Synthesis of oligosaccharide determinants with amide spacers of T-type antigens

The hapten of the T-antigen was synthesized with a peptide-like amide-spacer as 2-(4-methoxycarbonylbutanecarboxamido)ethyl 2-acetamido-2-deoxy-3-O-beta-D-galactopyranosyl-alpha-D-galactopyranoside and coupled with serum albumin to give a synthetic antigen. Other O-beta-D-galactopyranosyl haptens, 2-(4-methoxycarbonylbutanecarboxamido)ethyl 2-acetamido-2-deoxy-4-O-beta-D-galactopyranosyl-alpha-D-galactopyranoside, O-beta-D-galactopyranosyl-(1 leads to 3)-O-[beta-D-galactopyranosyl-(1 leads to 4)]-2-acetamido-2-deoxy-alpha-D-galactopyranoside, and 2-acetamido-2-deoxy-3-O-beta-D-galactopyranosyl-alpha-D-glucopyranoside, the last compound being the determinant of the Lewis Lec antigen, were also synthesized.     

Synthesis and glycan priming activity of acetylated disaccharides

Five disaccharides related in structure to the glycans of vertebrate mucins have been chemically synthesized using orthogonal blocking, coupling and deblocking techniques. These include 2-naphthylmethyl 3,4,6-tetra-O-acetyl-beta-D-galactopyranosyl-( 1 --> 4)-2-acetamido-3,6-di-O-acetyl-2-deoxy-beta-D-glucopyranoside (6), 2-naphthylmethyl 2-aceta-mido-3,4,6-tri-O-acetyl-2-deoxy-beta-D-glucopyranosyl-(1 --> 3)-2,4,6-tri-O-acetyl-beta-D-galactopyranoside (14), 2-naph-thylmethyl2,3,4,6-tetra-O-acetyl-beta-D-galactopyranosyl-(1 --> 3)-2-acetamido-4,6-di- O-acetyl-2-deoxy-alpha-D-galactopyranoside (20), 2-naphthylmethyl 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-beta-D-glucopyranosyl-(1 --> 3)-2-acetamido-4,6-di-O-acetyl-2-deoxy-alpha-D-galactopyranoside (23) and 2-naphthylmethyl 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-beta-D-glu-copyranosyl-(1 --> 6)-2-acetamido-3,4-di-O-acetyl-2-deoxy-alpha-D-galactopyranoside (27). These per-O-acetylated compounds were fed to U-937 cells to test their ability to prime oligosaccharide synthesis, inhibit glycoprotein biosynthesis and alter adhesion to E-selectin expressed on endothelial cells. The results show that 6, 14, and 20 served as substrates for oligosaccharide synthesis. The generation of glycoside-primed glycans altered the formation of glycoproteins on the cell surface and inhibited cell adhesion dependent on E-selectin.     

Monospecific antibodies against synthetic T-antigen. Characteristics of their specificity and use in the identification of T-antigenic determinants on the cell surface

