Showing papers on "Pyranose published in 2019"

One-Pot Bioconversion of l-Arabinose to l-Ribulose in an Enzymatic Cascade

Abstract: This work reports the one-pot enzymatic cascade that completely converts l-arabinose to l-ribulose using four reactions catalyzed by pyranose 2-oxidase (P2O), xylose reductase, formate dehydrogenase, and catalase. As wild-type P2O is specific for the oxidation of six-carbon sugars, a pool of P2O variants was generated based on rational design to change the specificity of the enzyme towards the oxidation of l-arabinose at the C2-position. The variant T169G was identified as the best candidate, and this had an approximately 40-fold higher rate constant for the flavin reduction (sugar oxidation) step, as compared to the wild-type enzyme. Computational calculations using quantum mechanics/molecular mechanics (QM/MM) molecular dynamics (MD) showed that this improvement is due to a decrease in the steric effects at the axial C4-OH of l-arabinose, which allows a reduction in the distance between the C2-H and flavin N5, facilitating hydride transfer and enabling flavin reduction.

A GROMOS Force Field for Furanose-Based Carbohydrates

Abstract: The article describes a GROMOS force field parameter set for molecular dynamics simulations of furanose carbohydrates. The proposed united-atom force field is designed and validated with respect to the conformational properties of furanose mono-, di-, oligo-, and polymers in aqueous solvent. The set accounts for the possibility of arbitrary glycosidic linkage connectivity between units, O-alkylation, as well as of different anomery. The compatibility with the already existing, pyranose-dedicated GROMOS 56A6CARBO/CARBO_R set allows one to use the presently proposed extension for studying more diverse and biologically relevant carbohydrates that exploit both pyranose and furanose units. The validation performed against the quantum-mechanical and experimental data concerning the structural and conformational features shows that the newly developed set is capable to reproduce conformational equilibrium within the furanose ring, relative free energies of anomers, hydroxymethyl rotamers, and glycosidic linkage ...

Versatile Oxidase and Dehydrogenase Activities of Bacterial Pyranose 2-Oxidase Facilitate Redox Cycling with Manganese Peroxidase In Vitro.

Abstract: Pyranose 2-oxidase (POx) has long been accredited a physiological role in lignin degradation, but evidence to provide insights into the biochemical mechanisms and interactions is insufficient. There are ample data in the literature on the oxidase and dehydrogenase activities of POx, yet the biological relevance of this duality could not be established conclusively. Here we present a comprehensive biochemical and phylogenetic characterization of a novel pyranose 2-oxidase from the actinomycetous bacterium Kitasatospora aureofaciens (KaPOx) as well as a possible biomolecular synergism of this enzyme with peroxidases using phenolic model substrates in vitro A phylogenetic analysis of both fungal and bacterial putative POx-encoding sequences revealed their close evolutionary relationship and supports a late horizontal gene transfer of ancestral POx sequences. We successfully expressed and characterized a novel bacterial POx gene from K. aureofaciens, one of the putative POx genes closely related to well-known fungal POx genes. Its biochemical characteristics comply with most of the classical hallmarks of known fungal pyranose 2-oxidases, i.e., reactivity with a range of different monosaccharides as electron donors as well as activity with oxygen, various quinones, and complexed metal ions as electron acceptors. Thus, KaPOx shows the pronounced duality of oxidase and dehydrogenase similar to that of fungal POx. We further performed efficient redox cycling of aromatic lignin model compounds between KaPOx and manganese peroxidase (MnP). In addition, we found a Mn(III) reduction activity in KaPOx, which, in combination with its ability to provide H2O2, implies this and potentially other POx as complementary enzymatic tools for oxidative lignin degradation by specialized peroxidases.IMPORTANCE Establishment of a mechanistic synergism between pyranose oxidase and (manganese) peroxidases represents a vital step in the course of elucidating microbial lignin degradation. Here, the comprehensive characterization of a bacterial pyranose 2-oxidase from Kitasatospora aureofaciens is of particular interest for several reasons. First, the phylogenetic analysis of putative pyranose oxidase genes reveals a widespread occurrence of highly similar enzymes in bacteria. Still, there is only a single report on a bacterial pyranose oxidase, stressing the need of closing this gap in the scientific literature. In addition, the relatively small K. aureofaciens proteome supposedly supplies a limited set of enzymatic functions to realize lignocellulosic biomass degradation. Both enzyme and organism therefore present a viable model to study the mechanisms of bacterial lignin decomposition, elucidate physiologically relevant interactions with specialized peroxidases, and potentially realize biotechnological applications.

Single-Molecule Studies Reveal That Water Is a Special Solvent for Amylose and Natural Cellulose

Abstract: It is generally accepted that water is deeply involved in the structures and functions of DNA and proteins. For polysaccharides, however, the role of water remains poorly understood. Amylose and natural cellulose (NC) are two polysaccharides with similar molecular structures but different linkages (α or β) between the pyranose rings. In this study, the effects of H-bonds on the single-molecule mechanics and affinity for water of amylose and NC are explored by single-molecule atomic force microscopy (AFM) and molecular dynamics (MD) simulations, respectively. The experimental results show that the single-molecule mechanics of both amylose and NC are dependent on the solvent polarity. Accordingly, the status of H-bonds of each polysaccharide can be inferred. We find that the two polysaccharides present the same status of H-bonds in a given organic solvent: the intrachain H-bonds can be formed in a nonpolar solvent (nonane), while they are completely prohibited in a highly polar solvent (dimethyl sulfoxide, ...

Structure and functional analysis of a bacterial adhesin sugar-binding domain.

