Photoaffinity labeling of the lysosomal neuraminidase from bovine testis

ASA-NeuAc2en, a photoreactive arylazide derivative of sialic acid, is shown to be a powerful competitive inhibitor of lysosomal neuraminidase from bovine testis (Ki approximately 21 microM). Photoaffinity labeling and partial purification of preparations containing this lysosomal neuraminidase activity result in specifically and non-specifically labeled polypeptides. Only labeling in a 55 kDa polypeptide is found to be specific, since it could be prevented by the competitive neuraminidase inhibitor NeuAc2en. We conclude that the 55 kDa polypeptide in the bovine testis beta-galactosidase/neuraminidase/protective protein complex contains the catalytic site of neuraminidase.     

Streptococcus pneumoniae NanC: STRUCTURAL INSIGHTS INTO THE SPECIFICITY AND MECHANISM OF A SIALIDASE THAT PRODUCES A SIALIDASE INHIBITOR*

Streptococcus pneumoniae is an important human pathogen that causes a range of disease states. Sialidases are important bacterial virulence factors. There are three pneumococcal sialidases: NanA, NanB, and NanC. NanC is an unusual sialidase in that its primary reaction product is 2-deoxy-2,3-didehydro-N-acetylneuraminic acid (Neu5Ac2en, also known as DANA), a nonspecific hydrolytic sialidase inhibitor. The production of Neu5Ac2en from α2–3-linked sialosides by the catalytic domain is confirmed within a crystal structure. A covalent complex with 3-fluoro-β-N-acetylneuraminic acid is also presented, suggesting a common mechanism with other sialidases up to the final step of product formation. A conformation change in an active site hydrophobic loop on ligand binding constricts the entrance to the active site. In addition, the distance between the catalytic acid/base (Asp-315) and the ligand anomeric carbon is unusually short. These features facilitate a novel sialidase reaction in which the final step of product formation is direct abstraction of the C3 proton by the active site aspartic acid, forming Neu5Ac2en. NanC also possesses a carbohydrate-binding module, which is shown to bind α2–3- and α2–6-linked sialosides, as well as N-acetylneuraminic acid, which is captured in the crystal structure following hydration of Neu5Ac2en by NanC. Overall, the pneumococcal sialidases show remarkable mechanistic diversity while maintaining a common structural scaffold.     

Differential functional relevance of a plasma membrane ganglioside sialidase in cholinergic and adrenergic neuroblastoma cell lines.

Gangliosides located in the outer leaflet of the plasma membrane are important modulators of cellular functions. Our previous work has shown that in cultured human SK-N-MC neuroblastoma cells a sialidase residing in the same membrane selectively desialylates gangliosides with terminal sialic acid residues, causing a shift from higher species to GM1 and a conversion of GM3 to lactosylceramide. Inhibition of this sialidase by 2-deoxy-2,3-dehydro-N-acetylneuraminic acid (NeuAc2en) resulted in increased cell proliferation and a loss of differentiation markers. In this study, we examined the occurrence and function of this ganglioside sialidase in other neuronal cells. Subcellular fractionation showed the sialidase to be located in the plasma membrane of all cell lines studied. The presence of the inhibitor NeuAc2en led to a profound decrease in the amount of the differentiation marker 200 kDa/70 kDa neurofilaments and an increase in cell proliferation in the cholinergic SK-N-MC and mixed cholinergic/adrenergic SK-N-FI and SK-N-DZ neuroblastoma lines, but had little or no effect in the human adrenergic SK-N-SH and SK-N-AS and the adrenergic/cholinergic PC12 cells from rat. The influence of the inhibitor on cell behaviour was paralleled by a diminished number of cholera toxin B-binding GM1 sites. The findings demonstrate that the plasma membrane ganglioside sialidase is an important element of proliferation and differentiation control in some, but not all, neuroblastoma cells and suggest that there might be a relationship between plasma membrane sialidase activity and cholinergic differentiation.     

On the inhibition mechanism of the sialidase activity from Newcastle disease virus.