Monospecific antibodies directed to a Thomsen-Friedenreich antigen (T-antigen) were obtained using artificial antigen. T-antigen immunodominant alpha-disaccharide Galbeta (1----3) GalNAc alpha 1-(T alpha) and its beta-anomer Gal beta (1----3) GalNAc beta 1-(T beta) were bound to bovine serum albumin (BSA) and cytochrome C (CCC) through a spacer (sp = -O(CH2)3NHCO (CH2)4CO-) by the azide method to give neoglycoproteins T alpha-sp-BSA, T alpha-sp-CCC and T beta-sp-BSA. Anti-T alpha antiserum was obtained by immunization of rabbits with T alpha-sp-BSA and then purified by sequential affinity chromatography on BSA-Sepharose and T alpha-sp-BSA-Sepharose to yield monospecific anti-T IgG antibodies. As elucidated by ELISA method, binding T alpha-sp-BSA to the antibodies was inhibited by T alpha-sp-CCC, T alpha-sp-OEt, asialofetuin, T alpha-OBzl, the activity of the inhibitors decreasing in the above order. Methyl beta-galactopyranoside, benzyl 2-acetamido-2-deoxy-alpha-D-galactopyranoside, disaccharide Gal beta (1----3) GalNAc and H-sp-BSA were inactive. The inhibitory analysis suggests that both disaccharide moiety T alpha- and a definite part of the spacer are important for the binding and that T alpha-OCH2 seems to be the minimal recognized structure. In immunoprecipitation tests the antibodies react with T alpha-sp-BSA but not with T beta-sp-BSA, whereas peanut (Arachis hypogaea) lectin (PNL) precipitated both T alpha- and T beta-sp-BSA. These data suggest the significance of the alpha-galactosaminide bond for the antibody recognition. Desialylated human erithrocites (natural T-antigen) were effectively agglutinated with the antibodies. Murine cortical thymocytes (obtained by agglutination-sedimentation method using PNL) were agglutinated with the antibodies only partially (67%), while these cells as well as the cells unaffected by the antibodies were completely agglutinated with PNL. These results indicate to different contents of glycoproteins (T alpha) and glycolipids (T beta) oligosaccharide determinants on the surface of cortical thymocytes species.     

Proton nuclear magnetic resonance studies of soybean lectin-monosaccharide interactions: computer analysis of complex binding behavior

1H NMR was used to quantify soybean lectin binding to monosaccharides, using presaturation of HOD plus a spin-echo sequence to observe sugar -NHCOCH3 and -OCH3 to below 0.01 mM. Binding is in the very-slow-exchange limit; there is no broadening or shifting and only unbound sugar is observed for pH 5 to 8 and 25 to 75 degrees C. Preliminary results were consistent with those previously reported for methyl 2-acetamido-2-deoxy-alpha-D-galactopyranoside (Me alpha-D-GalNAcp) (K = 3 X 10(4) liters mol-1 with four sites per tetramer). More detailed studies, however, gave concave Scatchard plots for methyl 2-acetamido-2-deoxy-alpha- and beta-D-galactopyranoside, (Me alpha- and Me beta-D-GalNAcp), best fitted using K1 values of (6-12) X 10(4) liters mol-1, K2 values less than or equal to 0.5 X 10(4) liters mol-1, and four sites of each type in D2O or 80% H2O at 25 degrees C and pH 7.2. Data for methyl alpha-D-galactopyranoside were fitted with K = 0.5 X 10(4) liters mol-1 and eight sites of the same K. Monosaccharides may be binding in the recently reported "hydrophobic sites" of soybean lectin. Both methyl 2-acetamido-2-deoxy-beta-D-galactofuranoside (Me beta-D-GalNAcf) and methyl 2-acetamido-2-deoxy-alpha-D-glucopyranoside (Me alpha-D-GlcNAcP) showed some binding by 1H NMR; K's were similar to those for the high-affinity sugars, but the occupancy was much lower. The soybean lectin in this study was saturated in Ca2+ (greater than or equal to 4 mol/tetramer), but low in Mn2+, with Mn2+ plus Mg2+ less than 4. We report new melting points for D-N-acetylgalactosamine, Me alpha-D-GlcNAcp, Me beta-D-GalNAcp, and Me beta-D-GalNAcf, and a fully listed program for fitting curved Scatchard plots using Apple IIc and IIe computers.     

