Dictyoglomus turgidum DSM 6724 α-Glucan Phosphorylase: Characterization and Its Application in Multi-enzyme Cascade Reaction for D-Tagatose Production.

Phosphorylase is a type of enzyme-producing sugar phosphates through the reversible phosphorolysis reactions of glycosides, which makes it an important starting enzyme in multi-enzyme systems for rare sugar biomanufacturing. To investigate its application in D-tagatose biosynthesis from maltodextrin using in vitro multi-enzyme cascade biosystem, the α-glucan phosphorylase (αGP; EC 2.4.1.1) from the thermophile D. turgidum DSM 6724 was prepared and characterized. It exhibited the specific activity of 30.28 U/mg at its optimal temperature of 70 °C. Thermostability results revealed that DituαGP could maintain more than 25% of initial activity for 4 h, even at 90 °C. The highest activity was observed at pH 5.5, and most divalent metal ions deactivated the enzyme. DituαGP exhibited great application potential in the multi-enzyme system that about 3.919 g/L of D-tagatose was produced from 150 g/L of maltodextrin within 36 h. DituαGP has played an important role in this biosystem and will also be applied in the synthesis of other rare sugars from maltodextrin.

Production of tagatose and talose through isomerization of galactose in a buffer solution under subcritical water conditions.

Galactose was isomerized in pure water or in 10 mmol/L sodium phosphate buffer at 160 °C under pressurized conditions. The isomerization of galactose to tagatose and talose in phosphate buffer resulted in 14% and 1.4% yields, respectively, which were significantly higher than those obtained in subcritical pure water (0.6% and <0.1%, respectively). The effect of the temperature on isomerization was examined between 100 and 160 °C. The most remarkable isomerization was observed at 120 °C or higher. The effect of the buffer solution type was also examined. The pH drop of the treated solution was lesser in MOPS and PIPES buffers than in the phosphate buffer; however, the isomerization was less likely to occur in MOPS and PIPES buffers. The relationship between the pH drop during the reaction in phosphate buffer and the yields of tagatose and talose revealed that the isomerization proceeded only when the pH was >6.3. Our results indicate that the galactose isomerization by Lobry de Bruyn-Alberda-van Ekenstein transformation rarely occurred at low pH due to the formation of organic acids.

d-Idose, d-Iduronic Acid, and d-Idonic Acid from d-Glucose via Seven-Carbon Sugars.

A practical synthesis of the very rare sugar d-idose and the stable building blocks for d-idose, d-iduronic, and d-idonic acids from ido-heptonic acid requires only isopropylidene protection, Shing silica gel-supported periodate cleavage of the C6-C7 bond of the heptonic acid, and selective reduction of C1 and/or C6. d-Idose is the most unstable of all the aldohexoses and a stable precursor which be stored and then converted under very mild conditions into d-idose is easily prepared.

Comparative Study of Aryl O-, C-, and S-Mannopyranosides as Potential Adhesion Inhibitors toward Uropathogenic E. coli FimH.

A set of three mannopyranoside possessing identical 1,1'-biphenyl glycosidic pharmacophore but different aglyconic atoms were synthesized using either a palladium-catalyzed Heck cross coupling reaction or a metathesis reaction between their corresponding allylic glycoside derivatives. Their X-ray structures, together with their calculated 3D structures, showed strong indicators to explain the observed relative binding abilities against E. coli FimH as measured by a improved surface plasmon resonance (SPR) method. Amongst the O-, C-, and S-linked analogs, the C-linked analog showed the best ability to become a lead candidate as antagonist against uropathogenic E. coli with a Kd of 11.45 nM.

Replacement of the L-iduronic acid unit of the anticoagulant pentasaccharide idraparinux by a 6-deoxy-L-talopyranose - Synthesis and conformational analysis.

