The deduced amino acid sequence was searched for potential N-linked glycosylation sites, and one potential site was identified at residue 64. The proteins were analyzed for their ability to bind various lectins as an assay for the presence of carbohydrates. The proteins were then digested with trypsin to facilitate the isolation of the potential glycosylation site. The resulting digestion products were subsequently analyzed by liquid chromatography/mass spectrometry using in-source collision induced dissociation to detect glycopeptides. Once the glycopeptide had been identified, treatment with an endoglycosidase both verified the location of glycosylation and identified the mass of the glycan. The Complex Carbohydrate Structural Database was searched for possible N-linked structures with the same mass, and the suggested primary sequence was confirmed by an exoglycosidase digestion. The data demonstrated that the larger recombinant protein contained a high mannose N-linked structure (Man(5)GlcNAc(2)) attached to N-64, while this site was not occupied in the smaller protein.     

Halobenzoyl groups in glycosylation: effect on stereoselectivity and reactivity of glycosyl donors

Described herein is the synthesis and evaluation of a series of glycosyl donors equipped with halobenzoyl substituents at O(4) and O(6) to study their properties in glycosylations. Among possible effects that may include carbonyl participation or H-bond mediated aglycone delivery, our results indicate that halobenzoyls act via a different mode.     

Efficient synthesis of lupane-type saponins via gold(I)-catalyzed glycosylation with glycosyl ortho-alkynylbenzoates as donors

Glycosylation of the acid labile betulin and betulinic acid derivatives was achieved with glycosyl ortho-hexynylbenzoates as donors under the catalysis of PPh(3)AuNTf(2); this enabled the efficient synthesis of lupane-type saponins, as exemplified by the total synthesis of the proposed betulinic acid trisaccharide from Bersama engleriana.     

Inositolphosphoglycan mediators structurally related to glycosyl phosphatidylinositol anchors: synthesis, structure and biological activity

The preparation of the pseudopentasaccharide 1a, an inositol-phosphoglycan (IPG) that contains the conserved linear structure of glycosyl phosphatidylinositol anchors (GPI anchors), was carried out by using a highly convergent 2+3-block synthesis approach which involves imidate and sulfoxide glycosylation reactions. The preferred solution conformation of this structure was determined by using NMR spectroscopy and molecular dynamics simulations prior to carrying out quantitative structure--activity relationship studies in connection with the insulin signalling process. The ability of 1a to stimulate lipogenesis in rat adipocytes as well as to inhibit cAMP dependent protein kinase and to activate pyruvate dehydrogenase phosphatase was investigated. Compound 1a did not show any significant activity, which may be taken as a strong indication that the GPI anchors are not the precursors of the IPG mediators.     

Significant role of bacterial undecaprenyl diphosphate (C55-UPP) for rubber synthesis by Hevea latex enzymes

Washed bottom fraction (BF) membrane-bound particles of centrifuged fresh Hevea latex were found to be very active in rubber biosynthesis (RB). The washed BF membrane (WBM) showed higher RB activity and is strongly stimulated by anionic surfactants--more by DOC than SDS. WBM enzymes system can synthesize rubber either with allylic isoprenes (higher RB) or without (lower RB). Washed rubber particles (WRP), used generally in RB assays, had very low RB activity compared to the much higher activity observed for WBM. Bacterial undecaprenyl diphoshate (C(55)-UPP) was very active allylic initiator for rubber synthesis by WBM. Comparisons of allylic UPP with the shorter ones (C(15)-FPP, C(20)-GGPP) showed that UPP was the most effective. WBM activity orders were UPP > GGPP > FPP. The DOC activated WBM synthesized less polyprenyl intermediates (butanol extractable) but more final rubber product (toluene/hexane extract), different than FPP and GGPP. WBM enzymes were highly versatile in using diverse different allylics, but UPP was most preferable. WRP was found a little active for UPP with DOC, but still much lower than WBM. Rubber product analysis by RP-TLC with acetone/hexane solvent system showed that WBM was mostly rubber, but WRP was mainly the intermediates. Quantitative analysis showed that WBM labeled rubber was confined to the origin spot, different than WRP as mainly labeled intermediates. It was thus confirmed that the WBM plays the key role in RB functions, and not WRP as mostly reported. WBM served as the actual rubber synthesis site, and bacterial UPP was very good RB initiator.     