An efficient access to protected disialylated glycohexaosyl threonine present on the leukosialin of activated T-lymphocytes

The total synthesis of the threonine-linked core 2 class disialylated hexasaccharide in a completely protected form was accomplished for the first time. The L-threonine conjugate, N-(9-fluorenylmethoxycarbonyl)-O-[(5-acetamido-4,7,8,9-tetra-O-ben zyl-3,5-dideoxy-D-glycero-alpha-D-galacto-2-nonulopyranosylonic acid)-(2-->3)-(2,6-di-O-benzyl-beta-D-galactopyranosyl)-(1-->4)-2-acetam ido-2-deoxy-3,6-di-O-benzyl-beta-D-glucopyranosyl-(1-->6)-[(5-acetamido- 4,7,8,9-tetra-O-benzyl-3,5-dideoxy-D-glycero-alpha-D-galacto-2-nonulo pyranosylonic acid)-(2-->3)-2,6-di-O-benzyl-beta-D-galactopyranosyl-(1-->3)]-2-acetami do-2-deoxy-alpha-D-galactopyranosyl-(1d-->4c:1f-->4e)-dilactone ]-L-threonine allyl ester was synthesized via stereocontrolled glycosylations employing readily accessible monosaccharidic blocks; t-butyl-diphenylsilyl-2-azido-2-deoxy-3,6-di-O-benzyl-beta-D-gluco pyranose, N-(9-fluorenylmethoxycarbonyl)-O-(2-azido-6-O-t-butyldimethylsilyl -2-deoxy-alpha-D-galactopyranosyl)-L-threonine allyl ester, 8, 9 and N-(9-fluorenylmethoxycarbonyl)-O-(2-azido-4,6-O-benzylidene-3-O-ch loroacetyl-2-deoxy-alpha-D-galactopyranosyl)-L-threonine allyl ester. For the introduction of the amino acid, the azide group was used to temporarily mask the amino group of GalNAc so as to obtain an alpha-glycosidic linkage without participation from the C-2 substituent. The threonine was attached to the sugar unit at the monosaccharide stage to avoid loss of oligosaccharide at a later stage. The Fmoc and allyl ester protected amino acid at the reducing end facilitates efficient glycopeptide synthesis on solid-phase support.     

Total synthesis of 3-O-[2-acetamido-6-O-(N-acetyl-alpha-D-neuraminyl-2-deoxy- alpha-D-galactosyl]-L-serine and a stereoisomer

O-(5-Acetamido-3,5-dideoxy-D-glycero-alpha-D-galacto-2- nonulopyranoxylonic acid)-(2----6)-O-(2-acetamido-2-deoxy-alpha-D-galactopyranosyl)-(1----3) -L-serine, a structural unit occurring in various submaxillary mucins, was synthesized for the first time by using O-[methyl (5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-alpha-D- galacto-2-nonulopyranosyl)onate]-(2----6)-3,4-di-O-acetyl-2- azido-2-deoxy-D- galactopyranosyl trichloroacetimidate (13) and N-(benzyloxycarbonyl)-L-serine benzyl ester as the key intermediates. The trichloroacetimidate 13 was prepared by starting from two monosaccharide synthons, namely, allyl 2-azido-2-deoxy-beta-D-galactopyranoside and methyl (5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-beta-D- galacto-2-nonulopyranosyl chloride)onate, which were coupled in the presence of silver triflate in tetrahydrofuran to give the desired alpha-(2----6)-linked disaccharide in moderate selectivity.     

Synthesis of neoglycoproteins containing O-methylated trisaccharides related to excretory/secretory antigens of Toxocara larvae

The disaccharides allyl beta-D-galactopyranosyl-(1-->3)-2-acetamido-2-deoxy-beta- and alpha-D-galactopyranoside 10a and 10b and the trisaccharides allyl 2-O-methyl-alpha-L-fucopyranosyl-(1-->2)-beta-D-galactopyranosyl-(1-->3)-2-acetamido-2-deoxy-beta- and alpha-D-galactopyranoside 18a and 18b have been prepared using stepwise assembly of the sugar units. The glycosidic linkages were formed employing the trichloroacetimidate procedure for the attachment of the galactopyranosyl residue and N-iodosuccinimide/triflic acid activation of an ethyl 1-thiofucopyranoside donor for fucosylation. Deprotection furnished the allyl glycosides which were converted into cysteamine-spacered ligands, activated with thiophosgene and subsequently linked to bovine serum albumin. The neoglycoproteins serve as immunoreagents to determine epitope specificities of monoclonal antibodies directed against highly immunogenic O-glycans located at the surface of Toxocara larvae.     

