Synthesis of sialyllactosamine clusters using carbosilane as core scaffolds by means of chemical and enzymatic approaches.

An efficient synthesis of sialyllactosamine (SiaLacNAc) clusters using carbosilanes as core scaffolds has been accomplished by means of chemical and enzymatic approaches. N-Acetyl-D-glucosamine (GlcNAc) clusters having O-glycosidic linkage or S-glycosidic linkage were chemically synthesized from known intermediates in high yields. The GlcNAc clusters were first used as substrates for beta1,4 galactosyl transferase using UDP-galactose (UDP-Gal) as a sugar source to provide corresponding N-acetyllactosamine clusters. Further sugar elongation of the LacNAc clusters was demonstrated using alpha2,3 sialyl transferase and CMP-neuraminic acid (CMP-NANA) to yield the corresponding SiaLacNAc clusters.

Systematic syntheses of influenza neuraminidase inhibitors: a series of carbosilane dendrimers uniformly functionalized with thioglycoside-type sialic acid moieties.

In order to develop novel influenza sialidase inhibitors, we constructed a library of glycoclusters composed of twelve types of sialylated dendrimers with thioglycosidic linkage that are resistant to hydrolysis by the sialidases. These sialodendrimers were synthesized by condensation reaction between a thiosialoside modified on the aglycon terminal end by a thioacetyl group and twelve types of carbosilane dendrimers having brominated terminal ends under deacetylation conditions, and temporal re-protection was performed for purification. Removal of all protection of the glycodendrimers was accomplished by transesterification and subsequent saponification to provide corresponding water-soluble glycodendrimers in good yields. For investigation of the structure-activity relationship, dendrimer scaffolds having differences in number of the sugar moieties, such as 3-, 4-, 6- and 12-functionalized dendrimers, and in linkage patterns, such as normal aliphatic linkage, ether- and amide-linkages. Biological evaluations of these glycodendrimers showed that all of the ether- and amide-elongated compounds had inhibitory potencies for the influenza sialidases in the mM range, while compounds having normal aliphatic linkage did not have any activities except for a 12-functionalized compound.

Syntheses and biological evaluations of carbosilane dendrimers uniformly functionalized with sialyl alpha(2-->3) lactose moieties as inhibitors for human influenza viruses.

A series of carbosilane dendrimers uniformly functionalized with sialyl lactose moieties (Neu5Ac alpha2-->3Gal beta1-->4Glc) was systematically synthesized, and biological evaluations for anti-influenza virus activity using the glycodendrimers were performed. The results suggested that the glycodendrimers had unique biological activities depending on the form of their core frame, and Dumbbell(1)6-amide type glycodendrimer 7 showed particularly strong inhibitory activities against human influenza viruses [A/PR/8/34 (H1N1) and A/Aichi/2/68 (H3N2)]. The results suggested that the structure-activity relationship (SAR) on the glycolibrary against various influenza viruses was observed, and dumbbell-shaped dendrimers as supporting carbohydrate moieties were found to be the most suitable core scaffolds in this study.

Synthesis and characterization of photo-responsive carbosilane dendrimers.

Preparation of photo-responsive carbosilane dendrimers bearing 4-phenylazo-benzonitrile units on their molecular surface has been accomplished, and their both photo and thermal behaviors have also been characterized. These functional dendrimers suggest that the apparent molecular sizes of the cis-isomers are smaller than those of the corresponding trans-isomers, since the molecular diameter of these dendrimers would be shorter on the basis of trans-->cis photo-isomerization of azobenzene.

Sialyl alpha(2-->3) lactose clusters using carbosilane dendrimer core scaffolds as influenza hemagglutinin blockers.

An efficient synthesis of a series of carbosilane dendrimers uniformly functionalized with sialyl alpha(2-->3) lactose (Neu5Acalpha(2-->3)Galbeta(1-->4)Glcbeta1-->) moieties was accomplished. The results of a preliminary study on biological responses against influenza virus hemagglutinin, using the sialyl lactose clusters showed unique biological activities on the basis of the structure-activity relationship according to the carbosilane scaffolds.

Characterization of short-chain poly3-hydroxybutyrate in baker's yeast.

A short-chain poly3-hydroxybutyrate including four comonomers, originating from a complex with calcium polyphosphate, was isolated from commercial baker's yeast cells (Saccharomyces cerevisiae) and characterized as the second complexed poly(3-hydroxyalkanoate) (cPHA) in eukaryotes. The number-average molecular weight of 4982.5 Da with a polydispersity index of 1.11 was much lower than that of beet cPHA previously isolated. End-group analysis suggested that at least 60% of the molecules form the cyclic structures. Here, the organism-dependent structural diversity of cPHAs was completely established. It was also found that a change of culture medium influences the molecular weight but not the polydispersity of baker's yeast cPHA.

