The 2-naphthylmethyl (NAP) group in carbohydrate synthesis: first total synthesis of the GlyCAM-1 oligosaccharide structures.

Total syntheses of the GlyCAM-1 (glycosylation-dependent cell adhesion molecule-1) oligosaccharide structures: [alpha-NeuAc-(2 --> 3)-beta-Gal-(1 --> 4)-[alpha-Fuc-(1 --> 3)]-beta-(6-O-SO3Na)-GlcNAc-(1 --> 6)]-[alpha-NeuAc-(2 --> 3)-beta-Gal-(1 --> 3)]-alpha-GalNAc-OMe (1) and [alpha-NeuAc-(2 --> 3)-beta-Gal-(1 --> 4)-[alpha-Fuc-(1 --> 3)]-beta-GlcNAc-(1 --> 6)]-[alpha-NeuAc-(2 3)-beta-Gal-(1 --> 3)]-alpha-GalNAc-OMe (2) through a novel sialyl LewisX tetrasaccharide donor are described. Employing sequential glycosylation strategy, the starting trisaccharide was regio- and stereoselectively constructed through coupling of a disaccharide imidate with the monosaccharide acceptor phenyl-6-O-naphthylmethyl-2-deoxy-2-phthalimido-1-thio-beta-D-glucopyranoside with TMSOTf as a catalyst without affecting the SPh group. The novel sialyl Lewisx tetrasaccharide donor 3 was then obtained by alpha-L-fucosylation of trisaccharide acceptor with the 2,3,4-tri-O-benzyl-1-thio-beta-L-fucoside donor. The structure of the novel sialyl Lewisx tetrasaccharide was established by a combination of 2D DQF-COSY and 2D ROESY experiments. Target oligosaccharides 1 and 2 were eventually constructed through heptasaccharide which was obtained by regioselective assembly of advanced sialyl Lewisx tetrasaccharide donor 3 and a sialylated trisaccharide acceptor in a predictable and controlled manner. Finally, target heptasaccharides 1 and 2 were fully characterized by 2D DQF-COSY, 2D ROESY, HSQC, HMBC experiments and FAB mass spectroscopy.

Chemical synthesis of sulfated oligosaccharides with a beta-D-Gal-(1-->3).

The syntheses of two sulfated pentasaccharides: beta-D-Gal6SO3Na-(1-->3)-[beta-D-Gal-(1-->4)-alpha-L-Fuc-(1-->3)-beta-D-Glc-NAc-(1-->6)]-alpha-D-GalNAc-->OMe (1) and beta-D-Gal6SO3Na-(1-->3)-[beta-D-Gal-(1-->4)-alpha-L-Fuc-(1-->3)-beta-D-Glc-NAc6SO3Na-(1-->6)]-alpha-D-GalNAc-->OMe (2) by using Lewisx trisaccharides 12 and 16 as glycosyl donors are described. Sulfated oligosaccharides 1-2 and intermediate compounds are fully characterized by 2D 1H-1H DQF-COSY and 2D ROESY experiments.

A convergent synthesis of trisaccharides with alpha-Neu5Ac-(2 --> 3)-beta-D-gal-(1 --> 4)-beta-D-GlcNAc and alpha-Neu5Ac-(2 --> 3)-beta-D-gal-(1 --> 3)-alpha-D-GalNAc sequences.

The syntheses of three trisaccharides: alpha-Neu5Ac-(2 --> 3)-beta-D-Gal-(1 --> 4)-beta-D-GlcNAc --> OMe, alpha-Neu5Ac-(2 --> 3)-beta-D-Gal6SO3Na-(1 --> 4)-beta-D-GlcNAc --> OMe, and alpha-Neu5Ac-(2 --> 3)-beta-D-Gal-(1 --> 3)-alpha-D-GalNAc --> OBn were accomplished by using either methyl (phenyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-2-thio-beta-D-glycero-D-g alacto-2-nonulopyranoside)onate or methyl (phenyl N-acetyl-5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-2-thio-beta-D-gl ycero-D-galacto-2-nonulopyranoside)onate as the sialyl donor. The N,N-diacetylamino sialyl donor appears to be more reactive than its parent acetamido sugar when allowed to react with an disaccharide acceptor under the same glycosylation conditions. The trisaccharides, as well as the intermediate products, were fully characterized by 2D DQF 1H-1H COSY and 2D ROESY spectroscopy.