N-Acetylneuraminic, 2-deoxy-2,3-didehydro-N-acetylneuraminic acid and the beta anomer of methoxyneuraminic acid (Neu5Ac, Neu5Ac2en, MeONeu) have been used as probes for the catalytic mechanism of the activities of the outer membrane-bound haemagglutinin-neuraminidase (HN) from newcastle disease virus (NDV). Neu5Ac and Neu5Ac2en produced a competitive inhibition of the sialidase (= neuraminidase) activity, whereas MeONeu had no effect on this activity. This lack of inhibition can be explained by the free amino-acid group lacking the acetyl substituent in the MeONeu. Neu5Ac2en produced the highest inhibition. Based on the effect of the inhibitors, a reaction mechanism is suggested. On the other hand, the above mentioned inhibitors of the sialidase activity had no effect on haemagglutinating activity, suggesting different active sites for the both activities.     

Characterization of a sialidase (neuraminidase) isolated from Clostridium chauvoei (Jakari strain)

A sialidase from Clostridium chauvoei (Jakari strain), an indigenous bacterial strain that causes blackleg in Nigerian cattle and other ruminants was isolated and partially purified by chromatography on DEAE cellulose, hydroxyapatite and phenyl agarose columns. The enzyme migrated as a 65-kDa protein after electrophoresis on sodium dodecyl sulphate polyacrylamide gels. It was optimally active at pH 4.5 and 40 degrees C with an activation energy (Ea) of 13.40 kJ mol(-1). It had Km and Vmax values of 170 microM and 200 micromole h(-1) mg(-1) respectively with fetuin as substrate. When sialyllactose (Neu5Ac2,3 lactose) was used as substrate the Km and Vmax values were 8 microM and 5 micromoles min(-1) mg(-1) respectively. The Clostridium chauvoei sialidase cleaved sialic acids from RBC ghosts of sheep, horse, goat, cattle, pig and mice as well as mouse brain cells, albeit at different rates. The enzyme was activated by Ca2+ and Mg2+ and inhibited by the group-specific reagents diethylpyrocarbonate (DEP) and N-ethylmalemide (NEM). The sialidase inhibitors, 2,3 didehydroneuraminic acid (Neu5Ac2,3en) and paranitrophenyl oxamic acid (pNPO) inhibited the enzyme competitively with Ki values of 40 and 30 microM respectively.     

Characterization of sialidase from bloodstream forms of Trypanosoma vivax

Sialidase (EC: 3.2.1.18) from Trypanosoma vivax (Agari Strain) was isolated from bloodstream forms of the parasite and purified to apparent electrophoretic homogeneity. The enzyme was purified 77-fold with a yield of 32% and co-eluted as a 66-kDa protein from a Sephadex G 110 column. The T. vivax sialidase was optimally active at 37 degrees C with an activation energy (E(a)) of 26.2 kJ mole(-1). The pH activity profile was broad with optimal activity at 6.5. The enzyme was activated by dithiothreitol and strongly inhibited by para-hydroxy mercuricbenzoate thus implicating a sulfhydryl group as a possible active site residue of the enzyme. Theenzyme hydrolysed Neu5Ac2,3lac and fetuin. It was inactive towards Neu5Ac2,6lac, colomic acid and the gangliosides GM1, and GDI. Initial velocity studies, for the determination of kinetic constants with fetuin as substrate gave a V(max) of 142.86 micromol h(-1) mg(-1) and a K(M) of 0.45 mM. The K(M) and V(max) with Neu5Ac-2,3lac were 0.17 mM and 840 micromole h(-1) mg(-1) respectively. The T. vivax sialidase was inhibited competitively by both 2,3 dideoxy neuraminic acid (Neu5Ac2,3en) and para-hydroxy oxamic acid. When ghost RBCs were used as substrates, the enzyme desialylated the RBCs from camel, goat, and zebu bull. The RBCs from dog, mouse and ndama bull were resistant to hydrolysis.     

Synthesis and evaluation of 4-O-alkylated 2-deoxy-2,3-didehydro-N-acetylneuraminic acid derivatives as inhibitors of human parainfluenza virus type-3 sialidase activity

The X-ray crystal structure of the paramyxoviral surface glycoprotein haemagglutinin-neuraminidase (HN) from Newcastle Disease virus was used as a template to design inhibitors of the HN from human parainfluenza virus type-3 (hPIV-3). 4-O-Alkylated derivatives of 2-deoxy-2,3-didehydro-N-acetylneuraminic acid (Neu5Ac2en), accessed from 8,9-O-isopropylidenated-Neu5Ac2en1Me, were found to inhibit the sialidase (neuraminidase) activity of hPIV-3 (strain C243) in the range of 3-30muM. This is comparable or improved activity compared to the parent 4-hydroxy compound.     