Fluorinated carbohydrates as potential plasma membrane modifiers and inhibitors. Synthesis of 2-acetamido-2,6-dideoxy-6-fluoro-D-galactose

Reaction of benzyl 2-acetamido-3,4-di-O-benzyl-2-deoxy-6-O-mesyl-alpha-D-galactopyran oside with cesium floride gave benzyl 2-acetamido-3,6-anhydro-4-O-benzyl-2-deoxy-alpha-D-galactopyranoside instead of the desired 6-fluoro derivative. Acetonation of benzyl 2-acetamido-2-deoxy-6-O-mesyl-alpha-D-galactopyranoside gave the corresponding 3,4-O-isopropylidene derivative. The 6-O-mesyl group was displaced by fluorine with cesium fluoride in boiling 1,2-ethanediol, and hydrolysis and subsequent N-acetylation gave the target compound. In another procedure, treatment of 2-acetamido-1,3,4-tri-O-acetyl-2-deoxy-alpha-D-galactose with N-(diethylamino)sulfur trifluoride gave 2-acetamido-1,3,4-tri-O-acetyl-2,6-dideoxy-6-fluoro-D-galactose which, on acid hydrolysis followed by N-acetylation, gave 2-acetamido-2,6-dideoxy-6-fluoro-D-galactose.     

Synthesis of the t [beta-D-Gal-(1 goes to 3)-alpha-D-GalNAc]-antigenic determinant in a form useful for the preparation of an effective artificial antigen and the corresponding immunoadsorbent

The T antigenic determinant was synthesized in the form 8-methoxycarbonyloctyl 2-acetamido-2-deoxy-3-O-(beta-D-galactopyranosyl)-alpha-D-galactopyranoside (6) (beta-D-Gal-(1 goes to 3)-alpha-D-GalNAcO(CH2)8CO2Me). This T-hapten was used to prepare a T-BSA artificial antigen (7) and an immunoadsorbent (8), which were shown to possess the expected immunological properties. Nuclear Overhauser enhancements of the signals for anti-periplanar H-2' and the syn-axial H-3' of the beta-D-galactopyranosyl group were observed on saturation of H-1'. The signal for H-3 of the 2-acetamido-2-deoxy-alpha-D-galactopyranoside residue was also enhanced.     

8-Amino-5-nitro-6-phenoxyquinolines: potential non-peptidic neuropeptide Y receptor ligands

The synthesis and pharmacological evaluation of analogues of PD 160170, a neuropeptide Y1 (NPY) receptor antagonist are reported. Phamacomodulation of this 8-amino-5-nitro-6-phenylsulfonylquinoline was carried out by replacing the sulfone moiety by oxygen. The corresponding ethers 11-16 were obtained by nucleophilic substitution of 8-acetamido-6-chloro-5-nitroquinoline 4 with phenols, followed by acidic hydrolysis of the intermediary amides 5-10. The test compounds 11-16 exerted no appreciable Y1 activity and they were also inactive in terms of Y5 receptor binding; their IC50 values were >1 microM and 10 microM, respectively. The dramatic decrease in potency resulting from replacement of the sulfone function by an ether was confirmed by IP administration of 16 to ob/ob mice; after a 4-day administration, no decrease in food consumption or weight was observed.     