Total synthesis of sialylated and sulfated oligosaccharide chains from respiratory mucins.

The total syntheses of several complex oligosaccharide moieties that occur in the core structure of sulfated mucins are reported. A trisaccharide acceptor was obtained through regio- and stereoselective sialylation of methyl (6-O-pivaloyl-beta-D-galactopyanosyl)(1-->3)-4,6-O-benzylidene-2-a cetamido-2-deoxy-alpha-D-galactopyranoside with a novel sialyl donor. A tetrasaccharide, pentasaccharide, and hexasaccharide were constructed in predictable and controlled manner with high regio- and stereoselectivity after the successful preparation and employment of a disaccharide donor, trisaccharide donor, disaccharide acceptor, and trisaccharide acceptor building blocks. Finally, a mild oxidative cleaving method was adopted for the selective removal of 2-naphthylmethyl (NAP) in the presence of benzyl groups.

N-p-nitrobenzyloxycarbonyl galactosamine imidate as a glycosyl donor for the efficient synthesis of mucin core-2 analogue.

An efficient synthesis of the mucin core-2 analogue 1a was accomplished using N-p-nitrobenzyloxycarbonyl(PNZ)-protected trichloroacetimidate 4 as a novel glycosyl donor.

An efficient synthesis of two monosulfated trisaccharides with the Galbeta1,3GlcNacbeta1,3Galbeta-O-allyl backbone.

The GlcNAcbeta(1-->3) Gal linked disaccharide 7 was synthesized as key building blocks for the construction of target monosulfated trisaccharides 1 and 2 using oxazoline 3 as glycosyl donor promoted by BF3 x Et2O.

Inhibition of L- and P-selectin by a rationally synthesized novel core 2-like branched structure containing GalNAc-Lewisx and Neu5Acalpha2-3Galbeta1-3GalNAc sequences.

The selectins interact in important normal and pathological situations with certain sialylated, fucosylated glycoconjugate ligands containing sialyl Lewisx(Neu5Acalpha2-3Galbeta1-4(Fucalpha1-3)GlcN Ac). Much effort has gone into the synthesis of sialylated and sulfated Lewisxanalogs as competitive ligands for the selectins. Since the natural selectin ligands GlyCAM-1 and PSGL-1 carry sialyl Lewisxas part of a branched Core 2 O-linked structure, we recently synthesized Galbeta1-4(Fucalpha1-3)GlcNAcbeta1-6(SE-3Galbeta1++ +-3)GalNAc1alphaOMe and found it to be a moderately superior ligand for L and P-selectin (Koenig et al. , Glycobiology 7, 79-93, 1997). Other studies have shown that sulfate esters can replace sialic acid in some selectin ligands (Yeun et al. , Biochemistry, 31, 9126-9131, 1992; Imai et al. , Nature, 361, 555, 1993). Based upon these observations, we hypothesized that Neu5Acalpha2-3Galbeta1-3GalNAc might have the capability of interacting with L- and P-selectin. To examine this hypothesis, we synthesized Galbeta1-4(Fucalpha1-3)GlcNAcbeta1-6(Neu5Acalpha2++ +-3Galbeta1-3)-GalNAc alpha1-OB, which was found to be 2- to 3-fold better than sialyl Lexfor P and L selectin, respectively. We also report the synthesis of an unusual structure GalNAcbeta1-4(Fucalpha1- 3)GlcNAcbeta1-OMe (GalNAc-Lewisx-O-methyl glycoside), which also proved to be a better inhibitor of L- and P-selectin than sialyl Lewisx-OMe. Combining this with our knowledge of Core 2 branched structures, we have synthesized a molecule that is 5- to 6-fold better at inhibiting L- and P-selectin than sialyl Lewisx-OMe, By contrast to unbranched structures, substitution of a sulfate ester group for a sialic acid residue in such a molecule resulted in a considerable loss of inhibition ability. Thus, the combination of a sialic acid residue on the primary (beta1-3) arm, and a modified Lexunit on the branched (beta1-6) arm on an O-linked Core 2 structure generated a monovalent synthetic oliogosaccharide inhibitor superior to SLexfor both L- and P-selectin.