One critical part of the synthesis of heparinoid anticoagulants is the creation of the L-iduronic acid building block featured with unique conformational plasticity which is crucial for the anticoagulant activity. Herein, we studied whether a much more easily synthesizable sugar, the 6-deoxy-L-talose, built in a heparinoid oligosaccharide, could show a similar conformational plasticity, thereby can be a potential substituent of the L-idose. Three pentasaccharides related to the synthetic anticoagulant pentasaccharide idraparinux were prepared, in which the L-iduronate was replaced by a 6-deoxy-L-talopyranoside unit. The talo-configured building block was formed by C4 epimerisation of the commercially available L-rhamnose with high efficacy at both the monosaccharide and the disaccharide level. The detailed conformational analysis of these new derivatives, differing only in their methylation pattern, was performed and the conformationally relevant NMR parameters, such as proton-proton coupling constants and interproton distances were compared to the corresponding ones measured in idraparinux. The lack of anticoagulant activity of these novel heparin analogues could be explained by the biologically not favorable 1C4 chair conformation of their 6-deoxy-L-talopyranoside residues.

Novel hamamelitannin analogues for the treatment of biofilm related MRSA infections-A scaffold hopping approach.

Antimicrobial research is increasingly being focused on the problem of resistance and biofilm formation. Hamamelitannin (HAM) was recently identified as an antimicrobial potentiator for conventional antibiotics towards Staphylococcus aureus. This paper describes the synthesis and biological evaluation of novel hamamelitannin analogues with alternative central scaffolds. Via a ligand-based approach, several interesting compounds with improved synthetic accessibility were identified as potentiators for vancomycin in the treatment of MRSA infections.

Shape-selective Valorization of Biomass-derived Glycolaldehyde using Tin-containing Zeolites.

A highly selective self-condensation of glycolaldehyde to different C4 molecules has been achieved using Lewis acidic stannosilicate catalysts in water at moderate temperatures (40-100 °C). The medium-sized zeolite pores (10-membered ring framework) in Sn-MFI facilitate the formation of tetrose sugars while hindering consecutive aldol reactions leading to hexose sugars. High yields of tetrose sugars (74 %) with minor amounts of vinyl glycolic acid (VGA), an α-hydroxyacid, are obtained using Sn-MFI with selectivities towards C4 products reaching 97 %. Tin catalysts having large pores or no pore structure (Sn-Beta, Sn-MCM-41, Sn-SBA-15, tin chloride) led to lower selectivities for C4 sugars due to formation of hexose sugars. In the case of Sn-Beta, VGA is the main product (30 %), illustrating differences in selectivity of the Sn sites in the different frameworks. Under optimized conditions, GA can undergo further conversion, leading to yields of up to 44 % of VGA using Sn-MFI in water. The use of Sn-MFI offers multiple possibilities for valorization of biomass-derived GA in water under mild conditions selectively producing C4 molecules.

Triacetonide of Glucoheptonic Acid in the Scalable Syntheses of d-Gulose, 6-Deoxy-d-gulose, l-Glucose, 6-Deoxy-l-glucose, and Related Sugars.

Ease of separation of petrol-soluble acetonides derived from the triacetonide of methyl glucoheptonate allows scalable syntheses of rare sugars containing the l-gluco or d-gulo structural motif with any oxidation level at the C6 or C1 position of the hexose, usually without chromatography: meso-d-glycero-d-guloheptitol available in two steps is an ideal entry point for the study of the biotechnological production of heptoses.

Characterization of an L-arabinose isomerase from Bacillus coagulans NL01 and its application for D-tagatose production.

