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1.
Bioorg Med Chem ; 27(12): 2345-2367, 2019 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-30606671

RESUMO

Novel carbohydrate mimics were designed which contain two 5a-carba-d-glucose residues, one each at reducing and nonreducing end, and thus these mimics are 5a,5a'-dicarba-d-glucobioses. Dicarbadisaccharides have attractive features such as stability against endogenous degradative enzymes and being resistant to glycation reactions such as the Maillard reaction. For the synthesis of dicarba-ß-d-isomaltose derivatives, the carbaglucosyl triflate locked in 4C1 conformation was synthesized by protecting with butane-2,3-diacetal group or benzylidene group. Then, 5a,5a'-dicarba-ß-d-maltose and 5a,5a'-dicarba-α,ß-d-trehalose were synthesized by the SN2-type inversion reaction using 4,6-O-benzylidene carbaglucosyl triflate with 4-OH and 1-OH carba-ß-d-glucose derivatives, respectively, and similarly 5a,5a'-dicarba-α-d-isomaltose with 6-OH carba-α-d-glucose derivative.


Assuntos
Cicloexanóis/síntese química , Dissacarídeos/síntese química , Mesilatos/química , Conformação Molecular
2.
Biosci Biotechnol Biochem ; 80(6): 1179-85, 2016 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-26856546

RESUMO

The antioxidant potencies of chondroitin sulfates (CSs) from shark cartilage, salmon cartilage, bovine trachea, and porcine intestinal mucosa were compared by three representative methods for the measurement of the antioxidant activity; DPPH radical scavenging activity, superoxide radical scavenging activity, and hydroxyl radical scavenging activity. CSs from salmon cartilage and bovine trachea showed higher potency in comparison with CSs from shark cartilage and porcine intestinal mucosa. Next, CS from salmon cartilage chelating with Ca(2+), Mg(2+), Mn(2+), or Zn(2+) were prepared, and their antioxidant potencies were compared. CS chelating with Ca(2+) or Mg(2+) ions showed rather decreased DPPH radical scavenging activity in comparison with CS of H(+) form. In contrast, CS chelating with Ca(2+) or Mg(2+) ion showed remarkably enhanced superoxide radical scavenging activity than CS of H(+) or Na(+) form. Moreover, CS chelating with divalent metal ions, Ca(2+), Mg(2+), Mn(2+), or Zn(2+), showed noticeably higher hydroxyl radical scavenging activity than CS of H(+) or Na(+) form. The present results revealed that the scavenging activities of, at least, superoxide radical and hydroxyl radical were enhanced by the chelation with divalent metal ions.


Assuntos
Antioxidantes/química , Compostos de Bifenilo/antagonistas & inibidores , Quelantes/química , Sulfatos de Condroitina/química , Radical Hidroxila/antagonistas & inibidores , Picratos/antagonistas & inibidores , Superóxidos/antagonistas & inibidores , Animais , Antioxidantes/isolamento & purificação , Compostos de Bifenilo/química , Cálcio/química , Cartilagem/química , Cátions Bivalentes , Bovinos , Quelantes/isolamento & purificação , Sulfatos de Condroitina/isolamento & purificação , Radical Hidroxila/química , Mucosa Intestinal/química , Magnésio/química , Manganês/química , Picratos/química , Salmonidae , Tubarões , Superóxidos/química , Suínos , Traqueia/química , Zinco/química
3.
Biosci Biotechnol Biochem ; 80(1): 128-34, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26613404

RESUMO

Manα1 → 2Man, Manα1 → 3Man, Manα1 → 4Man, and Manα1 → 6Man were converted to the glycosylamine derivatives. Then, they were mixed with monobenzyl succinic acid to obtain their amide derivatives. After removing the benzyl group by hydrogenation, the succinylamide derivatives were coupled with the hydrazino groups on BlotGlyco™ beads in the presence of water-soluble carbodiimide. d-Mannobiose-linked beads were incubated with fluorescence-labeled Escherichia coli with type 1 fimbria, and the number of the fluorescent dots associated with the beads was counted in order to determine the binding preference among d-mannobiose isomers. The results showed that the bacteria bind strongly to Manα1 → 2Man1 → beads, Manα1 → 3Man1 → beads, Manα1 → 4Man1 → beads, and Manα1 → 6Man1 → beads, in order. In the presence of 0.1 M methyl α-d-mannopyranoside, most of the bacteria failed to bind to these beads. These results indicate that E. coli with type 1 fimbria binds to all types of d-mannobiose isomers but preferentially to Manα1 → 2Man disaccharide.