Inhibitory activity of linoleic acid isolated from proso and Japanese millet toward histone deacetylase.

Linoleic acid was isolated from both the methanol extracts of proso and Japanese millet as a histone deacetylase inhibitor. It showed uncompetitive inhibitory activity toward histone deacetylase (IC(50)=0.51 mM) and potent cytotoxicity toward human leukemia K562 (IC(50)=68 microM) and prostate cancer LNCaP cells (IC(50)=193 microM). Millet containing linoleic acid might have anti-tumor activity.

Thiosialoside clusters using carbosilane dendrimer core scaffolds as a new class of influenza neuraminidase inhibitors.

An efficient synthesis of a series of carbosilane dendrimers uniformly functionalized with alpha-thioglycoside of sialic acid was accomplished. The results of a preliminary study on biological responses against influenza virus sialidases using thiosialoside clusters showed that some of the glycodendrimers have inhibitory potencies against the sialidases.

Vialinin B, a novel potent inhibitor of TNF-alpha production, isolated from an edible mushroom, Thelephora vialis.

A novel dibenzofuran compound designated vialinin B was isolated from dry fruiting bodies of an edible mushroom, Thelephora vialis, and potently inhibits TNF-alpha production in RBL-2H3 cells (IC(50)=0.02nM) and is a promising anti-allergic agent.

Carbosilane dendrimers bearing globotriaoses: syntheses of globotrioasyl derivative and introduction into carbosilane dendrimers.

As an application of a one-pot reaction involving Birch reduction and subsequent S(N)2 reaction in liquid ammonia, synthetic assembly of trisaccharidic moieties of globotriaosyl ceramide onto carbosilane dendrimers was accomplished using tris(3-bromopropyl)phenylsilane and tris[tris(3-bromopropyl)silylpropyl]phenylsilane as the core scaffolds. The common globotriaosyl derivative having benzylsulfide functionality at the terminal of the aglycon was efficiently prepared from d-galactose and d-lactose as starting materials. The glycosyl donor derived from galactose and the glycosyl acceptor derived from lactose were condensed in the presence of silver triflate as the best promoter to provide corresponding trisaccharide with newly formed alpha-1-4 linkages in 90% yield. Fully benzylated protection of the trisaccharide was deprotected under the Birch reduction condition followed by acetylation to give an acetate in which alkene was converted into benzyl sulfide by radical addition of alpha-toluenethiol in high yields. On the other hand, carbosilane dendrimers were prepared from appropriate chlorosilanes as starting materials by a combination of hydrosylation followed by alkenylation. The terminal C=C double bonds of the carbosilanes were converted into corresponding alcohols by means of the usual hydroboration reaction, and the alcohols underwent further chemical manipulation to give carbosilane dendrimers with peripheral bromine atoms.

Carbosilane dendrimers bearing globotriaoses: construction of a series of carbosilane dendrimers bearing globotriaoses.

To enhance biological activities on the basis of the sugar cluster effect, a series of carbosilane dendrimers as core scaffolds for the construction of glycodendrimers was systematically synthesized from appropriate chlorosilanes by a combination of alkenylation and hydrosylation reactions. Those carbosilane dendrimers having terminal C=C double bonds underwent general hydroboration reactions to give corresponding primary polyols. Further transformations of the alcohols were then performed by mesylation followed by a displacement with NaBr to provide corresponding dendrimers with 4 to 36 bromine atoms at each terminal end. Assembly of trisaccharide moieties of globotriaosyl ceramide using alkyl halide-type carbosilane dendrimers as the core frame was conducted in liquid ammonia by a one-pot reaction involving selective removal of a benzyl group under the Birch reduction condition and subsequent S(N)2 reaction to yield a series of carbosilane dendrimers having appropriate numbers of trisaccharide moieties. These dendrimers have unique shapes and adequate numbers of terminal trisaccharide moieties. Some of the dendrimers showed unique biological activity against Stxs, which were produced by pathogenic Escherichia coli O157:H7.

Syntheses of a series of lacto-N-neotetraose clusters using a carbosilane dendrimer scaffold.