Synthesis of Gal-beta-(1-->4)-GlcNac-beta-(1-->6)-[Gal-beta-(1-->3]-GalNAc-alpha- OBn oligosaccharides bearing O-methyl or O-sulfo groups at C-3 of the Gal residue: specific acceptors for Gal: 3-O-sulfotransferases.

Our recent studies have revealed the existence of two distinct Gal: 3-O-sulfotransferases capable of acting on the C-3 position of galactose in a Core 2 branched structure, e.g., Galbeta1-->4GlcNAcbeta1-->6(Galbeta1-->3)GalNacalph a1-->OBenzyl as acceptor to give 3-O-sulfoGalbeta1-->4GlcNAcbeta1-->3(Galbeta1-->3)G alNAcalpha1-->OB 20 and Galbeta1-->4GlcNAcbeta1-->6(3-O-sulfoGalbeta1-->3)G alNAcalpha1-->OB 23. We herein report the synthesis of these two compounds and also that of other modified analogs that are highly specific acceptors for the two sulfotransferases. Appropriately protected 1-thio-glycosides 7, 8, and 10 were employed as glycosyl donors for the synthesis of our target compounds.

Synthetic mucin fragments: synthesis of O-sulfo and O-methyl derivatives of allyl O-(beta-D-galactopyranosyl)-(1-->3)-2-acetamido-2-deoxy-alpha-D- galactopyranoside as potential compounds for sulfotransferases.

Allyl 2-acetamido-4,6-O-(4-methoxybenzylidene)-2-deoxy-alpha-D-galact opy ranoside (1) was condensed with either 2,3,4,6-tetra-O-acetyl-alpha-D-galactopyranosyl bromide (2) or 2,3,4-tri-O-benzoyl-6-O-bromoacetyl-alpha-D-galactopyranosyl bromide (14) in the presence of mercuric cyanide. Selective substitution with methyl, sulfo or both at desired positions, followed by the removal of protecting groups, afforded allyl O-(beta-D-galactopyranosyl)-(1-->3)-2-acetamido-2-deoxy-6-O-methyl-alpha -D- galactopyranoside (5), allyl O-(6-O-sulfo-beta-D-galactopyranosyl sodium salt)-(1-->3)-2-acetamido-2-deoxy-6- O-methyl-alpha-D-galactopyranoside (10), allyl O-(beta-D-galactopyranosyl)-(1-->3)-2-acetamido-2-deoxy-6-O-sulfo-alpha- D- galactopyranoside sodium salt (13), allyl O-(6-O-sulfo-beta-D-galactopyranosyl sodium salt)-(1-->3)-2-acetamido-2-deoxy- alpha-D-galactopyranoside (17) and allyl O-(3-O-sulfo-beta-D-galactopyranosyl sodium salt)-(1-->3)-2-acetamido-2-deoxy- alpha-D-galactopyranoside (22). The structures of compounds 5, 10, 13, 17 and 22 were established by 13C NMR and FAB mass spectroscopy.

A facile synthesis of nitrophenyl oligosaccharides containing the O-alpha-L-fucopyranosyl-(1----3)-2-acetamido-2-deoxy-beta-D-glucopyrano syl unit at their nonreducing end.

A facile approach towards the synthesis of 4-nitrophenyl O-alpha-L-fucopyranosyl-(1----3)-2-acetamido-2-deoxy-beta-D-glucopyra nos ide, 2-nitrophenyl O-alpha-L-fucopyranosyl-(1----3)-O-(2-acetamido-2-deoxy-beta-D-glucop yra nosyl)- (1----6)-2-acetamido-2-deoxy-alpha-D-galactopyranoside, 4-nitrophenyl O-alpha-L-fucopyranosyl-(1----3)-O-(2-acetamido-2-deoxy-beta-D-glucop yra nosyl)- (1----6)-alpha-D-mannopyranoside, and 4-nitrophenyl O-alpha-L-fucopyranosyl-(1----3)-O-(2-acetamido-2-deoxy-beta-D-glucop yra nosyl)-(1----6)-beta-D-galactopyranoside has been accomplished through the development and use of methyl 3,4-O-isopropylidene-2-O-(4-methoxybenzyl)-1-thio-beta-L-fucopyranoside as the glycosyl donor.

