Synthesis of selective inhibitors against V. cholerae sialidase and human cytosolic sialidase NEU2

Sialidases or neuraminidases catalyze the hydrolysis of terminal sialic acid residues from sialyl oligosaccharides and glycoconjugates. Despite successes in developing potent inhibitors specifically against influenza virus neuraminidases, the progress in designing and synthesizing selective inhibitors against bacterial and human sialidases has been slow. Guided by sialidase substrate specificity studies and sialidase crystal structural analysis, a number of 2-deoxy-2,3-dehydro-N-acetylneuraminic acid (DANA or Neu5Ac2en) analogues with modifications at C9 or at both C5 and C9 were synthesized. Inhibition studies of various bacterial sialidases and human cytosolic sialidase NEU2 revealed that Neu5Gc9N(3)2en and Neu5AcN(3)9N(3)2en are selective inhibitors against V. cholerae sialidase and human NEU2, respectively.     

Analysis of transmembrane dynamics of cholera toxin using photoreactive probes.

Using sodium dodecyl sulfate--polyacrylamide gel electrophoresis and autoradiography, we have shown that 125I-labeled cholera toxin binds to Newcastle disease virus. Pretreatment of Newcastle disease virus with "cold" cholera toxin (at 37 degrees C for 30 minutes) inhibits the binding of 125I-labeled toxin in a subsequent incubation (at 37 degrees C for 30 minutes). These results suggest that cholera toxin binds to Newcastle disease virus in a specific manner. The precise receptor for toxin is unknown in Newcastle disease virus but it is presumed to be the ganglioside GM1. We have previously shown that the photoreactive probe 12-(4-azido-2-nitrophenoxy)stearoylglucosamine[1-14C] labels the membrane proteins of Newcastle disease virus. Since the reactive group of the probe, ie, N3, resides within the membrane bilayer, studies were initiated to determined which, if any, of the subunits of cholera toxin cross the membrane of Newcastle disease virus and become radioactively labeled upon photoactivation of the probe at 360 nm. After a 15-minute incubation of cholera toxin with Newcastle disease virus containing the photoreactive probe, irradiation effected the 14C-labeling of the active A1 subunit of cholera toxin. Irradiation of cholera toxin in solution with an equivalent amount of probe but without virus resulted in no labeling of toxin subunits.     

Investigation of the stability of thiosialosides toward hydrolysis by sialidases using NMR spectroscopy

[formula: see text] 1H NMR spectroscopy has been used to investigate whether the alpha(2-->6)-linked thiosialoside 3 and the alpha(2-->3)-linked thiosialoside 9 are hydrolyzed in the presence of Vibrio cholerae sialidase. Similarly, the hydrolysis of the O-ketosides Neu5Ac-2-O-alpha-(2-->3)-Gal beta Me (4) and the alpha-(2-->6)-sialyllactoside 7, representing natural alpha(2-->3)- and alpha(2-->6)-linked sialosides, respectively, was investigated. The results of the 1H NMR experiments clearly demonstrate that the thiosialosides are not hydrolyzed by Vibrio cholerae sialidase. As expected, the O-sialosides are hydrolyzed to give N-acetyl-alpha-D-neuraminic acid as the first product of substrate cleavage.     

Synthesis of phosphorylated and sulfated glycosyl serines in the linkage region of the glycosaminoglycans

We synthesized novel acidic glycans having acidic groups located in the linkage region of the glycosaminoglycans (GAGs). The targeted compounds, beta-D-Xyl(2P)-Ser (1), beta-D-Gal(+/-6S)-(1-->4)-beta-D-Xyl(2P)-Ser (3 and 2), beta-D-Gal(+/-6S)-(1-->3)-beta-D-Gal-(1-->4)-beta-D-Xyl(2P)-Ser (5 and 4), and beta-D-Gal-(1-->3)-beta-D-Gal(6S)-(1-->4)-beta-D-Xyl(2P)-Ser (6) contain phosphate and/or sulfate at the specified positions. Some of them (3, 5, and 6) are the first synthesized examples of natural-type glycoconjugates that simultaneously possess phosphate and sulfate as well as carboxylic acid.     