The Aspergillus fumigatus sialidase is a 3-deoxy-D-glycero-D-galacto-2-nonulosonic acid hydrolase (KDNase): structural and mechanistic insights

Aspergillus fumigatus is a filamentous fungus that can cause severe respiratory disease in immunocompromised individuals. A putative sialidase from A. fumigatus was recently cloned and shown to be relatively poor in cleaving N-acetylneuraminic acid (Neu5Ac) in comparison with bacterial sialidases. Here we present the first crystal structure of a fungal sialidase. When the apo structure was compared with bacterial sialidase structures, the active site of the Aspergillus enzyme suggested that Neu5Ac would be a poor substrate because of a smaller pocket that normally accommodates the acetamido group of Neu5Ac in sialidases. A sialic acid with a hydroxyl in place of an acetamido group is 2-keto-3-deoxynononic acid (KDN). We show that KDN is the preferred substrate for the A. fumigatus sialidase and that A. fumigatus can utilize KDN as a sole carbon source. A 1.45-Å resolution crystal structure of the enzyme in complex with KDN reveals KDN in the active site in a boat conformation and nearby a second binding site occupied by KDN in a chair conformation, suggesting that polyKDN may be a natural substrate. The enzyme is not inhibited by the sialidase transition state analog 2-deoxy-2,3-dehydro-N-acetylneuraminic acid (Neu5Ac2en) but is inhibited by the related 2,3-didehydro-2,3-dideoxy-d-glycero-d-galacto-nonulosonic acid that we show bound to the enzyme in a 1.84-Å resolution crystal structure. Using a fluorinated KDN substrate, we present a 1.5-Å resolution structure of a covalently bound catalytic intermediate. The A. fumigatus sialidase is therefore a KDNase with a similar catalytic mechanism to Neu5Ac exosialidases, and this study represents the first structure of a KDNase.     

The structure of Clostridium perfringens NanI sialidase and its catalytic intermediates

Clostridium perfringens is a Gram-positive bacterium responsible for bacteremia, gas gangrene, and occasionally food poisoning. Its genome encodes three sialidases, nanH, nanI, and nanJ, that are involved in the removal of sialic acids from a variety of glycoconjugates and that play a role in bacterial nutrition and pathogenesis. Recent studies on trypanosomal (trans-) sialidases have suggested that catalysis in all sialidases may proceed via a covalent intermediate similar to that of other retaining glycosidases. Here we provide further evidence to support this suggestion by reporting the 0.97A resolution atomic structure of the catalytic domain of the C. perfringens NanI sialidase, and complexes with its substrate sialic acid (N-acetylneuramic acid) also to 0.97A resolution, with a transition-state analogue (2-deoxy-2,3-dehydro-N-acetylneuraminic acid) to 1.5A resolution, and with a covalent intermediate formed using a fluorinated sialic acid analogue to 1.2A resolution. Together, these structures provide high resolution snapshots along the catalytic pathway. The crystal structures suggested that NanI is able to hydrate 2-deoxy-2,3-dehydro-N-acetylneuraminic acid to N-acetylneuramic acid. This was confirmed by NMR, and a mechanism for this activity is suggested.     

Synthesis of polyhydroxy amino acids based on D- and L-alanine from D-glycero-D-gulo-heptono-1,4-lactone

2-Amino-2,3-dideoxy-D-manno-heptonic acid (7) has been synthesized from 2,5,6,7-tetra-O-acetyl-3-deoxy-D-gluco-heptono-1,4-lactone (1), which was readily prepared from D-glycero-D-gulo-heptono-1,4-lactone. O-Deacetylation of 1 followed by treatment with 13:1 (v/v) 2,2-dimethoxypropane/acetone in the presence of p-toluenesulfonic acid gave methyl 3-deoxy-4,5:6,7-di-O-isopropylidene-D-gluco-heptonate (3) as a crystalline product (80% yield). The free hydroxyl group (OH-2) of 3 was mesylated and substituted by azide to give the corresponding azide derivative 5. Hydrogenolysis and further hydrolysis of the ester function of 5 afforded alpha-amino acid 7 (43% overall yield from 1). Compound 7 is an analog of L-alanine having a polyhydroxy chain attached to C-3. The diastereoisomer of 7 at C-2, 2-amino-2,3-dideoxy-D-gluco-heptonic acid (12) was also prepared from 3, by a route that involved 2,3-dideoxy-2-iodo derivative 8 as a key intermediate.     