Traceless Staudinger ligation of glycosyl azides with triaryl phosphines: stereoselective synthesis of glycosyl amides

Alpha-glycosyl amides can be synthesized from the corresponding O-benzyl-alpha-glycosyl azides using a traceless Staudinger ligation with diphenylphosphanyl-phenyl esters 4. All the phosphines employed and their phenol precursors are stable to air at 4 degrees C for months. Fast intramolecular trapping of the reduction intermediates results in the direct formation of the amide link, which, in turn, prevents epimerisation and allows retention of configuration at the anomeric carbon. Yields and alpha-selectivity are high when the reaction is performed in polar aprotic solvents. Removal of the benzyl ether protecting groups is achieved by catalytic hydrogenation. Alpha-glycosyl amides represent a class of virtually unexplored nonhydrolyzable monosaccharide derivatives that may find a useful application as sugar mimics. Conformational studies by NMR spectroscopy confirm that deprotected alpha-glycosyl amides in the gluco, galacto, and fuco series retain the normal pyranose conformation of the monosaccharide. The reaction of phosphines 4 with tetra-O-acetyl-glycosyl azides is nonstereoconservative, and beta-glycosyl amides are obtained in good yields and with complete stereoselectivity starting from both alpha and beta azides.     

A glycosylation reaction: Conversion of methyl glicosides to glycosyl chlorides by boron trichloride

Methyl glycosides react readily with boron trichloride in dichloromethane solutions at −78 °C to give the corresponding glycosyl chlorides for subsequent use in glycosylation reactions. This reaction is compatible with the presence of glycosyl linkages in the molecule, as well as benzyl and acetyl protecting groups.     

2-(Hydroxycarbonyl)benzyl glycosides: a novel type of glycosyl donors for highly efficient beta-mannopyranosylation and oligosaccharide synthesis by latent-active glycosylation

2-(Benzyloxycarbonyl)benzyl (BCB) glycosides were prepared by coupling of the corresponding tetraacetylglycosyl bromides and benzyl 2-(hydroxymethyl)benzoate. The BCB glycosides were converted almost quantitatively into the corresponding 2-(hydroxycarbonyl)benzyl (HCB) glycosides by selective hydrogenolysis of the benzyl ester functionality without affecting the benzylidene acetal and the benzyl ether. Treatment of the HCB 4,6-O-benzylidenemannopyranoside 4 with triflic anhydride in the presence of di-tert-butylmethylpyridine and subsequent addition of the glycosyl acceptor having a primary hydroxyl group afforded exclusively the disaccharide of the beta-mannopyranosyl linkage. Glycosylation of the compound 4 with secondary and tertiary alcohols also provided beta-mannopyranosides as the major products. Glycosylation of the HCB 4,6-O-cyclohexylidenemannoside 5 with primary alcohols was also highly beta-selective, and the HCB 2,3-O-cyclohexylidenemannoside 6 exhibited the moderate beta-selectivity. On the other hand, unlike the HCB mannosides, the HCB 4,6-O-benzylideneglucoside 7 gave exclusively the disaccharides of the alpha-glycopyranosyl linkage in the glycosylation with primary alcohols. The latent BCB-disaccharide 23, which was obtained from the HCB mannoside 4 as the donor and the BCB glucoside 12 as the acceptor by the present glycosylation method, was converted into the active HCB-disaccharide 39 by selective hydrogenolysis. Repetitive glycosylation of the donor 39 with the same acceptor 12 afforded the BCB-trisaccharide 40. Other BCB-trisaccharides 42 and 46 were also efficiently synthesized by employing the present methodology.     

Molecular requirements of imino sugars for the selective control of N-linked glycosylation and glycosphingolipid biosynthesis

N-Butyl-deoxynojirimycin (NB-DNJ) has been approved for clinical trials as a potential therapy for Gaucher disease, a glycolipid lysosomal storage disorder. As this compound has both glycoprotein processing α-glucosidase and ceramide glucosyltransferase inhibitory activity, we have sought to determine the molecular basis for these two activities. NB-DNJ is known to resemble the positively charged oxocarbonium-like transition state for α-glucosidase I and the structure–function relationships we present now help to define the recognition epitope for the enzyme. Inhibition of ceramide glucosyltransferase by NB-DNJ was competitive for ceramide (K_i=7.4 μM) and non-competitive for UDP-glucose, indicating inhibitory activity is by ceramide mimicry. The presence of an N-alkyl chain was obligatory for transferase inhibition and increases in alkyl chain length provided a modest increase in inhibitory potency.By contrast, α-glucosidase inhibition was independent of the N-alkyl chain and changes in chain length. The effects of ring substitutions identified the C₃ hydroxyl group as being critical for both enzymes but C₁ and C₆ modifications led to a loss of transferase inhibition only. Attempts to rationalise these data for transferase inhibition using an energy minimised molecular model of NB-DNJ and ceramide predicted structural homology of three stereogenic centres and the N-alkyl chain of NB-DNJ, with the trans-alkenyl and N-acyl chain of ceramide. On the basis of these studies, modifications to imino sugar inhibitors can be suggested that allow a more selective approach for molecular inhibition of both ceramide glucosyltransferase and α-glucosidase I, leading to improved compounds for the potential treatment of lysosomal glycosphingolipid storage disorders and viral infections, respectively.     