BACKGROUND: L-arabinose isomerase (AI) is a crucial catalyst for the biotransformation of D-galactose to D-tagatose. In previous reports, AIs from thermophilic bacterial strains had been wildly researched, but the browning reaction and by-products formed at high temperatures restricted their applications. By contrast, AIs from mesophilic Bacillus strains have some different features including lower optimal temperatures and lower requirements of metallic cofactors. These characters will be beneficial to the development of a more energy-efficient and safer production process. However, the relevant data about the kinetics and reaction properties of Bacillus AIs in D-tagatose production are still insufficient. Thus, in order to support further applications of these AIs, a comprehensive characterization of a Bacillus AI is needed. RESULTS: The coding gene (1422 bp) of Bacillus coagulans NL01 AI (BCAI) was cloned and overexpressed in the Escherichia coli BL21 (DE3) strain. The enzymatic property test showed that the optimal temperature and pH of BCAI were 60 °C and 7.5 respectively. The raw purified BCAI originally showed high activity in absence of outsourcing metallic ions and its thermostability did not change in a low concentration (0.5 mM) of Mn(2+) at temperatures from 70 °C to 90 °C. Besides these, the catalytic efficiencies (k cat/K m) for L-arabinose and D-galactose were 8.7 mM(-1) min(-1) and 1.0 mM(-1) min(-1) respectively. Under optimal conditions, the recombinant E. coli cell containing BCAI could convert 150 g L(-1) and 250 g L(-1) D-galactose to D-tagatose with attractive conversion rates of 32 % (32 h) and 27 % (48 h). CONCLUSIONS: In this study, a novel AI from B. coagulans NL01was cloned, purified and characterized. Compared with other reported AIs, this AI could retain high proportions of activity at a broader range of temperatures and was less dependent on metallic cofactors such as Mn(2+). Its substrate specificity was understood deeply by carrying out molecular modelling and docking studies. When the recombinant E. coli expressing the AI was used as a biocatalyst, D-tagatose could be produced efficiently in a simple one-pot biotransformation system.

Synthesis and x-ray crystallographic analysis of 4,6-di-O-acetyl-2,3-dideoxy-α-D-threo-hexopyranosyl cyanide.

The glycopyranosyl cyanide 4,6-di-O-acetyl-2,3-dideoxy-α-D-threo-hexopyranosyl cyanide has been synthesized from tri-O-acetyl-D-galactal by reaction with trimethylsilyl cyanide in the presence of boron trifluoride diethyl etherate followed by catalytic hydrogenation. The synthesis provides the α-anomer stereoselectively, the structure of which was assigned based on 2D NMR techniques and x-ray crystallography.

Synthetic Approaches to L-Iduronic Acid and L-Idose: Key Building Blocks for the Preparation of Glycosaminoglycan Oligosaccharides.

L-Iduronic acid (IdoA) is an important monosaccharide component of glycosaminoglycans (GAGs) such as heparin, heparan sulfate and dermatan sulfate. GAGs are complex, highly sulfated polysaccharides that mediate a multitude of physiological and pathological processes via their interactions with a range of diverse proteins. The main challenge in the synthesis of GAG oligosaccharides is the efficient gram-scale preparation of IdoA building blocks since neither IdoA nor L-idose is commercially available or readily accessible from natural sources. In this review, the different synthetic approaches for the preparation of IdoA and its derivatives, including L-idose, are presented and discussed. Derivatives of the latter are often used in GAG synthesis and are elaborated to IdoA via selective oxidation at C-6 after incorporation into a GAG chain. Particular focus will be given to the preparation of IdoA synthons most commonly used for GAG oligosaccharide synthesis, and on the progress made since the last systematic review in this area.

Development of montelukast sodium loaded niosomal carriers by film hydration technique.

The aim of this study was to develop and characterize montelukast sodium loaded niosomal drug carrier systems. The vesicles were prepared by film hydration technique using different surfactants. The optimized formulation was selected on the basis of results obtained from drug entrapment, morphology and in vitro drug release studies, and further evaluated for possible drug-excipient interaction, thermal behavior and drug physical state, before and after formulation using Fourier transform infrared spectroscopy, differential scanning calorimetry and X-ray diffraction analysis methods, respectively. The morphological characterization of vesicles was done using Transmission electron microscopy. Energy-dispersive X-ray spectroscopy system was used for elemental and dimensional analysis of developed vesicles. The vesicle surface charge was determined using zeta potential measurements. The results suggested that the optimized formulation had small size (103±6.01 nm) and high drug entrapment (72.20±2.10%). No chemical interaction was observed between the drug and excipients. The study revealed that Span 60 is a good nonionic surfactant for vesicle formulation. After 3 months storage at 2-8°C, the optimized formulation preserved stability in terms of formulation colour, drug amount and percent drug release. After 3 months, flocculation occured and hard cake was not formed on the settlement of vesicles. The preliminary results of this study suggest that the designed vesicles could enhance drug entrapment, reduce the initial burst release of drug and modulate the drug release.