Assuntos
Aderência Bacteriana/fisiologia , Escherichia coli/metabolismo , Fímbrias Bacterianas/metabolismo , Mananas/química , Manose/química , Carbodi-Imidas/química , Configuração de Carboidratos , Escherichia coli/química , Fímbrias Bacterianas/química , Corantes Fluorescentes/química , Hidrogenação , Mananas/metabolismo , Manose/metabolismo , Microesferas , Estereoisomerismo , Succinatos/química
4.
J Appl Glycosci (1999) ; 63(2): 31-37, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-34354480

RESUMO

Five kinds of fucoidans from the brown seaweeds Cladosiphon okamuranus, Sargassum hornery, Kjellmaniella crassifolia (Saccharine sculpera), Nemacystus decipiens, and Fucus vesiculosus, were isolated according to a previously reported procedure with slight modification. The scavenging activities of DPPH radical, superoxide radical, and hydroxyl radical, as well as the ORAC value were measured for the isolated fucoidans. Fucoidans from S. hornery, F. vesiculosus, and K. crassifolia showed higher antioxidant activity than that from S. hornery and C. okamuranus, except for the hydroxyl radical scavenging activity. The relationship between the antioxidant activity and the structure was examined for each fucoidan. Fucoidans with high amount of sulfate groups did not necessarily result in increased antioxidant activity, although the sulfate group itself was essential for the antioxidant activity. Furthermore, the fucoidan linked to a side chain monosaccharide, such as GlcA, demonstrated similar antioxidant activity. The antioxidant activity of the fucoidans was possibly due to a combination of the factors involved, such as the amount of sulfate groups, the position of the sulfate groups, the kind of side chain sugar, the linkage of a side chain sugar, and the molecular weight.

5.
Carbohydr Res ; 347(1): 147-50, 2012 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-22115720

RESUMO

Instead of an enzyme-assisted reverse hydrolysis reaction for the synthesis of manno-oligosaccharides, we propose here a versatile new approach. By Fischer type glycosylation, a D-mannose solution of extremely high concentration (approximately 83% (w/w)) was incubated at 60°C for 65 h in 0.5 M HCl. After dilution and neutralization, the small amount of formed ß-linked oligosaccharides was hydrolyzed by ß-mannosidase. The yields of α-D-Manp-(1→2)-D-Manp (7.9%), α-D-Manp-(1→3)-D-Manp (7.9%), and α-D-Manp-(1→6)-D-Manp (29.1%) isolated by an activated carbon column chromatography were almost identical to those of the enzymatic reaction, but the yield of α-D-Manp-(1→3)-D-Manp increased enormously by the present method.


Assuntos
Mananas/química , Mananas/síntese química , Ácidos , Sequência de Carboidratos , Catálise , Hidrólise , Dados de Sequência Molecular
6.
Carbohydr Res ; 345(17): 2485-90, 2010 Nov 22.
Artigo em Inglês | MEDLINE | ID: mdl-20947069

RESUMO

We confirmed that a recombinant α-(2→3)-sialyltransferase cloned from Photobacterium sp. JT-ISH-224 recognizes inositols having a structure corresponding to the C-3 and C-4 of a galactopyranoside moiety, such as epi-, 1d-chiro, myo-, and muco-inositol, as acceptor substrates, and that the enzyme can transfer N-acetylneuraminic acid (Neu5Ac) from cytidine 5'-monophospho-N-acetylneuraminic acid (CMP-Neu5Ac) to them. After purifying the reaction products, the structures were confirmed by use of NMR spectroscopy and mass spectrometry. From these results, it was clearly shown that the α-(2→3)-sialyltransferase from Photobacterium sp. JT-ISH-224 recognizes acceptor substrates through the cis-diol structure corresponding to the 3- and 4-position of the galactopyranoside moiety.


Assuntos
Inositol/metabolismo , Ácido N-Acetilneuramínico/metabolismo , Photobacterium/enzimologia , Proteínas Recombinantes/metabolismo , Sialiltransferases/metabolismo , Inositol/química , Ácido N-Acetilneuramínico/química , Estereoisomerismo , Especificidade por Substrato , beta-Galactosídeo alfa-2,3-Sialiltransferase
7.
Methods Enzymol ; 480: 511-24, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-20816225