8-Amino-5-nitro-6-phenoxyquinolines: Potential Non-peptidic Neuropeptide Y Receptor Ligands

The synthesis and pharmacological evaluation of analogues of PD 160170, a neuropeptide Y1 (NPY) receptor antagonist are reported. Phamacomodulation of this 8-amino-5-nitro-6-phenylsulfonylquinoline was carried out by replacing the sulfone moiety by oxygen. The corresponding ethers 11 - 16 were obtained by nucleophilic substitution of 8-acetamido-6-chloro-5-nitroquinoline 4 with phenols, followed by acidic hydrolysis of the intermediary amides 5 - 10. The test compounds 11 - 16 exerted no appreciable Y1 activity and they were also inactive in terms of Y5 receptor binding; their IC 50 values were >1 μM and 10 μM, respectively. The dramatic decrease in potency resulting from replacement of the sulfone function by an ether was confirmed by IP administration of 16 to ob / ob mice; after a 4-day administration, no decrease in food consumption or weight was observed.     

Purification and characterization of a new lectin from the hard roe of skipjack tuna,Katsuwonus pelamis

Fish eggs are known as a rich source of lectins. In this study we purified and characterized a lectin from unfertilized Katsuwonus pelamis hard roe. K. pelamis lectin (KPL) was purified by separation into two fractions above and below the molecular weight of 10kDa using ultramembrane, gel filtration on a Sephadex G-100, and affinity chromatography on an asialofetuin-Sepharose 4B. KPL is a glycoprotein of 140kDa, composed mainly of aspartic acid, glycine, phenylalanine, glutamic acid, threonine and serine residues. Analysis of the carbohydrate composition by gas-liquid chromatography indicated that carbohydrates constituted 14% of the total weight and this 14% is comprised of mannose, galactose, N-acetyl-D-glucosamine, N-acetyl-D-galactosamine, fucose, arabinose and sialic acid. The lectin is comprised of four subunits. These subunits have a molecular mass corresponding to 35kDa. KPL specifically agglutinated human blood type A erythrocytes and, in a hemagglutination inhibitory test, the potent inhibitors were D-galactose, lactose, lactosamine, asialofetuin, N-acetyl-D-galactosamine, O-serinyl-2-acetamido-2-deoxy-alpha-D-galactopyranoside and O-serinyl-2-acetamido-2-deoxy-beta-D-galactopyranoside (O-serinyl-beta-D-GalNAc). The first 10 residues of the N-terminal region were determined as PVELCDAKCT. Furthermore it was determined that the hemagglutinating activity of KPL was dependent on divalent metal cations and that the optimum activity of KPL was exhibited at 40 degrees C and pH 6.0-8.5 in the presence of Ca2+.     

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.     

Multigram syntheses of the disaccharide repeating units of chondroitin 4- and 6-sulfates.

The multigram syntheses of beta-D-glucopyranosyluronic acid-(1-->3)-2-acetamido-2-deoxy-4- and 6-O-sulfo-D-galactopyranose disodium salt, the disaccharide repeating units of chondroitin 4- and 6-sulfates, are described. The disaccharide benzyl methyl 2,3,4-tri-O-benzoyl-beta-D-glucopyranosyluronate- (1-->3)-2-acetamido-2-deoxy-alpha-D-galactopyranoside was used as a common intermediate. Selective benzoylation at O-6 followed by O-sulfonation at C-4 of the aminosugar moiety, saponification and catalytic hydrogenation afforded the 4-O-sulfo derivative, whereas selective O-sulfonation at C-6 followed by similar deprotection steps provided the 6-O-sulfo derivative in high yield.     

An efficient synthesis of ganglioside GM3: highly stereocontrolled glycosylations by use of auxiliaries