Radioiodination of the envelope proteins of Newcastle disease virus.

Lactoperoxidase-catalyzed iodination selectively labels the two glycoproteins (VP1 and VP2) of Newcastle disease virus. The low-molecular-weight, nonglycosylated major viral protein, VP6, was not iodinated in the intact virus but was iodinated in disrupted virions, suggesting a localization on the inner, rather than the outer, envelope surface. Studies on the distribution of virion proteins labeled with 125-I and 3-H-isoleucine between detergent-soluble and detergent-insoluble fractions show that the virion proteins VP4, VP5, and VP6 are solubilized to a much lesser extent than are VP1 and VP2.     

Interdigitated array microelectrode based impedance immunosensor for detection of avian influenza virus H5N1

Continuous outbreaks of avian influenza (AI) in recent years with increasing threat to animals and human health have warranted the urgent need for rapid detection of pathogenic AI viruses. In this study, an impedance immunosensor based on an interdigitated array (IDA) microelectrode was developed as a new application for sensitive, specific and rapid detection of avian influenza virus H5N1. Polyclonal antibodies against AI virus H5N1 surface antigen HA (Hemagglutinin) were oriented on the gold microelectrode surface through protein A. Target H5N1 viruses were then captured by the immobilized antibody, resulting in a change in the impedance of the IDA microelectrode surface. Red blood cells (RBCs) were used as biolabels for further amplification of the binding reaction of the antibody-antigen (virus). The binding of target AI H5N1 onto the antibody-modified IDA microelectrode surface was further confirmed by atomic force microscopy. The impedance immunosensor could detect the target AI H5N1 virus at a titer higher than 10³ EID₅₀/ml (EID₅₀: 50% Egg Infective Dose) within 2 h. The response of the antibody-antigen (virus) interaction was shown to be virus titer-dependent, and a linear range for the titer of H5N1 virus was found between 10³ and 10⁷ EID₅₀/ml. Equivalent circuit analysis indicated that the electron transfer resistance of the redox probe [Fe(CN)₆]^3−/4− and the double layer capacitance were responsible for the impedance change due to the protein A modification, antibody immobilization, BSA (bovine serum albumin) blocking, H5N1 viruses binding and RBCs amplification. No significant interference was observed from non-target RNA viruses such as Newcastle disease virus and Infectious Bronchitis disease virus. (The H5N1 used in the study was inactivated virus.)     

2-(Trimethylsilyl)ethyl glycosides. Synthesis of the asialo-GM1-tetrasaccharide, spacer glycoside, and BSA and Sepharose glycoconjugates.

The tri and tetra-saccharide glycosides beta-D-GalNAc-(1-4)-beta-D-Gal-(1-4)-beta-D-Glc-1-OTMSEt and beta-D-Gal-(1-3)-beta-D-GalNAc-(1-4)-beta-D-Gal-(1-4)-beta-D-Glc-1-OT MSEt (asialo-GM1) have been synthesized by sequential glycosylations of a suitably protected 2-(trimethylsilyl)ethyl (TMSEt) lactoside with 3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-beta-D-galactopyranosyl chloride and 2,3,4,6-tetra-O-acetyl-beta-D-galactopyranosyl bromide. The tetrasaccharide glycoside was transformed into the corresponding hemiacetal sugar as well as the 1-chloro sugar. The latter was used for glycosylation of 2-bromoethanol. The resulting glycoside was used to alkylate methyl 3-mercaptopropionate and the resulting spacer glycoside was used for the preparation of the title glycoconjugates.     