Synthetic antigens as immunogens: Part IV. Specificity of antisera developed to Gal beta 1-3(GlcNAc beta 1-6)GalNAc and GlcNAc beta 1-6GalNAc.

Antisera to the chemically synthesized trisaccharide, Gal beta 1-3(GlcNAc beta 1-6)GalNAc, and disaccharide, GlcNAc beta 1-6GalNAc, were developed using the diazophenyl bovine serum albumin derivatives. The binding specificity of the antisera were analyzed by enzyme immunoassays with structurally related, chemically synthesized oligosaccharides. The anti-trisaccharide antibody showed no reactivity to T antigenic structures which bear the Gal beta 1-3GalNAc moiety. The immunodominant area of the Gal beta 1-3(GlcNAc beta 1-6)GalNAc was contained in the GlcNAc beta 1-6GalNAc region of the molecule. The reactivity pattern of the anti-trisaccharide antibody, although directed to the disaccharide, did not exactly duplicate the reactivity pattern of the anti-disaccharide.

Separation by liquid chromatography (under elevated pressure) of benzyl and nitrophenyl glycosides of oligosaccharides.

Liquid chromatography under elevated pressure (l.c.) was employed for the separation of some benzyl and nitrophenyl glycosides of a variety of mono-, di-, tri-, and tetra-saccharides. The separation was conducted on a Waters Carbohydrate Analysis column by use of a mixture of acetonitrile-water as the mobile phase. In general, monosaccharides emerged first from the column, followed sequentially by di-, tri-, and tetra-saccharides. It was observed that the pattern of substitution imparts a noticeable effect on the elution profiles of isomeric oligosaccharides. Also, substitution of a hydroxyl group with a methyl group, or its replacement with a fluorine atom, led to a substantial decrease in retention times of some oligosaccharides. Moreover, resolution was clearly enhanced, and retention times were congruently increased by decreasing the water content of the mobile phase.

Synthesis of 2-acetamido-2-deoxy-4-O-(2-O-methyl-beta-D-galactopyranosyl)- D-glucopyranose (N-acetyl-2'-O-methyllactosamine).

1,3,4,6-Tetra-O-acetyl-2-O-methyl-D-galactopyranose, prepared from known methyl 6-O-acetyl-3,4-O-isopropylidene-beta-D-galactopyranoside, was treated with hydrogen bromide in dichloromethane to afford 3,4,6-tri-O-acetl-2-O-methyl-alpha-D-galactopyranosyl bromide. Condensation with benzyl 2-acetamido-3,6-di-O-benzyl-2-deoxy-alpha-D-glucopyranoside in acetonitrile in the presence of mercuric cyanide gave an approximately 1:1 mixture of benzyl 2-acetamido-3, 6-di-O-benzyl-2-deoxy-4-O-(3,4,6-tri-O-acetyl-2-O-methyl-beta- (8) and -alpha-D-galactopyranosyl)-alpha-D-glucopyranoside. O-Deacetylation and catalytic hydrogenolysis of the benzyl group furnished 2-acetamido-2-deoxy-4-O-(2-O-methyl-beta- and alpha-D-galactopyranosyl)-D-glucopyranose. Alternatively, benzyl 2-acetamido-3,6-di-O-benzyl-2-deoxy-4-O-beta-D- galactopyranosyl-alpha-D-glucopyranoside was treated with tert-butyldiphenyl-chlorosilane in N,N-dimethylformamide, in the presence of imidazole, to give a 6'-O-tert-butyldiphenylsilyl intermediate that was in turn converted into its 3',4'-O-isopropylidene acetal. Methylation with methyl iodide-silver oxide in N,N-dimethylformamide, followed by removal of the silyl and isopropylidene groups gave benzyl 2-acetamido-3,6-di-O-benzyl-2-deoxy-4-O-(2-O-methyl-beta-D- galactopyranosyl)-alpha-D-glucopyranoside, which was further characterized as its triacetate 8.

Serum beta-(1----4)-galactosyltransferase activity with synthetic low molecular weight acceptor in human ovarian cancer.