Purification and characterization of sialidase fromClostridium sordellii G12

Sialidase secreted by the urease-positiveClostridium sordellii strain G12 was isolated from culture medium and purified to apparent homogeneity as estimated by Fast Protein Liquid Chromatography (FPLC) and sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE). For this purpose, ion-exchange chromatography, gel filtration, isoelectric focusing, and FPLC on ion-exchange resin and gel filtration materials were used. The sialidase was purified 159 300-fold from 5 l of culture medium, yielding 9 g of enzyme protein with a specific activity of 480 U/mg. For the denatured (SDS-PAGE) and native (FPLC) sialidase relative molecular masses of 40 000 and 38 500 Da, respectively, were estimated. The substrate specificity, kinetic data, and pH-optimum of the enzyme are similar to those of other bacterial sialidases. The influences of salt or serum proteins on enzyme activity are of interest.Abbreviations MU-Neu5Ac 4-methylumbelliferyl -d-N-acetylneuraminic acid - Ganglioside GD1a IV³NeuAc, ll³NeuAc-GgOse₄Cer - Neu5Ac2en 2-deoxy-2,3-didehydro-N-acetylneuraminic acid     

Structural parameters and natural occurrence of 2-deoxy-2,3-didehydro-N-glycoloylneuraminic acid

2-Deoxy-2,3-didehydro-N-glycoloylneuraminic acid has been found to occur in porcine, bovine and equine submandibular glands as well as in the urine of pig, horse and rat. This novel, unsaturated sialic acid was isolated by gel filtration and ion-exchange chromatography. Final purification was achieved by column chromatography or by preparative thin-layer chromatography on cellulose. The structural analysis was performed by combined capillary gas-liquid chromatography/mass spectrometry. The various data were compared with those from synthetic 2-deoxy-2,3-didehydro-N-glycoloylneuraminic acid. Besides of the unsaturated N-glycoloylated sialic acid, also the corresponding N-acetylated derivative was present in the materials analyzed. The inhibitory effect of 2-deoxy-2,3-didehydro-N-glycoloylneuraminic acid on Vibrio cholerae sialidase using N-acetylneuraminyl-(alpha 2----3)-lactose as substrate is slightly higher (50% inhibition at 10 microM) when compared with 2-deoxy-2,3-didehydro-N-acetylneuraminic acid (50% inhibition at 15 microM).     

Differential effect of various inhibitors on four types of rat sialidase

The inhibitory effect of various compounds on the activities of four types of rat sialidase was investigated. 2-Deoxy-2,3-dehydro-N-acetylneuraminic acid andN-acetylneuraminic acid were competitive inhibitors for the sialidases. The former was effective against cytosolic sialidase and intralysosomal sialidase more than two membrane-associated sialidases I and II, the latter being a much weaker inhibitor. A heavy metal ion such as Cu²⁺ (1mm) and thiol-modifying 4-hydroxymercuribenzoate (50 µm) caused complete inhibition of the activities of cytosolic sialidase and membrane sialidase I, while no decrease in the activities of intralysosomal sialidase and membrane sialidase II was observed. When 4-nitrophenyloxamic acid and siastatin B, inhibitors of bacterial sialidases, and synthetic thioglycoside GM3 analogue Neu5Ac-s-(2-6)Gal(1-4)Glc(1-1) ceramide, an inhibitor of influenza virus sialidase, were tested, they did not affect any activity of the rat sialidases. By the differential effect of these inhibitors, the four types of rat sialidase could be discriminated from one another and furthermore from viral and bacterial sialidases.Abbreviations Neu5Ac N-acetylneuraminic acid - Neu5Ac2en 2-deoxy-2,3-dehydro-N-acetylneuraminic acid - 4MU-Neu5Ac 4-methylumbelliferyl--N-acetyl-d-neuraminic acid     

Substrate specificity and inhibitor studies of a membrane-bound ganglioside sialidase isolated from human brain tissue