Structural assignment of disialylated biantennary N-glycan isomers derivatized with 2-aminopyridine using negative-ion multistage tandem mass spectral matching

To investigate the possibility of structural assignment based on negative-ion multistage tandem mass (MS(n)) spectral matching, four isomers of disialylated biantennary N-glycans (alpha2-6 and/or alpha2-3 linked sialic acid on alpha1-6 and alpha1-3 antennae) derivatized with 2-aminopyridine (PA) were analyzed by employing high-performance liquid chromatography/electrospray ionization linear ion trap time-of-flight mass spectrometry (HPLC/ESI-LIT-TOFMS), which uses helium gas for ion trapping and collision-induced dissociation (CID). It is shown that the MS(2) spectra derived from each precursor ion [M-2H](2-) are reproducible and useful for distinguishing the four isomers. Thus, they can be assigned by negative-ion MS(2) spectral matching based on correlation coefficients. In addition, MS(3) spectra derived from D-type fragment ions clearly differentiate the alpha2-3- or alpha2-6-linked sialic acid on the alpha1-6 antenna due to their characteristic spectral patterns. The C(4)-type fragment ions, which are produced from both the alpha1-6 and alpha1-3 antennae, show the characteristic MS(3) spectra reflecting alpha2-3- or alpha2-6- linkage type or a mixture of both types. Thus, the differentiation and assignment of these disialylated biantennary N-glycan isomers can also be supported with the MS(3) spectra of C(4)- and D-type ions.     

Structural assignment of isomeric 2-aminopyridine-derivatized monosialylated biantennary N-linked oligosaccharides using negative-ion multistage tandem mass spectral matching

To investigate the possibility of structural assignment based on negative-ion tandem multistage (MSn) mass spectral matching, four isomers of 2-aminopyridine (PA)-derivatized monosialylated oligosaccharides (i.e., complex-type N-glycans with an alpha2-3- or alpha2-6-linked sialic acid on alpha1-6 or alpha1-3 antennae) were analyzed using high-performance liquid chromatography/electrospray ion trap time-of-flight mass spectrometry (HPLC/ESI-IT-TOFMS). The negative ion [M-2H]2- is observed predominantly in the MS1 spectra without the loss of a sialic acid. The MS2 spectra derived from it are sufficiently reproducible that MS2 spectral matching based on correlation coefficients can be applied to the assignment of these isomers. The isomers containing a sialic acid on alpha1-6 or alpha1-3 antennae can be distinguished by MS2 spectral matching, but the alpha2-3 and alpha2-6 linkage types of sialic acid cannot be distinguished by their MS2 spectra. However, MS3 spectra derived from fragment ions containing a sialic acid (i.e., C4- and D-type ions) clearly differentiate the alpha2-3 and alpha2-6 linkage types of sialic acid in their MS3 spectral patterns. This difference might be rationalized in terms of a proton transfer from the reducing-end mannose to the negatively charged sialic acid. These two moieties are very close in the structural conformations of the precursor C4-type fragment ions of alpha2-6 linkage type, as predicted by molecular mechanics calculations. Thus, negative-ion MSn (n = 2, 3) spectral matching was demonstrated to be useful for the structural assignment of these four monosialylated PA N-glycan isomers.     

Synthesis and Characterization of the Crystalline Methyl α-Glycoside of the Repeating Unit of the O-Polysaccharide of Vibrio Cholerae O:1

Abstract

There are more than eighty serotypes of Vibrio cholerae, all causing disease with symptoms of Asian cholera. Systematic prevention of cholera by immunization has not yet been achieved because of a lack of a protective vaccine. Vibrio cholerae 0:1 Gramnegative bacteria occur as two immunologically distinct strains: Ogawa and Tnaba. The lipopolysaccharide (LPS) of both strains seem to contain the same 0-polysaccharide antigen consisting^3,4 of (1+2)-a-linked 4-amino-4,6-dideoxy-a-D-mannopyranosyl residues the amino groups of which are acylated with 3-deoxy-L-glycero-tetronic acid. Although the chemical structure of the 0-polysaccharides has been known5 since 1979, the synthesis of its monomeric repeating unit was reported only in 1988.     