A modified procedure was developed for the determination of UDP-galactose: 2-acetamido-2-deoxy-glucopyranoside beta-(1----4)-galactosyltransferase (GT) in human serum which employed the synthetic substrates p-nitrophenyl 6-0-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)-beta-D-galactopyranoside and p-nitrophenyl 6-0-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)-alpha-D- mannopyranoside as acceptors. The enzyme products were identified by thin layer chromatography with authentic reference compounds, and the galactosyl linkage was characterized by hydrolysis with beta-D-galactosidase from jack beans. The diagnostic value of this GT for ovarian cancer was tested by measuring the serum enzyme activity in 28 ovarian cancer patients with disease, 20 ovarian cancer patients with no clinical evidence of disease, and 22 healthy females. Although the level of the enzyme activity was significantly higher (P less than 0.002) in the serum of patients with active disease when compared to healthy controls, an appreciable overlap of enzyme activity was found between them. Also, no correlation was found between enzyme activity and tumor size. Differences in methodology and selection of patients makes it difficult to compare results from other reports. However, based on our improved assay procedure, we suggest caution should be exercised in evaluating the merits of GT as a diagnostic marker for ovarian cancer.

Synthesis of benzyl 2-acetamido-2-deoxy-3-O-beta-D-fucopyranosyl-alpha-D-galactopyranoside and benzyl 2-acetamido-6-O-(2-acetamido-2-deoxy-beta- D-glucopyranosyl)-2-deoxy-3-O-beta-D-fucopyranosyl-alpha-D- galactopyranoside.

Condensation of benzyl 2-acetamido-4,6-O-benzylidene-2-deoxy-alpha-D- galactopyranoside with 2,3,4-tri-O-acetyl-alpha-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-beta-D- fucopyranosyl)-alpha-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-alpha-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 some oligosaccharides containing the O-alpha-L-fucopyranosyl-(1----2)-D-galactopyranosyl unit.

A practical approach for the synthesis of oligosaccharides containing the O-alpha-L-fucopyranosyl-(1----2)-D-galactopyranosyl unit has been developed by utilizing the readily accessible 3,4,6-tri-O-acetyl-2-O-(2,3,4-tri-O-acetyl-alpha-L- fucopyranosyl)-alpha-D-galactopyranosyl bromide (1). Thus, condensation of bromide 1 with 8-(methoxycarbonyl)octanol in benzene, in the presence of mercuric cyanide, afforded, after column-chromatographic separation, the anomers (3 and 4) of 8-(methoxycarbonyl)octyl 3,4,6-tri-O-acetyl-2-O-(2,3,4-tri-O-acetyl-alpha-L-fucopyranosyl)-D- galactopyranoside in the ratio of 13:4. On the basis of their specific rotations, compounds 3 and 4 were tentatively assigned the alpha and beta configuration, respectively. Similarly, bromide 1 was allowed to react with some other, appropriately protected sugars having a free alcohol group, to afford the corresponding alpha and beta anomers of a galactopyranosyl residue having an alpha-L-fucopyranosyl substituent at O-2. Column-chromatographic separation of the anomeric mixtures, followed by systematic removal of the protecting groups, then provided the final oligosaccharides desired. In this manner, the synthesis of the following oligosaccharides has been accomplished: alpha-L-Fuc-(1----2)-alpha-D-Gal-1----O(CH2)8CO2Me (5), alpha-L-Fuc-(1----2)-beta-D-Gal-1----O(CH2)8CO2Me (6), alpha-L-Fuc-(1----2)-alpha-D-Gal-(1----3)-beta-D-GlcNAc-1----OC6H4NO2 -p (11), alpha-L-Fuc-(1----2)-beta-D-Gal-(1----3)-beta-D-GlcNAc-1----OC6H4NO2+ ++-p (13), alpha-L-Fuc-(1----2)-alpha-D-Gal-(1----3)-alpha-D-GalNAc-1----OC6H4NO 2-o, and alpha-L-Fuc-(1----2)-beta-D-Gal-(1----3)-alpha-D-GalNAc-1----OC6H4NO2 -o (18).(ABSTRACT TRUNCATED AT 250 WORDS)