Synthesis of benzyl 2-acetamido-2-deoxy-3-O-β-d-fucopyranosyl-α-d-galactopyranoside and benzyl 2-acetamido-6-O-(2-acetamido-2-deoxy-β-d-glucopyranosyl)-2-deoxy-3-O-β-d-fucopyranosyl-α-d-galactopyranoside

Condensation of benzyl 2-acetamido-4,6-O-benzylidene-2-deoxy-α-d-galactopyranoside with 2,3,4-tri-O-acetyl-α-d-fucopyranosyl bromide in 1:1 nitromethane-benzene, in the presence of powdered mercuric cyanide, afforded benzyl 2-acetamido-4,6-O-benzylidene-2-deoxy-3-O-(2,3,4-tri-O-acetyl-β-d-fucopyranosyl)-α-d-galactopyranoside (3). Cleavage of the benzylidene group of 3 with hot, 60% aqueous acetic acid afforded diol 4, which, on deacetylation, furnished the disaccharide 5. Condensation of diol 4 with 2-methyl-(3,4,6-tri-O-acetyl-1,2-di-deoxy-α-d-glucopyrano)-[2,1-d]-2-oxazoline in 1,2-dichloroethane afforded the trisaccharide derivative (7). Deacetylation of 7 with Amberlyst A-26 (OH^?) anion-exchange resin in methanol gave the title trisaccharide (8). The structures of 5 and 8 were confirmed by ¹³C-n.m.r. spectroscopy.     

Synthesis of 3-O-(2-acetamido-2-deoxy-3-O-β-d-galactopyranosyl-β-d-galactopyranosyl)-1,2-di-O-tetradecyl-sn-glycerol

Stereo- and regio-selective synthesis of 3-O-(2-acetamido-2-deoxy-3-O-β-d- galactopyranosyl-β-d-galactopyranosyl)-1,2-di-O-tetradecyl-sn-glycerol by use of 1,2-di-O-tetradecyl-3-O-(3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-β-d-galactopyranosyl)-sn-glycerol as a key intermediate is described.     

Synthesis of 2-acetamido-2-deoxy-3-O-β-d-mannopyranosyl-d-glucose

Condensation of 4,6-di-O-acetyl-2,3-O-carbonyl-α-d-mannopyranosyl bromide with benzyl 2-acetamido-4,6-O-benzylidene-2-deoxy-α-d-glucopyranoside (2) gave an α-d-linked disaccharide, further transformed by removal of the carbonyl and benzylidene groups and acetylation into the previously reported benzyl 2-acetamido-4,6-O-benzylidene-2-deoxy-3-O-(2,3,4,6-tetra-O-acetyl-α-d-mannopyranosyl)-α-d-glucopyranoside. Condensation of 3,4,6-tri-O-benzyl-1,2-O-(1-ethoxyethylidene)-α-d-glucopyranose or 2-O-acetyl-3,4,6-tri-O-benzyl-α-d-glucopyranosyl bromide with 2 gave benzyl 2-acetamido-3-O-(2-O-acetyl-3,4,6-tri-O-benzyl-β-d-glucopyranosyl)-4,6-O-benzylidene-2-deoxy-α-d-glucopyranoside. Removal of the acetyl group at O-2, followed by oxidation with acetic anhydride-dimethyl sulfoxide, gave the β-d-arabino-hexosid-2-ulose 14. Reduction with sodium borohydride, and removal of the protective groups, gave 2-acetamido-2-deoxy-3-O-β-d-mannopyranosyl-d-glucose, which was characterized as the heptaacetate. The anomeric configuration of the glycosidic linkage was ascertained by comparison with the α-d-linked analog.     

Synthesis of methyl 2-acetamido-2-deoxy-3-O-(β-d-galactopyranosyl)-α-d-galactopyranoside from a corresponding thioglycoside derivative

The title disaccharide glycoside was synthesized by halide ion-promoted glycosidation, using methanol and the disaccharide bromide derived from methyl 2-azido-3-O-(2,3,4,6-tetra-O-benzoyl--d-galactopyranosyl)-4,6-O-benzylidene-2-deoxy-1-thio--d-galactopyranoside. This derivative in turn was prepared by silver triflate-promoted condensation of monosaccharide derivatives.     