Synthesis and X-ray studies of novel 3-C-nitromethyl-hexofuranoses.

A practical method for the synthesis of three novel 3-C-nitromethyl-hexofuranoses is reported. The Henry reaction on a 1,2:5,6-di-O-isopropylidene-α-d-gulofuranose-derived ketone provided a 3-C-branched gulo-isomer as the sole reaction product. The dehydration-rehydration of the latter yielded an isopropylidene-protected 3-C-nitromethyl-galactofuranose. The reaction sequence can be also used for the synthesis of a 3-deoxy-3-C-nitromethyl-hexofuranose derivative with a gulo-configuration. Two of the newly obtained carbohydrate derivatives were characterized by X-ray crystallography.

A new HPLC-based assay for the measurement of fructosamine-3-kinase (FN3K) and FN3K-related protein activity in human erythrocytes.

BACKGROUND: An impact on glycation, and possibly on diabetic complications, is attributed to fructosamine-3-kinase (FN3K) and its related protein (FN3K-RP) because they degrade Amadori compounds in vivo. Little is known about individual differences in FN3K-RP activity, which might contribute to an individual risk for diabetic complications. METHODS: An HPLC-based activity assay for FN3K-RP in erythrocytes with the substrate N-α-hippuryl-N-ε-psicosyllysine was developed. The activities of FN3K and FN3K-RP were also analysed in erythrocytes of 103 consecutive participants of a health-care survey amongst a high-risk group for diabetes. The potential associations of these activities with the subjects' health background (anthropometric data, glucose tolerance and HbA1c, blood lipids, history of metabolic diseases in the subjects and their families, and medication) were examined. RESULTS: The interindividual variability of FN3K-RP is less pronounced than that of FN3K [60-135 vs. 2.8-12.5 mU/g haemoglobin (Hb)]. No correlations with age, sex, body weight, blood cholesterol, or plasma glucose in an oral glucose tolerance test were observed. Subjects with kidney disease had higher activity of mainly FN3K-RP [111±15 vs. 98±18 mU/g Hb, mean±standard deviations (SDs), n=16 vs. 87, p=0.009], whereas subjects whose parents or siblings had a stroke showed lower FN3K activity (6.2±1.6 vs. 7.1±1.8 mU/g Hb, mean±SD, n=24 vs. 66, p=0.040). CONCLUSIONS: There is a likely impact of FN3K and FN3K-RP on the glycation cascade in vivo with potential positive and negative effects. The new screening method enables further studies to elucidate the function and importance of FN3K-RP.

Glycosylation mediated-BAIL in aqueous solution.

The use of Brønsted acid ionic liquid (BAIL) as a catalyst for the activation of unreactive and unprotected glycosyl donors has been demonstrated for the first time in aqueous solution.

Syntheses of carbocyclic analogues of α-D-glucosamine, α-D-mannose, α-D-mannuronic acid, ß-L-idosamine, and ß-L-gulose.

A versatile synthesis of orthogonally protected derivatives of carba-α-D-glucosamine, carba-α-D-mannose, carba-α-D-mannuronic acid, carba-ß-L-idosamine, and carba-ß-L-gulose from methyl α-D-mannoside is described. Our synthetic strategy utilizes the palladium-promoted Ferrier carbocyclization and persistent butane-2,3-diacetal protection to produce a key chiral cyclohexanone intermediate, from which all five carbasugar derivatives can readily be obtained.

The asymmetric syntheses of methyl D-digitoxoside, L-oleandrose and L-cymarose from methyl sorbate, an achiral precursor.