RESUMO

Helicobacter pylori is known to bind with sugar chains possessing Lewis b structure. We are trying to combine oligosaccharides containing Lewis b sugar chain to water insoluble polysaccharide through some linker. Lacto-N-difucohexaose I (LNDFH I; Fucalpha1-->2Galbeta1-->3[Fucalpha1-->4]GlcNAcbeta1-->3Galbeta1-->4Glc) fits for that purpose, since it consists of Lewis b tetrasaccharide and lactose whose d-glucose residue can be utilized as a linker. We thus developed a method to synthesize this hexaose enzymatically. First, beta-1,3-N-acetylglucosaminyltransferase (beta-1,3-GnT) was partially purified from bovine blood by an established method. Using this enzyme preparation, d-GlcNAc was attached to the d-galactose residue of lactose with a beta-1,3-linkage to produce lacto-N-triose II at 44% yield. The low yield was thought to be due to contaminating N-acetylglucosaminidase that would have hydrolyzed the product, lacto-N-triose II. Next, d-galactose was attached by transglycosylation using ortho-nitrophenyl beta-d-galactopyranoside as a donor with the aid of recombinant beta-1,3-galactosidase from Bacillus circulans to generate lacto-N-tetraose (LNT) at 22% yield. l-Fucose was then linked to the d-galactose residue of LNT via an alpha-1,2-linkage using recombinant human fucosyltransferase I (FUT1) expressed in a baculovirus system (71% yield). The obtained pentasaccharide was subsequently incubated with GDP-beta-l-fucose and commercial fucosyltransferase III (FUT3) to attach l-fucose to the d-GlcNAc residue of LNT with an alpha-1,4-linkage. After purification with an activated carbon column chromatography, 1.7 mg of LNDFH I was obtained (85% yield). We thus produced LNDFH I over four enzymatic steps with a yield of 6%.


Assuntos
Sítios de Ligação Microbiológicos , Helicobacter pylori/metabolismo , Oligossacarídeos/biossíntese , Oligossacarídeos/metabolismo , Animais , Sítios de Ligação , Sequência de Carboidratos , Galactosidases/genética , Galactosidases/metabolismo , Helicobacter pylori/fisiologia , Humanos , Modelos Biológicos , Dados de Sequência Molecular , N-Acetilglucosaminiltransferases/genética , N-Acetilglucosaminiltransferases/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
8.
Carbohydr Res ; 345(10): 1417-21, 2010 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-20413108

RESUMO

We investigated the acceptor substrate specificities of marine bacterial alpha-(2-->3)-sialyltransferase cloned from Photobacterium sp. JT-ISH-224 and alpha-(2-->6)-sialyltransferase cloned from Photobacterium damselae JT0160 using several saccharides as acceptor substrates. After purifying the enzymatic reaction products, we confirmed their structure by NMR spectroscopy. The alpha-(2-->3)-sialyltransferase transferred N-acetylneuraminic acid (Neu5Ac) from cytidine 5'-monophospho-N-acetylneuraminic acid (CMP-Neu5Ac) to the beta-anomeric hydroxyl groups of mannose (Man) and alpha-Manp-(1-->6)-Manp, and alpha-(2-->6)-sialyltransferase transferred N-acetylneuraminic acid to the 6-OH groups of the non-reducing end galactose residues in beta-Galp-(1-->3)-GlcpNAc and beta-Galp-(1-->6)-GlcpNAc.


Assuntos
Oligossacarídeos/síntese química , Photobacterium/enzimologia , Sialiltransferases/metabolismo , Sequência de Carboidratos , Dados de Sequência Molecular , Oligossacarídeos/química , beta-D-Galactosídeo alfa 2-6-Sialiltransferase , beta-Galactosídeo alfa-2,3-Sialiltransferase
9.
J Agric Food Chem ; 57(8): 3102-7, 2009 Apr 22.
Artigo em Inglês | MEDLINE | ID: mdl-19309147

RESUMO

A detailed analysis of the antioxidative activity of 12 carbohydrates including chondroitin sulfate, fucoidan, agaro-oligosaccharide, 2-deoxy-scyllo-inosose (DOI), Galbeta1-4DOI, D-glucuronic acid, chitobiose, D-mannosamine, D-galactosamine, D-glucosamine, heparin, and colominic acid was performed using four established methods: 2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay, ferric reducing antioxidant power (FRAP) assay, superoxide dismutase (SOD) activity assay, and the deoxyribose method. Ascorbic acid and/or catechin were used as positive standards. In the DPPH radical scavenging activity measurements, fucoidan, DOI, and Galbeta1-4DOI showed remarkable levels of activity, although at lower levels than ascorbic acid. The SOD assay revealed that DOI, Galbeta1-4DOI, and agaro-oligosaccharide had high antioxidant activity, with DOI and Galbeta1-4DOI notably showing almost half of the antioxidative potency of ascorbic acid. Using the deoxyribose method, chitobiose and heparin showed the highest hydroxyl radical scavenging activity, followed by chondroitin sulfate, colominic acid, Galbeta1-4DOI, and d-glucosamine. Given that 11 of the carbohydrates analyzed share a common structure, agaro-oligosaccharide being the exception, we propose from our current results that at least one amino, carboxyl, carbonyl, or sulfonyl group is required, but is not in itself sufficient, for carbohydrates to function as antioxidants.