Complete 1H and 13C NMR assignments of three new polyhydroxylated sterols from the South China Sea gorgonian Subergorgia suberosa

Three new polyhydroxylated sterols, 3beta,6alpha,11,20beta,24-pentahydroxy- 9,11-seco-5alpha-24-ethylcholest-7,28-diene-9-one (1), 3-(1',2'-ethandiol)-24- methylcholest-8(9),22E-diene-3beta,5alpha,6alpha,7alpha,11alpha-pentaol (2), 24-methylcholest-7,22 E-diene-3beta,5alpha,6beta,25-tetraol (3) together with five known sterols, were isolated from the EtOH/CH2Cl2 extract of the South China Sea gorgonian Subergorgia suberosa. The complete assignments of the 1H and 13C NMR chemical shifts for these new compounds were achieved by means of 1D and 2D NMR techniques, including HSQC, HMBC, 1H--1H COSY, and NOESY spectra.     

Porritoxins, metabolites of Alternaria porri, as anti-tumor-promoting active compounds

To search for possible cancer chemopreventive agents from natural sources, we performed primary screening of metabolites of Alternaria porri by examining their possible inhibitory effects on Epstein-Barr virus early antigen (EBV-EA) activation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) in Raji cells. The ethyl acetate extract of A. porri showed the inhibitory effect on EBV-EA activation. Three porritoxins (1-3) were obtained as inhibitory active compounds for EBV-EA from ethyl acetate extract. 6-(3',3'-Dimethylallyloxy)-4-methoxy-5-methylphthalide (2) showed the strongest activity among them. Inhibitory effect of porritoxin (1) and (2) was superior to that of beta-carotene, a well-known anti-tumor promoter. Furthermore, the structure-activity correlation of porritoxins and their related compounds were discussed.     

A new pyrrolidine derivative and steroids from an algicolous Gibberella zeae strain

A new pyrrolidine derivative, 3-hydroxy-5-(hydroxymethyl)-4-(4'-hydroxyphenoxy)pyrrolidin-2-one (1), and eight known steroids, (22E,24R)-7beta,8beta-epoxy-3beta,5alpha,9alpha-trihydroxyergosta-22-en-6-one (2, a reassigned structure of (22E,24R)-5alpha,6alpha-epoxy-3beta,8beta,14alpha-trihydroxyergosta-22-en-7-one), (22E,24R)-3beta,5alpha,9alpha-trihydroxyergosta-7,22-dien-6-one (3), (22E,24R)-3beta,5alpha-dihydroxyergosta-7,22-dien-6-one (4), (22E,24R)-ergosta-7,22-dien-3beta/,5alpha,6beta-triol (5), (22E,24R)-ergosta-5,22-dien-3beta-ol (6), (22E,24R)-5alpha,8alpha-epidioxyergosta-6,22-dien-3beta-ol (7), (22E,24R)-5alpha,8alpha-epidioxyergosta-6,9(11),22-trien-3beta-ol (8), and (22E,24R)-1(10 --> 6)-abeo-ergosta-5,7,9,22-tetraen-3alpha-ol (9), were isolated from the cultures of Gibberella zeae, an endophytic fungus isolated from the marine green alga Codium fragile. Their structures and relative stereochemistry were elucidated by 1D, 2D NMR and mass spectroscopic techniques. Compound 1 showed cytotoxicity against A-549 and BEL-7402 cell lines.     

Synthesis of a sialic acid alpha(2-3) galactose building block and its use in a linear synthesis of sialyl Lewis X

[reaction: see text] The ubiquity of the sialic acid alpha(2-3) galactose linkage in oligosaccharides of biological relevance necessitates a building block for the incorporation of this motif into oligosaccharides prepared by modular synthesis. The linear synthesis of the sialyl Lewis X tumor-associated antigen (1) has been accomplished in good yield using a sialic acid alpha(2-3) galactose disaccharide building block. The disaccharide building block was synthesized efficiently from readily available galactal by a high-yielding and selective sialylation reaction.     