A ganglioside-specific sialidase that controls cellular functions such as growth, differentiation, and adhesion has been observed in a variety of cells, but its characterization proved difficult due to firm membrane attachment and lability of the purified enzyme. Here we report on the specificity toward gangliosides and susceptibility to certain inhibitors of a ganglioside sialidase solubilized and purified 5100-fold from human brain. The sialidase removed terminal sialic acids from gangliosides GM3, GM4, GD3, GD2, GD1 a, GD1 b, GT1 b and GQ1 b, but was inactive toward gangliosides with sialic acid in a branching position (as in GM1 and GM2). Lyso-GM3 and -GD1a were good substrates, too, whereas O-acetylation of the sialic acid as in 9-O-acetyl-GD3 caused strongly reduced cleavage. The new influenza virus drug 4-guanidino-2-deoxy-2,3-dehydro-N-acetylneuraminic acid (Zanamivir) exhibited an IC50 value of about 7 x 10(-5) M that was in the range of the 'classical' sialidase inhibitor 2-deoxy-2,3-dehydro-N-acetylneuraminic acid; the bacterial sialidase inhibitor 4-nitrophenyloxamic acid, however, was ineffective. The glycosaminoglycans heparan sulfate, heparin, chondroitin sulfates A and B, as well as dextran sulfate and suramin, were all strongly inhibitory, suggesting that glycosaminoglycans present on the cell surface or in the extracellular matrix may influence the ability of the sialidase to alter the ganglioside composition of the membrane.     

Diversity of expressed vlhA adhesin sequences and intermediate hemagglutination phenotypes in Mycoplasma synoviae

A reservoir of pseudogene alleles encoding the primary adhesin VlhA occurs in the avian pathogen Mycoplasma synoviae. Recombination between this reservoir and its single expression site was predicted to result in lineages of M. synoviae that each express a different vlhA allele as a consequence of host immune responses to those antigens. Such interstrain diversity at the vlhA expression site, including major differences in the predicted secondary structures of their expressed adhesins, was confirmed in 14 specimens of M. synoviae. Corresponding functional differences in the extent to which they agglutinated erythrocytes, a quantitative proxy for VlhA-mediated cytadherence, were also evident. There was a >20-fold difference between the highest- and lowest-agglutinating strains and a rheostatic distribution of intermediate phenotypes among the others (Tukey-Kramer honestly significant difference [HSD], P < 0.001). Coincubation with the sialic acid analog 2-deoxy-2,3-didehydro-N-acetylneuraminate inhibited hemagglutination in a pattern correlated with endogenous sialidase activity (r = 0.91, P < 0.001), although not consistently to the same extent that erythrocyte pretreatment with sialidase purified from Clostridium perfringens did (P < 0.05). The striking correlation between the ranked hemagglutination and endogenous sialidase activities of these strains (Spearman's r = 0.874, P < 0.001) is evidence that host-induced vlhA allele switching indirectly drives sequence diversity in the passenger sialidase gene of M. synoviae.     

Crystal structure of the NanB sialidase from Streptococcus pneumoniae

The Streptococcus pneumoniae genomes encode up to three sialidases (or neuraminidases), NanA, NanB and NanC, which are believed to be involved in removing sialic acid from host cell surface glycans, thereby promoting colonization of the upper respiratory tract. Here, we present the crystal structure of NanB to 1.7 Å resolution derived from a crystal grown in the presence of the buffer Ches (2-N-cyclohexylaminoethanesulfonic acid). Serendipitously, Ches was found bound to NanB at the enzyme active site, and was found to inhibit NanB with a K_i of ∼ 0.5 mM. In addition, we present the structure to 2.4 Å resolution of NanB in complex with the transition-state analogue Neu5Ac2en (2-deoxy-2,3-dehydro-N-acetyl neuraminic acid), which inhibits NanB with a K_i of ∼ 0.3 mM. The sulphonic acid group of Ches and carboxylic acid group of Neu5Ac2en interact with the arginine triad of the active site. The cyclohexyl group of Ches binds in the hydrophobic pocket of NanB occupied by the acetamidomethyl group of Neu5Ac2en. The topology around the NanB active site suggests that the enzyme would have a preference for α2,3-linked sialoglycoconjugates, which is confirmed by a kinetic analysis of substrate binding. NMR studies also confirm this preference and show that, like the leech sialidase, NanB acts as an intramolecular trans-sialidase releasing Neu2,7-anhydro5Ac. All three pneumoccocal sialidases possess a carbohydrate-binding domain that is predicted to bind sialic acid. These studies provide support for a possible differential role for NanB compared to NanA in pneumococcal virulence.     