Assuntos
Antioxidantes/farmacologia , Carboidratos/farmacologia , Polissacarídeos/farmacologia , Compostos de Bifenilo , Carboidratos/química , Desoxirribose , Compostos Férricos/química , Sequestradores de Radicais Livres/farmacologia , Picratos , Polissacarídeos/química , Relação Estrutura-Atividade , Superóxido Dismutase
10.
Biosci Biotechnol Biochem ; 68(10): 2086-90, 2004 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-15502353

RESUMO

The regioselectivity of beta-galactosidase derived from Bacillus circulans ATCC 31382 (beta-1,3-galactosidase) in transgalactosylation reactions using D-mannose as an acceptor was investigated. This D-mannose associated regioselectivity was found to be different from reactions using either GlcNAc or GalNAc as acceptors, not only for beta-1,3-galactosidase but also for beta-galactosidases of different origins. The relative hydrolysis rate of Gal beta-pNP and D-galactosyl-D-mannoses, of various linkages, was also measured in the presence of beta-1,3-galactosidase and was found to correlate well with the ratio of disaccharides formed by transglycosylation. The unexpected regioselectivity using D-mannose can therefore be explained by an anomalous specificity in the hydrolysis reaction. By utilizing the identified characteristics of both regioselectivity and hydrolysis specificity using D-mannose, an efficient method for enzymatic synthesis of beta-1,3-, beta-1,4- and beta-1,6-linked D-galactosyl-D-mannose was subsequently established.


Assuntos
Bacillus/química , Dissacarídeos/química , Dissacarídeos/síntese química , Manose/síntese química , beta-Galactosidase/química
11.
Biosci Biotechnol Biochem ; 67(3): 646-50, 2003 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-12723619

RESUMO

We isolated a beta-N-acetylglucosaminidase encoding gene from the filamentous fungus Aspergillus oryzae, and designated it nagA. The nagA gene encoded a polypeptide of 600 amino acids with significant similarity to glucosaminidases and hexosaminidases of various eukaryotes. A. oryzae strain carrying the nagA gene under the control of the improved glaA promoter produced large amounts of beta-N-acetylglucosaminidase in a wheat bran solid culture. The beta-N-acetylglucosaminidase was purified from crude extracts of the solid culture by column chromatographies on Q-Sepharose and Sephacryl S-200. This enzyme was used for synthesis of lacto-N-triose II, which is contained in human milk. By reverse hydrolysis reaction, lacto-N-triose II and its positional isomer were synthesized from lactose and D-N-acetylglucosamine in 0.21% and 0.15% yield, respectively.


Assuntos
Acetilglucosaminidase/genética , Acetilglucosaminidase/metabolismo , Aspergillus oryzae/enzimologia , Aspergillus oryzae/genética , Leite Humano/metabolismo , Trissacarídeos/biossíntese , Acetilglucosamina/metabolismo , Acetilglucosaminidase/biossíntese , Acetilglucosaminidase/isolamento & purificação , Sequência de Aminoácidos , Sequência de Carboidratos , Catálise , Clonagem Molecular , Genes Fúngicos/genética , Humanos , Hidrólise , Isomerismo , Lactose/metabolismo , Dados de Sequência Molecular , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Trissacarídeos/química
12.
Biosci Biotechnol Biochem ; 66(10): 2168-75, 2002 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-12450128

RESUMO

We isolated a beta-N-acetylglucosaminidase encoding gene and its cDNA from the filamentous fungus Aspergillus nidulans, and designated it nagA. The nagA gene contained no intron and encoded a polypeptide of 603 amino acids with a putative 19-amino acid signal sequence. The deduced amino acid sequence was very similar to the sequence of Candida albicans Hex1 and Trichoderma harzianum Nag1. Yeast cells containing the nagA cDNA under the control of the GAL1 promoter expressed beta-N-acetylglucosaminidase activity. The chromosomal nagA gene of A. nidulans was disrupted by replacement with the argB marker gene. The disruptant strains expressed low levels of beta-N-acetylglucosaminidase activity and showed poor growth on a medium containing chitobiose as a carbon source. Aspergillus oryzae strain carrying the nagA gene under the control of the improved glaA promoter produced large amounts of beta-N-acetylglucosaminidase in a wheat bran solid culture.


Assuntos
Acetilglucosaminidase/genética , Aspergillus nidulans/enzimologia , Aspergillus nidulans/genética , Aspergillus oryzae/enzimologia , Aspergillus oryzae/genética , Regulação Enzimológica da Expressão Gênica/genética , Regulação Fúngica da Expressão Gênica/genética , Genes Fúngicos/genética , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Meios de Cultura , Fibras na Dieta , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Escherichia coli/metabolismo , Dados de Sequência Molecular , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo
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