Use of 3-O-(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-d-glucopyranosyl)-2,4,6-tri-O-acetyl-α-d-galactopyranosyl bromide as a glycosyl donor: Synthesis of p-nitrophenyl 3-O-(2-acetamido-2-deoxy-β-d-glucopyranosyl)-β-d-galactopyranoside

Acetolysis of methyl 3-O-(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-d-glucopyranosyl)-2,4,6-tri-O-acetyl-α-d-galactopyranoside afforded 3-O-(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-d-glucopyranosyl)-1,2,4,6-tetra-O-acetyl-d-galactopyranose (2). Treatment of 2 in dichloromethane with hydrogen bromide in glacial acetic acid gave 3-O-(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-d-glucopyranosyl)- 2,4,6-tri-O-acetyl-α-d-galactopyranosyl bromide (3). The α configuration of 3 was indicated by its high, positive, specific rotation, and supported by its ¹H-n.m.r. spectrum. Reaction of 3 with Amberlyst A-26-p-nitrophenoxide resin in 1:4 dichloromethane-2-propanol furnished p-nitrophenyl 3-O-(2-acetamido-3,4,6- tri-O-acetyl-2-deoxy-β-d-glucopyranosyl)-2,4,6-tri-O-acetyl-β-d-galactopyranoside (7). Compound 7 was also obtained by the condensation (catalyzed by silver trifluoromethanesulfonate-2,4,6-trimethylpyridine) of 3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-β-d-glucopyranosyl bromide with p-nitrophenyl 2,4,6-tri-O-acetyl-β-d-galactopyranoside, followed by the usual deacylation-peracetylation procedure. O-Deacetylation of 7 in methanolic sodium methoxide furnished the title disaccharide (8). The structure of 8 was established by ¹³C-n.m.r. spectroscopy.     

Synthesis of methyl O-(3-deoxy-3-fluoro-β-d-galactopyranosyl)-(1→6)-β-d-galactopyranoside and methyl O-(3-deoxy-3-fluoro-β-d-galactopyranosyl)-(1→6)-O-β-d-galactopyranosyl-(1→6)-β-d-galactopyranoside

Condensation of 2,4,6-tri-O-acetyl-3-deoxy-3-fluoro-α-d-galactopyranosyl bromide (3) with methyl 2,3,4-tri-O-acetyl-β-d-galactopyranoside (4) gave a fully acetylated (1→6)-β-d-galactobiose fluorinated at the 3′-position which was deacetylated to give the title disaccharide. The corresponding trisaccharide was obtained by reaction of 4 with 2,3,4-tri-O-acetyl-6-O-chloroacetyl-α-d-galactopyranosyl bromide (5), dechloroacetylation of the formed methyl O-(2,3,4-tri-O-acetyl-6-O-chloroacetyl-β-d-galactopyranosyl)-(1→6)- 2,3,4-tri-O-acetyl-β-d-galactopyranoside to give methyl O-(2,3,4-tri-O-acetyl-β-d-galactopyranosyl)-(1→6)-2,3,4-tri-O-acetyl-β-d-galactopyranoside (14), condensation with 3, and deacetylation. Dechloroacetylation of methyl O-(2,3,4-tri-O-acetyl-6-O-chloroacetyl-β-d-galactopyranosyl)-(1→6)-O-(2,3,4-tri-O-acetyl- β-d-galactopyranosyl)-(1→6)-2,3,4-tri-O-acetyl-β-d-galactopyranoside, obtained by condensation of disaccharide 14 with bromide 5, was accompanied by extensive acetyl migration giving a mixture of products. These were deacetylated to give, crystalline for the first time, the methyl β-glycoside of (1→6)-β-d-galactotriose in high yield. The structures of the target compounds were confirmed by 500-MHz, 2D, ¹H- and conventional ¹³C- and ¹⁹F-n.m.r. spectroscopy.     

Syntheses of 2-acetamido-2-deoxy-4-O-βD-galacto-pyranosyl-D-mannose and -D-glucose and of 2-acetamido-2-deoxy-4-O-βD-mannopyranosyl-D-mannose and -D-glucose

2-acetamido-2-deoxy-4-O-β-D-galactopyranosyl-D-mannose (6) and -D-glucose (7) were prepared by addition of nitromethane to 3-O-β-D-galactopyranosyl-D-arabinose, followed by acetylation, ammonolysis, and application of the Nef reaction. Similarly, 2-acetamido-2-deoxy-4-O-β-D-mannopyranosyl-D-mannose (14) and -D-glucose (15) were prepared by the same scheme from 3-O-β-D-mannopyranosyl-D-arabinose. In the two series of experiments, 6 and 14 were the respective major products. Epimerization of the 2-acetamido-2-deoxy-D-mannose residue in 6 and 14 yielded 7 and 15, respectively.     

