7216-73-1

The cepA putative gene encoding a cellobiose phosphorylase of Thermotoga maritima MSB8 was cloned, expressed in Escherichia coli BL21-codonplus-RIL and characterized in detail. The maximal enzyme activity was observed at pH 6.2 and 80°C. The energy of activation was 74 kJ/mol. The enzyme was stable for 30 min at 70°C in the pH range of 6-8. The enzyme phosphorolyzed cellobiose in an random-ordered bi bi mechanism with the random binding of cellobiose and phosphate followed by the ordered release of D-glucose and α-D-glucose-1- phosphate. The Km for cellobiose and phosphate were 0.29 and 0.15 mM respectively, and the kcat was 5.4 s-1. In the synthetic reaction, D-glucose, D-mannose, 2-deoxy-D-glucose, D-glucosamine, D-xylose, and 6-deoxy-D-glucose were found to act as glucosyl acceptors. Methyl-β-D- glucoside also acted as a substrate for the enzyme and is reported here for the first time as a substrate for cellobiose phosphorylases. D-Xylose had the highest (40 s-1) kcat followed by 6-deoxy-D-glucose (17 s-1) and 2-deoxy-D-glucose (16 s-1). The natural substrate, D-glucose with the kcat of 8.0 s-1 had the highest (1.1 × 104 M-1 s-1) k cat/Km compared with other glucosyl acceptors. D-Glucose, a substrate of cellobiose phosphorylase, acted as a competitive inhibitor of the other substrate, α-D-glucose-1-phosphate, at higher concentrations.

-

The present invention relates to compounds with high chemical stability and methods for inhibiting the pathological activity of NETs in a subject. In particular, the invention relates to compounds with high chemical stability, uses thereof and methods for inhibiting or ameliorating NET mediated ailments (such as, for example, sepsis, systemic immune response syndrome (SIRS) and ischemia reperfusion injury (IRI)). More particularly, the invention relates to methods and uses of a polyanionic sulfated cellobioside modified with a small uncharged glycosidically linked substituent at its reducing terminus, wherein the presence of the substituent results in a molecule with high chemical stability without affecting the ability of the molecule to be effective in the therapy of NET mediated ailments. For example, the present invention relates to methods and uses of β-O-methyl cellobioside sulfate (mCBS) or a pharmaceutically acceptable salt thereof (e.g., mCBS.Na), in the therapy of a range of NET mediated ailments in subjects.

There are provided, inter alia, methods for decreasing mucus elasticity or decreasing mucus viscosity in a subject in need thereof, the methods including administering to the subject an effective amount of a dithiolsaccharide mucolytic agent, and compounds and pharmaceutical compositions useful for the methods.

The present invention relates to compounds with high chemical stability and methods for inhibiting the pathological activity of extracellular histones in a subject. In particular, the invention relates to compounds with high chemical stability, uses thereof and methods for inhibiting or ameliorating extracellular histone mediated ailments (such as, for example, sepsis, systemic immune response syndrome (SIRS) and ischemia reperfusion injury (IRI)). More particularly, the invention relates to methods and uses of a polyanionic sulfated cellobioside modified with a small uncharged glycosidically linked substituent at its reducing terminus, wherein the presence of the substituent results in a molecule with high chemical stability without affecting the ability of the molecule to be effective in the therapy of extracellular histone mediated ailments. For example, the present invention relates to methods and uses of β-O-methyl cellobioside sulfate (mCBS) or a pharmaceutically acceptable salt thereof (e.g., mCBS.Na), in the therapy of a range of extracellular histone mediated ailments in subjects.

4,6-Dimethoxy-1,3,5-triazin-2-yl glycosides, glycosyl donors prepared in one step from free saccharides without protection of the hydroxy groups, were stereoselectively and equivalently converted to the corresponding 1,2-cis-glycosides by using a catalytic amount of metal catalyst. This reaction was successfully applied not only to monosaccharides, but also to di- and oligosaccharides.

A completely β-selective glycosylation that does not rely on neighboring group participation is described. The novelty of this work is the design of the glycosyl donor locked into the axial-rich form by the o-xylylene bridge between the 3-O and 6-O of d-glucopyranose. The synthesized 2,4-di-O-benzyl-3,6-O-(o-xylyene)glucopyranosyl fluoride could efficiently react with various alcohols in a SnCl2-AgB(C6F 5)4 catalytic system. The mechanism composed of the glycosylation and isomerization cycles was revealed through comparative experiments using acidic and basic molecular sieves. The achieved perfect stereocontrol is attributed to the synergy of the axial-rich conformation and convergent isomerization caused by HB(C6F5)4 generated in situ.