An efficiently stereocontrolled total synthesis of GM3 alpha-D-Neup5Ac-(2----3)-beta-D-Galp-(1----4)-beta-D-Glcp-(1----1) -Cer was achieved by employing both methyl 5-acetamido-4,7,8,9-tetra-O-benzyl-2-bromo-2,3,5-trideoxy-3- phenylthio-D-erythro-beta-L-gluco-2-nonulopyranosonate for the key sialylation step, and O-[methyl(5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-alpha -D-galacto-2-nonulopyranosyl)onate]-(2----3)-O-(2,4,6-tri-O- acetyl-beta-D-galactopyranosyl-(1----4)-3,6-di-O-acetyl-2-O-pivaloyl- alpha-D-glucopyranosyl trichloroacetimidate and fluoride for the key coupling step with a ceramide derivative. These two steps were significantly altered and improved in comparison with our previous synthesis that had been executed without use of stereocontrolling auxiliaries. GM3 was obtained in 4.5% overall yield in 19 steps starting from allyl O-(2,6-di-O-acetyl-3,4-O-isopropylidene-beta-D-galactopyranosyl)-(1----4 )-2,3,6-tri-O-acetyl-beta-D-glucopyranoside.     

Synthesis of 3-(2-aminoethylthio)propyl glycosides

Anomeric pairs of 3-(2-aminoethylthio)propyl d-galactopyranoside (4, 4a), d-glucopyranoside (5, 5a), and 2-acetamido-2-deoxy-d-glucopyranoside (6, 6a) were prepared by addition of 2-aminoethanethiol to the corresponding, anomeric, allyl glycosides. The allyl α-glycosides were prepared by refluxing the sugars with allyl alcohol in the presence of an acid catalyst; the allyl β-glycosides were prepared by the reaction of acetylated glycosyl bromides with allyl alcohol in the presence of mercuric cyanide, followed by O-deacetylation. The rate of thiol addition to the allylic group was found to be different for each glycoside.     

Conversion of allyl 2-acetamido-2-deoxy-β-d-glucopyranoside into 2-methyl-(3,4,6-tri-O-benzoyl-1,2-dideoxy-α-d- galactopyrano)-[2′,1′:4,5]-2-oxazoline

2-Methyl-(3,4,6-tri-O-benzoyl-1,2-dideoxy-α-d-galactopyrano)-[2′,1′:4,5]-2-oxazoline (7) was prepared from 1-propenyl 2-acetamido-3,4,6-tri-O-benzoyl-2- deoxy-β-d-galactopyranoside (6). The latter was prepared from allyl 2-acetamido-2-deoxy-β-d-glucopyranoside (1) through selective benzoylation at O-3 and O-6, conversion into the 4-p-bromobenzenesulfonate 4, inversion of configuration at C-4 to afford allyl 2-acetamido-3,4,6-tri-O-benzoyl-β-d-galactopyranoside (5), and subsequent isomerization with palladium-charcoal to give 6.     

Synthesis of methyl (or propyl) 2-acetamido-2-deoxy-α-D-glucopyranoside 6-(α-D-glucopyranosyl phosphate) and derivatives for the study of the phosphoric ester linkage in the Micrococcus lysodeikticus cell-wall

Methyl 2-acetamido-3-O-allyl-2-deoxy-4-O-methyl-α-D-glucopyranoside, methyl 2-acetamido-2-deoxy-4-O-methyl-α-D-glucopyranoside, and methyl 2-acetamido-3,4-di-O-allyl-2-deoxy-α-D-glucopyranoside, prepared from methyl 2-acetamido-2-deoxy-α-D-glucopyranoside, were coupled with 2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl phosphate (13), to give the phosphoric esters methyl 2-acetamido-3-O-allyl-2-deoxy-4-O-methyl-α-D-glucopyranoside 6-(2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl phosphate) (16), methyl 2-acetamido-2-deoxy-4-O-methyl-α-D-glucopyranoside 6-(2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl phosphate) (23), and methyl 2-acetamido-3,4-di-O-allyl-2-deoxy-α-D-glucopyranoside 6-(2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl phosphate) (17). Compound 13 was prepared from penta-O-acetyl-β-D-glucopyranose by the phosphoric acid procedure, or by acetylation of α-D-glucopyranosyl phosphate. Removal of the allyl groups from 16 and 17 gave 23 and methyl 2-acetamido-2-deoxy-α-D-glucopyranoside 6-(2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl phosphate) (19), respectively. O-Deacetylation of 23 gave methyl 2-acetamido-2-deoxy-4-O-methyl-α-D-glucopyranoside 6-(α-D-glucopyranosyl phosphate) (26) and O-deacetylation of 19 gave methyl 2-acetamido-2-deoxy-α-D-glucopyranoside 6-(α-D-glucopyranosyl phosphate) (24). Propyl 2-acetamido-2-deoxy-α-D-glucopyranoside 6-(α-D-glucopyranosyl phosphate) (25) was prepared by coupling 13 with allyl 2-acetamido-3,4-di-O-benzyl-2-deoxy-α-D-glucopyranoside, followed by catalytic hydrogenation of the product to give the propyl glycoside, which was then O-deacetylated. Compounds 24, 25, and 26 are being employed in structural studies of the Micrococcus lysodeikticus cell-wall.     