A new geranylated aromatic compound from the mushroom Hericium erinaceum

A new geranylated aromatic compound, 5-[(2'E)-3',7'-dimethyl-2',6'-octadienyl]-4-hydroxy-6-methoxy-1-isoindolinone (1), was isolated from the fruiting bodies of the mushroom Hericium erinaceum (Bull.: Fr.) Pers. (Hericiaceae) together with three known sterols, 5alpha,6alpha-epoxy-(22E)-ergosta-8(14),22-diene-3beta,7alpha-diol (2), (22E)-ergosta-7,9(11),22-triene-3beta,5alpha,6beta-triol (3) and (22E)-ergosta-7,22-diene-3beta,5alpha,6alpha,9alpha-tetrol (4). The structure of the new compound was elucidated on the basis of spectral data.     

Four new 3,5-cyclosteroidal saponins from Dracaena surculosa

Further search for steroidal compounds contained in Dracaena surculosa (Agavaceae) led to the isolation of two new 3,5-cyclospirostanol saponins (1, 2) and two new 3,5-cyclofurostanol saponins (3, 4). Their structural assignment was established by spectroscopic analysis and a few chemical transformations as (24S,25R)-1beta-[(beta-D-fucopyranosyl)oxy]-6beta-hydroxy-3alpha,5alpha-cyclospirostan-24-yl beta-D-glucopyranoside (1), (24S,25R)-1beta-[(beta-D-glucopyranosyl)oxy]-6beta-hydroxy-3alpha,5alpha-cyclospirostan-24-yl beta-D-glucopyranoside (2), (25S)-1beta-[(beta-D-glucopyranosyl)oxy]-6beta-hydroxy-22alpha-methoxy-3alpha,5alpha-cyclofurostan-26-yl beta-D-glucopyranoside (3), and (25S)-1beta-[(beta-D-fucopyranosyl)oxy]-6beta-hydroxy-22alpha-methoxy-3alpha,5alpha-cyclofurostan-26-yl beta-D-glucopyranoside (4), respectively.     

Studies on the Thioglycosides of N-Acetylneuraminic Acid 7: Synthesis of S-(α-Sialyl)-(2↠6)-β-D-Hexopyranosyl and -(2↠6)-β-D-Lactosyl Ceramides and their Biological Activity

ABSTRACT

The coupling of the sodium salt of methyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3, 5-dideoxy-2-thio-D-glycero-α-D-galacto-2-nonulopyranosonate (17) with 2-(trimethylsilyl)ethyl 2,3,4-tri-O-acetyl-6-bromo-6-deoxy-β-D-galactopyranoside (5), glucopyranoside (10), and 2-(trimethylsilyl)ethyl 2,3,6,2′,3′,4′-hexa-O-acetyl-6′-bromo-6′-deoxy-β-D-lactoside (16), gave the corresponding α-thioglycosides 18, 21, and 24 of the 2-thio-N-acetyl-neuraminic acid derivative in good yields, which were converted, via selective removal of the 2-(trimethylsilyl)ethyl group, trichloroacetimidation, and coupling with (2S,3R,4E)-2-azido-3-O-benzoyl-4-octadecene-1,3-diol (27), into the ß-glycosides 28, 32, and 36, respectively.

Compounds 28, 32, and 36 were transformed, via selective reduction of the azide group, coupling with octadecanoic acid, O-deacetylation, and de-esterification, into the title compounds 31, 35, and 39, which showed potent inhibitory effect for sialidases from influenza and other viruses.     