Direct glycosylation: synthesis of alpha-indoline ribonucleosides

A selective synthesis of α-anomers of indoline nucleosides is described. Ribonucleosides of indoline, dimethylindoline and 5-bromoindoline are readily prepared in good yield by reacting indoline bases directly with the protected sugar, 2,3-O-(1-methylethylidene) 5-O-(triphenylmethyl)-D-ribofuranose in dry ethanol or methylene chloride in presence of molecular sieves at 40-60 °C.     

Synthesis of glycosyl phosphates from 1,2-orthoesters and application to in situ glycosylation reactions

[reaction: see text] A series of glycosyl phosphates were prepared in high yield by treatment of the corresponding 1,2-orthoesters with dibutyl phosphate. Glycosyl phosphates are efficient glycosylating agents even when used in crude form or when generated in situ. The immunodominant epitope trirhamnoside of group B Streptococcus was prepared to demonstrate the synthetic utility of the method.     

Linear dependence of the glycosylation stereoselectivity ofO-trityl ethers by carbohydrate 1,2-O-cyanoalkylidene derivatives on anion concentrations. Effect of substituents in the glycosyl acceptor and a new mechanism of 1,2-cis-glycosides formation

The dependence of the stereochemical outcome of trityl-cyanoalkylidene condensation (/-ratio of disaccharides) on the concentration of a catalyst (TrClO₄) was studied. The dependence was shown to be linear over a wide range of concentrations of the catalyst. The mechanism of the reaction and the effect of the nature of protective groups in the glycosyl acceptor on the stereochemistry of glycosylation are discussed. A new mechanism of 1,2-cis-glycosides formation is proposed.Translated fromIzvestiya Akademii Nauk. Seriya Khimicheskaya, No. 6, pp. 1158–1163, June, 1995.The present work was partially supported by the Russian Foundation for Basic Research (Project No. 93-03-18196) and the International Science Foundation (Grant MBV 000).     

A new method for the synthesis of glycosyl fluorides

Neat (diethylamino)sulfur trifluoride (DAST) has been found to react with the free anomeric hydroxyl of suitably protected aldoses and ketoses to afford glycosyl fluorides.     

Reactions of difluoroenoxysilanes with glycosyl donors: synthesis of difluoro-C-glycosides and difluoro-C-disaccharides

Difluoroenoxysilanes, prepared from acylsilanes and trifluoromethyltrimethylsilane under fluoride activation, were glycosylated with some glycosyl donors (acylglycosides, glycals) to yield difluoro-C-glycosides with a difluoromethylene group in the place of the anomeric oxygen. This reaction strongly depends on the substituent in the 2-position of the glycosyl donor. Application of this methodology to a xylose-derived acylsilane led to the formation of difluoro-C-disaccharides as an isosteric O-glycosyl mimetic.     

Donor–acceptor ferrocenyl triazines: synthesis and properties

A series of star shaped donor–π–acceptor type symmetrical triazine was designed and synthesized by the Pd-catalysed Sonogshira cross coupling reaction. The ferrocenyl moiety with different spacer groups acts as donor and 1,3,5-triazine as acceptor. The photophysical results show intramolecular charge transfer from ferrocene to the 1,3,5-triazine unit. The cyclic voltammetric analysis reveals substantial donor–acceptor interaction. The triazines exhibit good thermal stability with high decomposition temperature.     

Amberlyst 15 as a mild and effective activator for the glycosylation with disarmed glycosyl trichloroacetimidate donors

Amberlyst 15 acidic resin has been shown to be a mild and effective activator for the glycosylation with commonly used disarmed glycosyl trichloroacetimidate donors. Glucosylation, galactosylation, rhamnosylation, and lactosylation of a panel of representative alcohol and thiol acceptors promoted by Amberlyst 15 allowed for the formation of structurally diverse O- or S-linked oligosaccharides and glycosylated amino acids in moderate to excellent yields.     

Expedient synthesis of triazole-linked glycosyl amino acids and peptides

[structure: see text] An expedient, high-yielding synthesis of two types of triazole-linked glycopeptides is described. These novel and stable glycopeptide mimics were prepared via Cu(I)-catalyzed [3 + 2] cycloaddition of either azide-functionalized glycosides and acetylenic amino acids or acetylenic glycosides and azide-containing amino acids.