The synthesis of 2-acetamido-2-deoxy-6-O-β-d-mannopyranosyl-d-glucose

4,6-Di-O-acetyl-2,3-O-carbonyl-α-d-mannopyranosyl bromide was condensed with benzyl 2-acetamido-3,4-di-O-acetyl-2-deoxy-α-d-glucopyranoside in the presence of silver carbonate to give crystalline benzyl 2-acetamido-3,4-di-O-acetyl-2-deoxy-6-O-(4,6-di-O-acetyl-2,3-O-carbonyl-β-d-mannopyranosyl)-α-d-glucopyranoside in 32% yield. Removal of the protective O-acetyl and cyclic carbonate groups gave the crystalline benzyl α-glycoside of the disaccharide, which was catalytically hydrogenolyzed to yield the crystalline, title compound. Proof of the anomeric configuration of the interglycosidic linkage was obtained by comparison of the physical, spectral, and chromatographic properties of the disaccharide and its derivatives with those of the previously prepared α-d-linked analog.     

Synthesis of 2-(p-trifluoroacetamidophenyl)ethylO-α-l-fucopyranosyl-(1–3)-O-(2-acetamido-2-deoxy-β-d-glucopyranosyl) (1–3)-O-β-d-galactopyranosyl-(1–4)-β-d-glucopyranoside,a tetrasaccharide fragment of a tumour-associated glycosphingolipid

The tetrasaccharide 2-(p-trifluoroacetamidophenyl)ethylO-α-l-fucopyranosyl-(1–3)-O-(2-acetamido-2-deoxy-β-d-glucopyranosyl)-(1–3)-O-β-d-galactopyranosyl-(1–4)-β-d-glucopyranoside was synthesized from thioglycoside intermediates. The key step was a methyl triflate promoted glycosidation of a lactose-derived 3′,4′-diol with a disaccharide thioglycoside to give a β(1–3)-linked tetrasaccharide derivative in 67% yield.     

Synthesis of 2-O-(2-acetamido-2-deoxy-β-D-glucopyranosyl)-D-mannose,and its interaction with D-mannose-specific lectins

Condensation of 3,4:5,6-di-O-isopropylidene-D-mannose dimethyl acetal with 2-methyl-(3,4,6-tri-O-acetyl- 1,2-dideoxy-α-D-glucopyrano)-[2′, 1′:4,5]-2-oxazoline in the presence of a catalytic amount of p-toluenesulfonic acid afforded crystalline 2-O-(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopyranosyl)-3,4:5,6-di-O-isopropylidene-D-mannose dimethyl acetal (3) in 25% yield. Catalytic deacetylation of 3 with sodium methoxide, followed by hydrolysis with dilute sulfuric acid, gave 2-O-(2-acetamido-2-deoxy-α-D-glucopyranosyl)-D-mannose (4). Treatment of 3 with boiling 0.5% methanolic hydrogen chloride under reflux gave methyl 2-O-(2-acetamido-2-deoxy-β-D-glucopyranosyl)-α-D-mannopyranoside (5) and methyl 2-O-(2-acetamido-2-deoxy-β-D-glucopyranosyl)-α-D-mannofuranoside (6). The inhibitory activities of 4, 5, and 6 against the hemagglutinating and mitogenic activities of Lens culinaris and Pisum sativum lectins and concanavalin A were assayed. From the results of these hapten inhibition studies, subtle differences of specificity between these D-mannose-specific lectins were confirmed.     

Synthesis of methyl O-(3-deoxy-3-fluoro-β-d-galactopyranosyl)-(1→6)-O-β-d-galactopyranosyl-(1→6)-3-deoxy-3-fluoro -β-d-galactopyranoside and related n.m.r. studies

Sequential tritylation, benzoylation, and detritylation of methyl 3-deoxy-3-fluoro-β-d-galactopyranoside gave crystalline methyl 2,4-di-O-benzoyl-3-deoxy-3-fluoro-β-d-galactopyranoside (9), which was used as the initial nucleophile in the synthesis of the target oligosaccharide (16). Treatment of 9 with 2,3,4-tri-O-benzoyl-6-O-bromoacetyl-α-d-galactopyranosyl bromide gave the corresponding disaccharide derivative 13, having a selectively removable blocking group at O-6′. Debromoacetylation of 13 afforded the disaccharide nucleophile 14 which, when treated with 2,4,6-tri-O-benzoyl-3-deoxy-3-fluoro-α-d-galactopyranosyl bromide, gave the fully protected trisaccharide 15. Debenzoylation of 15 gave the title glycoside 16. Condensation reactions were performed with silver trifluoromethane-sulfonate as a promoter in the presence of sym-collidine under base-deficient conditions, and gave excellent yields of the desired β-(trans)-products. Analyses of the ¹H- and ¹³C-n.m.r. spectra, as well as determination of the J_CF and J_HF coupling constants, were made by using various one- and two-dimensional n.m.r. techniques.     