The addition of 4 eq of chloral to osmundalactone (4S,5R)-4 gave quantitative formation of the hemiacetal derivative (4S,5R)-8, which was treated with methane sulfonic acid to afford the intramolecular Micheal addition product (+)-(3S,4S,5R)-9 possessing a 3,4-cis-dihydroxy-δ-lactone in 78% overall yield from (4S,5R)-4. The obtained (+)-(3S,4S,5R)-9 was subsequently converted to methyl D-digitoxoside (pyranoside) (12) in 13% overall yield and methyl D-digitoxoside (furanoside) (12) in 20% overall yield. The reaction of benzyl-osmundalactone (4R,5S)-3 and MeOH in the presence of Amberlyst A-26 as a basic catalyst gave 3,4-trans-δ-lactone (-)-(3S,4R,5S)-20 in 28% yield and 3,4-cis-δ-lactone (-)-(3R,4R,5S)-21 in 45% yield. Dibal-H reduction of (-)-(3S,4R,5S)-20 followed by catalytic hydrogenation gave L-oleandrose (6) in 86% overall yield, while Dibal-H reduction of (-)-(3R,4R,5S)-21 followed by catalytic hydrogenation provided L-cymarose (7) in 85% overall yield.

Enhancement of ethanol production by simultaneous saccharification and fermentation (SSF) of rice straw using ethoxylated span 20.

In this work, four nonionic surfactants based on sorbitan monolaurate (Span 20) were synthesized by introducing ethylene oxide gas (n = 20, 40, 60, 80 ethylene oxide units) into Span 20 to give four new surfactants with different hydrophilic-lipophilic balance (HLB), namely, E(20), E(40), E(60), and E(80). The structures of the prepared nonionic surfactants were elucidated using Fourier-transform infrared (FT-IR) and (1)H-nuclear magnetic resonance (NMR) spectroscopy. The surface-tension measurements were recorded. The effects of the prepared nonionic surfactants on the simultaneous saccharification and fermentation (SSF) of microwave/alkali-pretreated rice straw to produce ethanol were investigated. From the obtained data, it was found that the addition of the nonionic surfactants at 2.5 g/L had a positive effect on SSF. The maximum ethanol yield (76 and 55%) was obtained after 72 hr for rice straw using Kluyveromyces marxianus and Saccharomyces cerevisiae, respectively. Also, it was found that the ethanol yield increases with increasing HLB of the prepared nonionic surfactants by increasing ethylene oxide units. The adsorption of nonionic surfactants on lignocelluloses is proposed to be due to hydrophobic and hydrogen bonding interactions between nonionic surfactants and the lignin part in the lignocelulose. It can be concluded that additions of surface-active compounds, such as nonionic surfactants, increase enzymatic conversion of rice straw for bioethanol purposes.

Preparation and properties of the aldohexofuranose pentaacetates.

The preparation of one isomer of each of the 16 enantiomeric pairs of the aldohexofuranose pentaacetates is described together with (1)H and (13)C NMR data. Eight of the isomers have been obtained crystalline. Equilibrium values for the anomeric pairs have been determined.

Development of a chemical strategy to produce rare aldohexoses from ketohexoses using 2-aminopyridine.

Rare sugars are monosaccharides that are found in relatively low abundance in nature. Herein, we describe a strategy for producing rare aldohexoses from ketohexoses using the classical Lobry de Bruyn-Alberda van Ekenstein transformation. Upon Schiff-base formation of keto sugars, a fluorescence-labeling reagent, 2-aminopyridine (2-AP), was used. While acting as a base catalyst, 2-AP efficiently promoted the ketose-to-aldose transformation, and acting as a Schiff-base reagent, it effectively froze the ketose-aldose equilibrium. We could also separate a mixture of Sor, Gul, and Ido in their Schiff-base forms using a normal-phase HPLC separation system. Although Gul and Ido represent the most unstable aldohexoses, our method provides a practical way to rapidly obtain these rare aldohexoses as needed.