Inhibition of human parainfluenza virus type 1 sialidase by analogs of 2-deoxy-2,3-didehydro-N-acetylneuraminic acid

Eleven novel analogs of 2-deoxy-2,3-didehydro-N-acetylneuraminic acid (Neu5Ac2en) modified at the C-4 and C-9 positions were designed and tested for their ability to inhibit sialidase of human parainfluenza virus type 1 (hPIV-1). The analogs modified by the cyanomethyl, amidinomethyl, and thiocarbamoylmethyl groups at the C-4 position exhibited potent inhibition against hPIV-1 sialidase compared with Neu5Ac2en. The most effective compound was thiocarbamoylmethyl analog (4-O-thiocarbamoylmethyl-Neu5Ac2en). The activity of 4-O-thiocarbamoylmethyl-Neu5Ac2en causing 50% enzyme inhibition at a concentration of approximately 1.0×10^–5M was 30-fold larger than Neu5Ac2en. While, the analogs of Neu5Ac2en modified by the azido and N-acetyl groups at the C-9 showed a decrease in inhibition of sialidase compared with the 9-hydroxy analogs. In addition, 4-O-thiocarbamoylmethyl-Neu5Ac2en strongly inhibited hPIV-1 infections of Lewis lung carcinoma-monkey kidney cells in comparison with Neu5Ac2en. The present findings would provide useful information for the development of anti-human parainfluenza virus compounds.     

Structural and biochemical characterization of the broad substrate specificity of Bacteroides thetaiotaomicron commensal sialidase

Sialidases release the terminal sialic acid residue from a wide range of sialic acid-containing polysaccharides. Bacteroides thetaiotaomicron, a symbiotic commensal microbe, resides in and dominates the human intestinal tract. We characterized the recombinant sialidase from B. thetaiotaomicron (BTSA) and demonstrated that it has broad substrate specificity with a relative activity of 97, 100 and 64 for 2,3-, 2,6- and 2,8-linked sialic substrates, respectively. The hydrolysis activity of BTSA was inhibited by a transition state analogue, 2-deoxy-2,3-dehydro-N-acetyl neuraminic acid, by competitive inhibition with a K_i value of 35 μM. The structure of BSTA was determined at a resolution of 2.3 Å. This structure exhibited a unique carbohydrate-binding domain (CBM) at its N-terminus (a.a. 23–190) that is adjacent to the catalytic domain (a.a. 191–535). The catalytic domain has a conserved arginine triad with a wide-open entrance for the substrate that exposes the catalytic residue to the surface. Unlike other pathogenic sialidases, the polysaccharide-binding site in the CBM is near the active site and possibly holds and positions the polysaccharide substrate directly at the active site. The structural feature of a wide substrate-binding groove and closer proximity of the polysaccharide-binding site to the active site could be a unique signature of the commensal sialidase BTSA and provide a molecular basis for its pharmaceutical application.     

Synthesis of aminoethylglycosides with oligosaccharide GM1 and asialo-GM1 ganglioside chains