The synthesis of C7- to C16-alkyl maltosides in the presence of tin(IV) chloride as Lewis acid catalyst was performed. The characterization of the products and theoretical investigation of the crucial step in the synthesis were carried out. The preparation of the β-maltosides required reaction time of 1 h, and that of the α-maltosides was 72 h. The side products were the α-D-maltosidechloride and 2-hydroxy-β-maltoside, respectively. The PM3 calculation confirmed the formation of the kinetically controlled β-product.

The conversion of phenylboronic acid (PBA) with methyl-β-d-cellobioside (Me-β-d-clb) and cellodextrins (DPw 12) was investigated to gain a basic understanding of the interactions of boric acid derivatives with oligo- and polyglucans. By means of MS and NMR experiments, it was possible to show a first stage formation of a six-membered ring at C-4 and C-6 of the non-reducing glucose occurs as in the case of monosaccharides. If the amount of reagent is increased the formation of seven-membered rings at the secondary OH moieties is observed. Even the existence of two of these large ring-systems in the direct neighborhood was found. Application of an excess of boronation reagent led to dimerization reactions of Me-β-d-clb via the primary reducing glucose residue as confirmed by DOSY NMR studies. Preliminary 13C NMR studies for the interaction of cellodextrins with PBA in DMSO solution confirmed a functionalization at the trans-1,2-diol moieties of these oligomers. The amount of reagent applied may either was shown to lead to soluble products or to insoluble cross-linked material.

The present invention relates to the use of an alkyl glycoside or of a mixture of at least two alkyl glycosides as agent intended for inhibiting microbial growth, in particular in a cosmetic, pharmaceutical or food composition.

Equation presented The 2-O[3-(2′-benzyloxyphenyl)-3,3- dimethylpropanoate] and 2-O-[3-(2′-benzyloxy-4′,6′- dimethylphenyl)-3,3-dimethylpropanoate] esters enable the synthesis of a range of β-glucosides and α-mannosides through neighboring participation in excellent yield, and are removed by hydrogenolysis in concert with the cleavage of benzyl esters in the presence of other esters making them particularly well suited to the stereocontrolled synthesis of glycosyl esters.

Bacillus macerans cyclomaltodextrin glucanyltransferase (CGTase, EC 2.4.1.19), in reaction with cyclomaltohexaose and methyl α-d- glucopyranoside, methyl β-d-glucopyranoside, phenyl α-d- glucopyranoside, and phenyl β-d-glucopyranoside gave four kinds of maltodextrin glycosides. The reactions were optimized by using different ratios of the individual d-glucopyranosides to cyclomaltohexaose, from 0.5 to 5.0, to obtain the maximum molar percent yields of products, which were from 68.3% to 78.6%, depending on the particular d-glucopyranoside, and also to obtain different maltodextrin chain lengths. The lower ratios of 0.5-1.0 gave a wide range of sizes from d.p. 2-17 and higher. As the molar ratio was increased from 1.0 to 3.0, the larger sizes, d.p. 9-17, decreased, and the small and intermediate sizes, d.p. 2-8, increased; as the molar ratios were increased further from 3.0 to 5.0, the large sizes completely disappeared, the intermediate sizes, d.p. 4-8, decreased, and the small sizes, d.p. 2 and 3 became predominant. A comparison is made with the synthesis of maltodextrins by the reaction of CGTase with different molar ratios of d-glucose to cyclomaltohexaose.

Alkyl, cycloalkyl, allyl, 4-pentenyl, and benzyl α-glycosides of maltose, cellobiose, and lactose were prepared via direct reaction of the free bioses with a binary AcBr-AcOH system, followed by glycosidation with alcohol using FeCl3 in MeNO2 or CH2Cl2, Zemple?n deacetylation, and the chromatographic resolution of the mixture. The respective β-biosides were obtained via the glycosidation in MeCN. Alkyl, cycloalkyl, allyl, 4-pentenyl, and benzyl α-glycosides of maltose, cellobiose, and lactose were prepared (17-77% yield; α/β = 70/30-96/4) via a direct reaction of the free disaccharides with a binary AcBr-AcOH mixture, followed by glycosidation with alcohol using FeCl3 in MeNO2 or CH2Cl2, Zemple?n deacetylation, and resolution of the anomeric mixture of glycosides by chromatography. Using MeCN as solvent for the glycosidation step, the corresponding β-biosides were also prepared (16-61% yield; α/β = 25/75-5/95).