Synthesis of phenyl 2-acetamido-2-deoxy-3-O-α-l-fucopyranosyl-β-d-glucopyranoside and related compounds

The reaction of phenyl 2-acetamido-2-deoxy-4,6- O-(p-methoxybenzylidene)-β-d-glucopyranoside with 2,3,4-tri-O-benzyl-α-l-fucopyranosyl bromide under halide ion-catalyzed conditions proceeded readily, to give phenyl 2-acetamido-2-deoxy-4,6-O-(p-methoxybenzylidene)-3-O-(2,3,4-tri-O-benzyl-α-l-fucopyranosyl)-β-d-glucopyranoside (8). Mild treatment of 8 with acid, followed by hydrogenolysis, provided the disaccharide phenyl 2-acetamido-2-deoxy-3-O-α-l-fucopyranosyl-β-d-glucopyranoside. Starting from 6-(trifluoroacetamido)hexyl 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-d-glucopyranoside, the synthesis of 6-(trifluoroacetamido)hexyl 2-acetamido-2-deoxy-3-O-β-l-fucopyranosyl-β-d-glucopyranoside has been accomplished by a similar reaction-sequence. On acetolysis, methyl 2-acetamido-2-deoxy-3-O-α-l-fucopyranosyl-α-d-glucopyranoside gave 2-methyl-[4,6-di-O-acetyl-1,2-dideoxy-3-O-(2,3,4-tri-O-acetyl-α-l-fucopyranosyl)-α-d-glucopyrano]-[2, 1-d]-2-oxazoline as the major product.     

Synthesis of 2-acetamido-2-deoxy-4-O-β-d-galactopyranosyl-3-O-methyl-d-glucopyranose (N-acetyl-3-O-methyllactosamine) and its benzyl α-glycoside

Benzyl 2-acetamido-2-deoxy-3-O-methyl-α-d-glucopyranoside (3) was obtained by deacetalation of its 4,6-O-benzylidene derivative (2). Compound 2 was prepared by methylation of benzyl 2-acetamido-4,6-O-benzylidene-2-deoxy-α-d-glucopyranoside with methyl iodide-silver oxide in N,N-dimethylformamide. Diol 3 was selectively benzoylated and p-toluenesulfonylated, to give the 6-benzoic and 6-p-toluenesulfonic esters (4 and 5, respectively). Displacement of the sulfonyl group of 5 with sodium benzoxide in benzyl alcohol afforded the 6-O-benzyl derivative (6). Glycosylation of 4 with 2,3,4,6-tetra-O-acetyl-α-d-galactopyranosyl bromide (7) in dichloromethane, in the presence of 1,1,3,3-tetramethylurea, furnished the disaccharide derivative 8. Similar glycosylation of compound 6 with bromide 7 gave the disaccharide derivative 10. O-Deacetylation of 8 and 10 afforded disaccharides 9 and 11. The structure of compound 9 was established by ¹³C-n.m.r. spectroscopy. Hydrogenolysis of the benzyl groups of 11 furnished the disaccharide 2-acetamido-2-deoxy-4-O-β-d-galactopyranosyl-3-O-methyl-d-glucopyranose (N-acetyl-3-O-methyllactosamine).     

Preparation, characterization, and biological evaluation of 6(I),6(IV)-di-O-[α-l-fucopyranosyl-(1→6)-2-acetamido-2-deoxy-β-d-glucopyranosyl]-cyclomaltoheptaose and 6-O-[α-l-fucopyranosyl-(1→6)-2-acetamido-2-deoxy-β-d-glucopyranosyl]-cyclomaltoheptaose