4'-O-Glycosylation of 2-azidoethyl 2,3,6-tri-O-benzyl-4-O-(2,3-di-O- benzyl-6-O-benzoyl-beta-D-galactopyranosyl)-beta-D-glucopyranoside with a disaccharide donor, 4-trichloroacetamidophenyl 4,6-di-O-acetyl-2-deoxy-3-O-(2,3,4,6-tetra-O-acetyl-beta-D- galactopyranosyl)-1-thio-2-trichloroacetamido-beta-D-galactopyranoside, in dichloromethane in the presence of N-iodosuccinimide and trifluoromethanesulfonic acid resulted in a tetrasaccharide, 2-azidoethyl (2,3,4,6-tetra-O-acetyl-beta-D-galactopyranosyl)-(1-->3)- (4,6-di-O-acetyl-2-deoxy-2-trichloroacetamido-beta-D-galactopyranosyl)- (1-->4)-(2,3-di-O-benzyl-6-O-benzoyl-beta-D-galactopyranosyl)- (1-->4)-2,3,6-tri-O-benzyl-beta-D-glucopyranoside, in 69% yield. The complete removal of O-protecting groups in the tetrasaccharide, the replacement of N-trichloroacetyl by N-acetyl group, and the reduction of the aglycone azide group to amine led to the target aminoethyl glycoside of beta-D-Gal- (1-->3)-beta-D-GalNAc-(1-->4)-beta-D-Gal-(1-->4)-beta-D-Glc-OCH2CH2NH2 containing the oligosaccharide chain of asialo-GM1 ganglioside in 72% overall yield. Selective 3'-O-glycosylation of 2-azidoethyl 2,3,6-tri-O- benzyl-4-O-(2,6-di-O-benzyl-beta-D-galactopyranosyl)-beta-D-glucopyranoside with thioglycoside methyl (ethyl 5-acetamido-4,7,8,9-tetra-O- acetyl-3,5-dideoxy-2-thio-D-glycero-alpha-D-galacto-2-nonulopyranosyl)oate in acetonitrile in the presence of N-iodosuccinimide and trifluoroacetic acid afforded 2-azidoethyl [methyl (5-acetamido-4,7,8,9-tetra-O-acetyl- 3,5-dideoxy-D-glycero-alpha-D-galacto-2-nonulopyranosyl)oate in acetonitrile in the presence of N-iodosuccinimide and tri-fluoracetic acid afforded 2-azidoethyl[methyl (5-acetamido-4,7,8,9-tetra-O-acetyl- 3,5-dideoxy-D-glycero-alpha-D-galacto-2-nonulopyranosyl) (2,6-di-O-benzyl-beta-D-galactopyranosyl)-(1-->4)-2,3,6-tri-O-benzyl-beta-D- glucopyranoside, the selectively protected derivative of the oligosaccharide chain of GM3 ganglioside, in 79% yield. Its 4'-O-glycosylation with a disaccharide glycosyl donor, (4-trichloroacetophenyl-4,6-di-O-acetyl-2-deoxy-3-O-(2,3,4,6-tetra-O- acetyl-beta-D-galactopyranosyl) 1-thio-2-trichloroacetamido-beta-D-galactopyranoside in dichloromethane in the presence of N-iodosuccinimide and trifluoroacetic acid gave 2-azidoethyl (2,3,4,6-tetra-O-acetyl-beta-D-galactopyranosyl)- (1-->3)-(4,6-di-O-acetyl-2-deoxy-2-trichloroacetamido-beta-D- galactopyranosyl)-(1-->4)-[[methyl (5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-D-glycero-alpha-D- galacto-2-nonulopyranosyl)onate]-(2-->3)]-(2,6-di-O-benzyl-beta-D- galactopyranosyl)-(1-->4)-2,3,6-tri-O-benzyl-beta-D-glucopyranoside in 85% yield. The resulting pentasaccharide was O-deprotected, its N-trichloroacetyl group was replaced by N-acetyl group, and the aglycone azide group was reduced to afford in 85% overall yield aminoethyl glycoside of beta-D-Gal-(1-->3)-beta-D-GalNAc-(1-->4)-[alpha-D-Neu5Ac-(2-->3)]- beta-D-Gal-(1-->4)-beta-D-Glc-OCH2CH2NH2 containing the oligosaccharide chain of GM1 ganglioside. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 1; see also http://www.maik.ru.     

Design of N-acetyl-6-sulfo-beta-d-glucosaminide-based inhibitors of influenza virus sialidase

Biological activity of N-acetyl-6-sulfo-beta-d-glucosaminides (6-sulfo-GlcNAc 1) having a structural homology to N-acetylneuraminic acid (Neu5Ac 2) and 2-deoxy-2,3-dehydro-N-acetylneuraminic acid (Neu5Ac2en 3) was examined in terms of inhibitory activity against influenza virus sialidase (influenza, A/Memphis/1/71 H3N2). pNP 6-Sulfo-GlcNAc 1a was proved to show substantial activity to inhibit the virus sialidase (IC(50)=2.8 mM), though p-nitrophenyl (pNP) GlcNAc without 6-sulfo group and pNP 6-sulfo-GlcNH(3)(+) 1b without 2-NHAc showed little activity (IC(50) >50 mM). The activity was enhanced nearly 100-fold when the pNP group of 1a was converted to p-acetamidophenyl one 5 (IC(50)=30 microM) or replaced with 1-naphthyl 6 (IC(50)=10 microM) or n-propyl one 8 (IC(50)=11 microM).