Efficient synthesis of 2-modified 1alpha,25-dihydroxy-19-norvitamin D3 with Julia olefination: high potency in induction of differentiation on HL-60 cells

Six novel 2-substituted analogues of 1alpha,25-dihydroxy-19-norvitamin D(3), 6a,b-8a,b, were efficiently synthesized utilizing (-)-quinic acid as the A-ring precursor. The C2-modified A-rings were prepared as 4-alkylated (3R,5R)-3,5-dihydroxycyclohexanones 12-15 from (-)-quinic acid based on radical allylation at the C4 position of methyl (-)-quinicate. The new type of the CD-ring coupling partner 23 was synthesized from 25-hydroxy Grundmann's ketone 19 to apply to the modified Julia olefination to construct a diene unit between the A-ring and the CD-ring. The coupling yields, including a deprotection step, were 47-62%. After the separation of the diastereomers based on C2 stereochemistry, the structure (2alpha or 2beta) was determined by (1)H NMR experiments and compared to DeLuca's 2-methyl- and 2-ethyl-1alpha,25-dihydroxy-19-norvitamin D(3). Thus, the synthesized 2alpha-(3-hydroxypropyl)-1alpha,25-dihydroxy-19-norvitamin D(3) (8a) showed almost the same potency in binding to the bovine thymus vitamin D receptor (VDR) as the natural hormone 1, while its beta-isomer 8b had only a 3% affinity. Both 2alpha-allyl- and 2alpha-propyl-1alpha,25-dihydroxy-19-norvitamin D(3) (6a and 7a) and their 2beta-analogues (6b and 7b) possessed a weak affinity for the VDR. The strong VDR ligand 8a was ca. 36-fold more potent in induction of HL-60 cell differentiation than 1, and interestingly, even the weaker ligand 8b showed a 6.7-fold higher potency in the cell differentiation activity than that of 1.     

New triterpenoid saponins from Stelmatocrypton khasianum

Four new triterpenoid saponins, designated as stelmatotriterpenosides E-H (1-4), together with three known compounds, asterbatanoside B (5), 2alpha,3beta,19alpha,23-tetrahydroxy-olean-12-en-28-oic acid-3-O-beta-D-glucopyranosyl-28-O-beta-D-glucopyranosyl ester (6) and 2alpha,3beta,19alpha,23-tetrahydroxy-urs-12-en-28-oic acid-3-O-beta-D-glucopyranosyl-28-O-beta-D-glucopyranosyl ester (7), were isolated from the stems of Stelmatocrypton khasianum. On the basis of chemical and spectral evidence, the structures of 1-4 were established as 2alpha,3beta,23-trihydroxy-olean-12-en-28-oic acid-3-O-beta-D-glucopyranosyl-28-O-beta-D-glucopyranosyl-(1-->6)-beta-D-glucopyranosyl ester (1), 2alpha,3beta,23-trihydroxy-urs-12-en-28-oic acid-3-O-beta-D-glucopyranosyl-28-O-beta-D-glucopyranosyl-(1-->6)-beta-D-glucopyranosyl ester (2), 2alpha,3beta,19alpha-trihydroxy-urs-12-en-28-oic acid-3-O-beta-D-glucopyranosyl-28-O-beta-D-glucopyranosyl-(1-->2)-beta-D-glucopyranosyl ester (3), and 2beta,3beta,19alpha-trihydroxy-urs-12-en-24,28-dioic acid-24-O-beta-D-glucopyranosyl-28-O-beta-D-glucopyranosyl diester (4).     

Multivalency and the mode of action of bacterial sialidases

Although complex modular proteins are encountered frequently in a variety of biological systems, their occurrence in biocatalysis has not been widely appreciated. Here, we describe that bacterial sialidases, which have both a catalytic and carbohydrate-binding domain, can hydrolyze polyvalent substrates with much greater catalytic efficiency than their monovalent counterparts. The enhancement of catalytic efficiency was due to a much smaller Michaelis constant and rationalized by a model in which the catalytic and lectin domains interact simultaneously with the polyvalent substrate, leading to an enhancement of affinity. Inhibition studies have shown that galactosides released by the action of the sialidase can act as the ligand for the lectin domain. This knowledge has been exploited in the design of a potent polyvalent inhibitor of the sialidase of Vibrio cholerae, which displayed exquisite selectivities for sialidases that have a lectin domain.