The m-xylylene moiety was employed as rigid spacer in intramolecular glycoside bond formation. Fifteen-membered macrocycle formation starting from 6-O-linked donor and 6- and 4-O-linked acceptor (5a,b, 6b) led exclusively to β(1-4)- and β(1-6)-linked compounds 7β and 8β, respectively, which gave cellobioside and gentiobioside derivatives. The glycosylation yields could be improved by 14-membered macrocycle formation. In the four cases studied, the donor was 6-O-linked to the spacer. For the acceptor linkage to the spacer and the accepting hydroxy group, relative D-/L-threo- and D-/L-erythro-arrangements were chosen. Standard glycosylation conditions led in three cases (13, 14, 23) only to β-linkage in high yield (16β, 17β, 25β). For the transformation of 24, having a D-erythro-arrangement in the acceptor moiety, the α-anomer 26α was preferentially obtained. Limitation of the conformational space of the donor and the acceptor as in 31, which is stereochemically identical with 24, led to the corresponding α-glycoside 32α in 87% yield. Synthesis of a pseudo mirror image of 23 [having 6-(D)/3-(D-threo)-arrangement], namely 35, having 3(L)/6-(L-threo)-arrangement of the donor and acceptor moieties, expectedly gave only α-glycoside 36α in very high yield. Thus, the efficiency and versatility of this conceptual approach to intramolecular glycoside bond formation is exhibited.

α,α-Dibromo-m-xylene gave compounds 5a,b and 7 which contain a glycosyl donor and acceptor moiety. Activation of 5a,b with NIS/TMSOTf as promoter system afforded β(1-4)-linkage leading to 6 as the only monomeric reaction product. Activation of 7 led also to β(1-4)-linkage affording 8 in very high yield.

Methyl 4-O-(α-D-glucopyranosyl)-α-D-glucopyranoside (methyl α-maltoside), a model compound for amylose, has been synthesized in four steps and 63% overall yield from relatively inexpensive D-(+)-maltose.

1H and 13C N.m.r. studies and conformational analysis have been performed on eight 1,4-linked disaccharides in which the glycosidic linkages are in different stereochemical environments.The disaccharide glycosides have been divided into two groups, one containing α-DD-, β-LD-, and β-DL- glycosides, and one containing β-DD-, α-LD-, and α-DL-glycosides with typical chemical shift differences for each group.The conformational analysis, using the HSEA-approach, indicates a number of proton-oxygen and proton-proton interactions resulting in downfield and upfield shifts of the proton signals, respectively.The 13C n.m.r. glycosylation shifts obtained have been used to simulate spectra of polysaccharides containing 1,4-linkages.

The synthesis of maltose derivatives substituted in the 6-position with F, I, N3, NH2, NHAc, COOH or COOMe, and having a 5-6 double bond, are described together with the preparation of the known 6-Cl and 6-Br derivatives using improved synthetic procedures.Furthermore, the 6'-Br and -F derivatives have been prepared.The identities of the deprotected methyl glycosides have in all cases been established by 1H and 13 C NMR spectroscopy.The synthetic compounds have been tested as substrates for the enzyme amyloglucosidase (AMG) and has been found that compounds with a charged group in the 6-position, such as amino or carboxylate, or with substituents in the 6'-position are not substrates for the enzyme; all the other compounds can be hydrolysed by the enzyme, although at widely differing rates.The 6-halo compounds proved in competition experiments to be potent enzyme inhibitors.

Condensation of 2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl bromide (1) with benzyl 2,3,6-tri-O-benzyl-β-D-glucopyranoside (6) in 1:1 benzene-nitromethane in the presence of mercuric cyanide gave, in 86percent yield after O-deacetylation followed by column chromatography, benzyl 2,3,6-tri-O-benzyl-β-cellobioside, which was catalytically hydrogenolyzed to afford cellobiose.In a similar way, methyl-α-cellobioside cellotriose, methyl-α- and β-cellotriosides, cellotetraose, lactose, and methyl α-lactoside were synthesized with high stereospecificity and in good yield by the coupling reaction, using methyl 2,3,6-tri-O-benzyl-α- and -β-D-glucopyranosyde, 6, and benzyl 2,3,6,2',3',6'-hexa-O-benzyl-β-cellobioside as the glycosyl acceptors, and 1, 2,3,4,6-tetra-O-acetyl-α-D-galactopyranosyl bromide, and hepta-O-acetyl-α-cellobiosyl bromide as the glycosyl donors.

-

-