Circular dichroism studies on alpha- and beta-ketosides of 5-acetamido-3,5-dideoxy-D-glycero-D-galacto- nonulopyranosonic acid (N-acetylneuraminic acid) and of some of its derivatives.

The circular dichroism spectra of a number of N-acetylneuraminic acid derivatives in aqueous solution were studied. For all compounds, the Cotton effects were found to be in the spectral range of the acetamido and carboxyl chromophores. The c.d. curves of the methy, ethyl, and allyl alpha-D-ketosides are characterized by a broad, positive band centered at lambda similar to 195 nm with a slight skew towards the higher wavelengths and weak bands between lambda 225 and 255 nm, whereas the methyl beta-D-ketoside and the corresponding methyl ester show only an intense positive band with a broad shoulder in the same spectral range. 5-Acetamido-3,5-dideoxy-D-glycero-beta-D-galacto-nonulopyranose, its methyl beta-D-ketoside, and 5-acetamido-3,5-dideoxy-D-glycero-D-galacto-nonulopyranosonamide containing only the acetamido chromophore showed one single positive Cotton effect centered at lambda similar to 192 nm. The c.d. spectrum of 5-acetamido-3,5-dideoxy-D-glycero-D-galacto-nonulopyranosonic acid confirms the beta-D configuration of the free acid in aqueous solution, whereas the shape of the c.d. curve of O-(N-acetyl-alpha-D-neuraminopyranosyl)-(2yields3)-O-beta-D-galactopyranosyl-(1 yields 4)-D-glucopyranose resembles that of the methyl, ethyl, and allyl alpha-D-ketosides 2-4.     

The linkage of 4-O-methyl-L-galactose in the sulphated polysaccharide of Aeodes ulvoidea

Methyl 2-acetamido-5,6-di-O-benzyl-2-deoxy-β-d-glucofuranoside (11) was obtained in six steps from the known methyl 3-O-allyl-2-benzamido-2-deoxy-5,6-O-isopropylidene-β-d-glucofuranoside. Mild acid hydrolysis, followed by benzylation gave the 5,6-dibenzyl ether. The benzamido group was exchanged for an acetamido group by strong alkaline hydrolysis, followed by N-acetylation, and the allyl group was isomerized into a 1-propenyl group that was hydrolyzed with mercuric chloride. Treatment of 11 with l-α-chloropropionic acid and with diazomethabe gave methyl 2-acetamido-5,6-di-O-benzyl-2-deoxy-3-O-[d-1-(methoxycarbonyl)ethyl]-β-d-glucofuranoside which formed on mercaptolysis the internal ester 16, further converted into 2-acetamido-4-O-acetyl-5,6-di-O-benzyl-2-deoxy-3-O-[d-1-(methoxycarbonyl)ethyl]-d-glucose diethyl dithioacetal (18) by alkaline treatment followed by esterification with diazomethane and acetylation. Attempts to remove the O-acetyl group of the corresponding dimethyl acetal 20 with sodium methoxide in mild conditions were not successful.