4-Pentenyl (2,3,4,6-tetra-O-acetyl-beta-d-galactopyranosyl)-(1-->4)-(3,6-di-O-acetyl-2-deoxy-2-phthalimido-beta-d-glucopyranosyl)-(1-->3)-(2,6-di-O-benzoyl-beta-d-galactopyranosyl)-(1-->4)-2,3,6-tri-O-benzoyl-beta-d-glucopyranoside (4) was synthesized by regioselective glycosylation of 4-pentenyl (2,6,-di-O-benzoyl-beta-d-galactopyranosyl)-(1-->4)-2,3,6-tri-O-benzoyl-beta-d-glucopyranoside and (2,3,4,6-tetra-O-acetyl-beta-d-galactopyranosyl)-(1-->4)-3,6-di-O-acetyl-2-deoxy-2-phthalimido-beta-d-glucopyranosyl chloride. By conversion of the protecting groups followed by thioacetylation, 4 was transformed into the corresponding lacto-N-neotetraose derivative, 5-(acetylthio)pentenyl (2,3,4,6-tetra-O-acetyl-beta-d-galactopyranosyl)-(1-->4)-O-(3,6-di-O-acetyl-2-acetamido-2-deoxy-beta-d-glucopyranosyl)-(1-->3)-(2,4,6-di-O-acetyl-beta-d-galactopyranosyl)-(1-->4)-2,3,6-tri-O-acetyl-beta-d-glucopyranoside (6). The lacto-N-neotetraose derivative 6 was introduced into carbosilane dendrimer cores of three shapes, and three kinds of new carbosilane dendrimers peripherally functionalized by lacto-N-neotetraose were obtained.

Vialinin A, a novel 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenger from an edible mushroom in China.

While screening for bioactive compounds from edible mushrooms, a new potent antioxidant, vialinin A (1), together with a known compound, ganbajunin B (2), and a mixture of ganbajunins D (3) and E (4), were isolated from the dry fruiting bodies of Thelephora vialis. The structure of 1, 5',6'-bis(phenylacetoxy)-1,1':4',1''-terphenyl-2',3',4,4''-tetraol, was elucidated by spectroscopic and chemical methods. This compound had strong 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical-scavenging activity with an EC(50) value of 14.0 microM, nearly equal to that of butylated hydroxytoluene (BHT; EC(50) = 10.0 microM). A radical scavenging experiment using 1 and DPPH radicals indicated that 1 donated two hydrogen atoms to two molecules of the DPPH radical under hydrophobic conditions.

Isolation and structure determination of complexed poly(3-hydroxyalkanoate) from beet (Beta vulgaris L.).

Complexed poly(3-hydroxyalkanoate)s (cPHAs), one of two types of natural PHAs, occur in both prokaryotes and eukaryotes as a complex with biomacromolecules and could be involved in various physiological functions. In this study, a cPHA-component derived from a complex with calcium polyphosphate was isolated from sugar beet (Beta vulgaris L.) and determined to be a homopolymer composed of 3-hydroxybutyrate. MALDI MS provided the number-average molecular weight (Mn = 9,124 Da) and polydispersity index (PDI = 1.01), showing that beet cPHA has a slightly lower molecular mass than the known Escherichia coli cPHA. In addition, the structural analysis of both end groups showed that (i) 100 mol-% of the carboxyl end is free, while about 30 mol-% of the hydroxyl end is free and about 70 mol-% masked and (ii) the end hydroxyl group is masked by at least six identified short-chain alkanoic and alkanedioic acids. Based on such end-group characteristics, the polymerization mechanism of beet cPHA is discussed.

Further studies of lipid droplets in the bombykol-producing pheromone gland of Bombyx mori.

Lipid droplets are abundant in the pheromone-producing cells of B. mori at adult eclosion, followed by daily fluctuations in both their size and number. Their dynamics are related to PBAN-stimulated de novo bombykol production. To elucidate associated events, we performed the following: (1) extraction, purification, and partial characterization of lipid droplet-associated proteins found on their surface since their function could possibly be to transport and/or dock putative lipases that are responsible for the lipolysis of triglycerides in them; (2) separation, purification, and initial analysis of lipids carried by lipophorins and lipid transfer particles originating from pupal and adult hemolymph because of their role in the formation and accumulation of lipid droplets.

Isolation and characterization of a humoral factor that stimulates transcription of the acyl-CoA-binding protein in the pheromone gland of the silkmoth, Bombyx mori.