6(I),6(IV)-Di-O-[α-l-fucopyranosyl-(1→6)-2-acetamido-2-deoxy-β-d-glucopyranosyl]-cyclomaltoheptaose (βCD) {6(I),6(IV)-di-O-[α-l-Fuc-(1→6)-β-d-GlcNAc]-βCD (5)} and 6-O-[α-l-fucopyranosyl-(1→6)-2-acetamido-2-deoxy-β-d-glucopyranosyl]-βCD {6-O-[α-l-Fuc-(1→6)-β-d-GlcNAc]-βCD (6)} were chemically synthesized using the corresponding authentic compounds, bis(2,3-di-O-acetyl)-pentakis(2,3,6-tri-O-acetyl)-βCD as the glycosyl acceptor and 2,3,4-tri-O-benzyl-α-l-fucopyranosyl-(1→6)-3,4-di-O-acetyl-2-deoxy-2-(2,2,2-trichloroethoxycarbonylamino)-d-glucopyranosyl trichloroacetimidate as the fuco-glucosaminyl donor. NMR confirmed that α-l-Fuc-(1→6)-d-GlcNAc was bonded by β-linking to the βCD ring. To evaluate biological efficiency, the biological activities of the new branched βCDs were examined. The cell detachment activity of 5 was lower than that of 6 in real-time cell sensing (RT-CES) assay, indicating that 5 has lower toxicity. In SPR analysis, 5 had a higher special binding with AAL, a fucose-recognizing lectin. These results suggest that 5 could be an efficient drug carrier directed at cells expressing fucose-binding proteins.     

Synthesis of 2-acetamido-1-N[N-(tert-butoxycarbonyl)-l-aspart-1-oyl-(l-phenylalanyl-l-serine methyl ester)-4-oyl]-2-deoxy-β-d-glucopyranosylamine and analogs

2-Acetamido-1-N-[N-(tert-butoxycarbonyl)-l-aspart-1-oyl-(l-phenylalanyl-l-serine methyl ester)-4-oyl]-2-deoxy-β-d-glucopyranosylamine and analogs containing d-glucopyranosyl, 4-O-β-d-glucopyranosyl-d-glucopyranosyl, l-Phe-l-Ala, and d-Phe-l-Ser were synthesized by condensation of glycosylamines having free hydroxyl groups with tripeptide esters activated with N-hydroxysuccinimide.     

The synthesis of O-(2-acetamido-2-deoxy-β-d-glucopyranosyl)-(1→4)-N-acetylnormuramoyl-l-α-aminobutanoyl-d-isoglutamine

Benzyl 2-acetamido-3-O-allyl-6-O-benzyl-2-deoxy-4-O-(3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-β-d-glucopyranosyl)- α-d-glucopyranoside (4) was obtained in high yield on using the silver triflate method in the absence of base. Compound 4 was converted in six steps into benzyl 2-acetamido-4-O-(2-acetamido-3,4,6-tri-O-benzyl-2-deoxy-β-d-glucopyranosyl)-6-O-benzyl-3-O-(carboxymethyl)-2-deoxy-α-d- glucopyranoside, which was coupled with the benzyl ester of l-α-aminobutanoyl-d-isoglutamine and the product hydrogenolyzed to afford the title compound. O-Benzylation of benzyl 2-acetamido-4-O-(2-acetamido-2-deoxy-β-d-glucopyranosyl)-3-O-allyl-6-O-benzyl-2-deoxy-α-d-glucopyranoside with benzyl bromide and barium hydroxide in N,N-dimethylformamide is strongly exhanced by sonication of the reaction mixture.     

Synthesis and X-ray crystallographic investigation of N-(3-deoxy-3-acetamido-��-D-glycopyranosyl)alkanamides as analogs of N-glycoprotein linkage region

As part of our ongoing program aimed at understanding the structural significance of GlcNAcβAsn linkage conserved in all eukaryotic N-glycoproteins, the present study reports on the synthesis and X-ray crystal structures of N-(3-deoxy-3-acetamido-β-D-glycopyranosyl)acetamide (Glc3NAcβNHAc) and the corresponding propionamide (Glc3NAcβNHPr). Comparative analysis of these structures with those of the corresponding GlcNAc (C2 acetamido) compounds showed that the bifurcated anti-parallel pattern involving N-H…O and C-H…O hydrogen bonds, the hallmark feature of the N-glycoprotein models, GlcNAcβNHAc and GlcNAcβAsn, is absent in both the C3 acetamido analogs. The extended (anti) conformation of the amido aglycon moiety as defined by χ(2) seen in the case of C2 acetamido derivative, GlcNAcβNHPr, turns into gauche for the C3 acetamido analog (Glc3NAcβNHPr). This observation is consistent with the earlier work on the critical role of the C2-NHAc group of GlcNAcβAsn in controlling χ(2) at the linkage region of N-glycoproteins.