Acyl-CoA binding protein (ACBP) is a highly conserved 10-kDa intracellular lipid-binding protein that binds straight-chain (C14-C22) acyl-CoA esters with high affinity and is expressed in a wide variety of species ranging from yeast to mammals. Functionally, ACBP can act as an acyl-CoA carrier or as an acyl-CoA pool maker within the cell. Much work on the biochemical properties regarding the ACBP has been performed using various vertebrate and plant tissues, as well as different types of cells in culture, the regulatory mechanisms underlying ACBP gene expression have remained poorly understood. By exploiting the unique sex pheromone production system in the moth pheromone gland (PG), we report that transcription of a specific ACBP termed pheromone gland ACBP is triggered by a hemolymph-based humoral factor. Following purification and structure elucidation by means of high resolution electrospray ionization mass spectrometry and NMR analyses, in conjunction with stereochemical analyses using acid hydrolysates, the humoral factor was identified to be beta-D-glucosyl-O-L-tyrosine. Examination of the hemolymph titers during development revealed that the amount of beta-D-glucosyl-O-L-tyrosine dramatically rose prior to eclosion and reached a maximum of 5 mg/ml (about 1 mg/pupa) on the day preceding eclosion, which was consistent with the effective dose of beta-D-glucosyl-O-L-tyrosine in stimulating pheromone gland ACBP transcription in vivo. Furthermore, in vitro assays using trimmed PG indicated that beta-D-glucosyl-O-L-tyrosine acts directly on the PG. These results provide the first evidence that transcription of some ACBPs can be triggered by specific humoral factors.

5-n-Alkylresorcinols from the nitrogen-fixing soil bacterium Azotobacter chroococcum Az12.

A mixture of five saturated 5-n-alkylresorcinol homologues was isolated from vegetative cells of the nitrogen-fixing soil bacterium Azotobacter chroococcum Az12. Their structures were established by spectrometry (1H NMR, EI-MS, FAB-MS, FAB-MS/MS) and chromatography (GC, TLC) means.

Biosynthesis of 5-alkylresorcinol in rice: incorporation of a putative fatty acid unit in the 5-alkylresorcinol carbon chain.

A previously unspecified "starter" unit in the predicted biosynthesis pathway of 5-alkylresorcinols has now identified as a fatty acid or its equivalent, using an efficient 5-alkylresorcinol production system of etiolated rice seedlings. Feeding saturated, odd-carbon fatty acid ester substrates from C11 to C19 specifically and markedly increased the amount of the corresponding 5-alkylresorcinol homologs with even-carbon chains that are shorter by one carbon than those of the supplied fatty acids. The amount of these homologs depended on substrate concentration. Some of the homologs whose amounts increased had linear carbon chains and the dodecyl homolog was shown to be 5-n-dodecylresorcinol. Moreover, the 13C label in the dodecyl homolog that was biosynthesized from the [1-13C]tridecanoate substrate was localized on the C-5 carbon of the resorcinol ring. These results obviously show that the fatty acid unit acts as a direct precursor and forms the side-chain moiety of 5-n-alkylresorcinol via the predicted biosynthesis pathway.

SNA-60-367 components, new peptide enzyme inhibitors of aromatase: structure of the fatty acid side chain and amino acid sequence by mass spectrometry.

SNA-60-367 components, new peptide enzyme inhibitors of aromatase, were isolated from the culture broth of soil bacterium, Bacillus sp. SNA-60-367. These inhibitors are a family of acylated decapeptides that differ from each other in terms of amino acid composition and the nature of the fatty acid side chain. The structures of the fatty acid moieties were shown to be (3-hydroxy)heptadecanoic acid and (3-hydroxy)hexadecanoic acid that possess normal-, iso- or anteiso-type alkyl groups. The amino acid sequence of the open form of the lactone ring of the acylpeptides is RCO-L-Glu-D-Orn-L(or D)-Tyr3-D-allo-Thr-L-Glu-D-X1 (Ala, Aba or Val)-L-Pro-L-Gln-D(or L)-Tyr-L-X2(10)(Ile or Val)-OH. The lactone ring of SNA-60-367 components is formed between Tyr3 and X2(10).

Control of liposidomycin production through precursor-directed biosynthesis.

Liposidomycin, a potent inhibitor of bacterial peptidoglycan biosynthesis, represents the main component of a group of over 24 closely related, fatty acyl nucleoside antibiotics produced by Streptomyces. Exogeneously supplied myristic acid or palmitic acid resulted in the almost exclusive production of liposidomycin C-(III) and/or M-(III). Exogeneously supplied [1-13C]-palmitic acid was incorporated directly into liposidomycin M-(III) as judged by the FAB-MS and 13C NMR spectra.