Abstract: Bacterial adhesins attach their hosts to surfaces through one or more ligand-binding domains. In RTX adhesins, which are localized to the outer membrane of many Gram-negative bacteria via the type I secretion system, we see several examples of a putative sugar-binding domain. Here we have recombinantly expressed one such ~20-kDa domain from the ~340-kDa adhesin found in Marinobacter hydrocarbonoclasticus, an oil-degrading bacterium. The sugar-binding domain was purified from E. coli with a yield of 100 mg/L of culture. Circular dichroism analysis showed that the protein was rich in beta-structure, was moderately heat resistant, and required Ca2+ for proper folding. A crystal structure was obtained in Ca2+ at 1.2-A resolution, which showed the presence of three Ca2+ ions, two of which were needed for structural integrity and one for binding sugars. Glucose was soaked into the crystal, where it bound to the sugar’s two vicinal hydroxyl groups attached to the first and second (C1 and C2) carbons in the pyranose ring. This attraction to glucose caused the protein to bind certain polysaccharide-based column matrices and was used in a simple competitive binding assay to assess the relative affinity of sugars for the protein’s ligand-binding site. Fucose, glucose and N-acetylglucosamine bound most tightly, and N-acetylgalactosamine hardly bound at all. Isothermal titration calorimetry was used to determine specific binding affinities, which lie in the 100-μM range. Glycan arrays were tested to expand the range of ligand sugars assayed, and showed that MhPA14 bound preferentially to branched polymers containing terminal sugars highlighted as strong binders in the competitive binding assay. Some of these binders have vicinal hydroxyl groups attached to the C3 and C4 carbons that are sterically equivalent to those presented by the C1 and C2 carbons of glucose.

The Mechanism of Sugar C−H Bond Oxidation by a Flavoprotein Oxidase Occurs by a Hydride Transfer Before Proton Abstraction

Abstract: Understanding the reaction mechanism underlying the functionalization of C-H bonds by an enzymatic process is one of the most challenging issues in catalysis. Here, combined approaches using density functional theory (DFT) analysis and transient kinetics were employed to investigate the reaction mechanism of C-H bond oxidation in d-glucose, catalyzed by the enzyme pyranose 2-oxidase (P2O). Unlike the mechanisms that have been conventionally proposed, our findings show that the first step of the C-H bond oxidation reaction is a hydride transfer from the C2 position of d-glucose to N5 of the flavin to generate a protonated ketone sugar intermediate. The proton is then transferred from the protonated ketone intermediate to a conserved residue, His548. The results show for the first time how specific interactions around the sugar binding site promote the hydride transfer and formation of the protonated ketone intermediate. The DFT results are also consistent with experimental results including the enthalpy of activation obtained from Eyring plots, as well as the results of kinetic isotope effect and site-directed mutagenesis studies. The mechanistic model obtained from this work may also be relevant to other reactions of various flavoenzyme oxidases that are generally used as biocatalysts in biotechnology applications.

Spectroscopic diagnostic for the ring-size of carbohydrates in the gas phase: furanose and pyranose forms of GalNAc

Abstract: Hexoses are mainly found in nature in the pyranose form (6-membered ring). Yet, furanose forms (5-membered ring) are observed in some rare polysaccharides. Using IRMPD spectroscopy (InfraRed Multiple Photon Dissociation), we propose a straightforward diagnostic of the ring-size of N-acetyl galactosamine ions. The furanose form of N-acetyl galactosamine was synthesized and its protonated ion was isolated in an ion trap to measure its gas phase vibrational spectrum by IRMPD. Comparison with the IRMPD spectrum of its pyranose counterpart reveals that they have distinctive optical fingerprints. This new MS-based diagnostic opens the way to facile identification of the ring-size in oligosaccharides. Our experimental data also provide new insights to support the theoretical description of the conformational behavior of the furanose ring, which is notoriously more flexible than the pyranose form but remains difficult to assess.

Synthesis and Biological Evaluation of 6-[(1R)-1-Hydroxyethyl]-2,4a(R),6(S),8a(R)-tetrahydropyrano-[3,2-b]-pyran-2-one and Structural Analogues of the Putative Structure of Diplopyrone

Abstract: The phytotoxin diplopyrone is considered to be the main phytotoxin in a fungus that is responsible for cork oak decline. A carbohydrate-based synthesis of the enantiomer of the structure proposed for diplopyrone has been developed from a commercially available derivative of d-galactose. Key steps in the synthesis are a highly stereoselective pyranose chain-extension based on methyltitanium, preparation of a vinyl glycoside via Isobe C-alkynylation-rearrangement/reduction, and RCM-based pyranopyran construction. Crystallographic and NMR analysis confirms an earlier report that the structure originally proposed for diplopyrone may require revision. Structural analogues were prepared for biological evaluation, the most promising being a pyranopyran nitrile synthesized from tri-O-acetyl-d-galactal by Ferrier cyanoglycosidation, Wittig chain extension, and lactonization. Biological assays revealed potent antibacterial activity for the nitrile analogue against common bacterial pathogens Edwardsiella ictaluri an...

Synthesis of vicinal dideoxy-difluorinated galactoses.

Abstract: Fluorinated carbohydrates have been employed as probes for fundamental studies of protein–carbohydrate interactions, but also in the development of mechanism-based enzyme inhibitors. There is a continuing demand for novel fluorinated carbohydrate probes. Whereas most examples so far involved monodeoxyfluorinated sugars, multiply deoxyfluorinated sugars have gained much interest. Here we report the synthesis and characterisation of novel vicinal dideoxy-difluorinated D-galactoses with fluorination at the 3,4-positions, and at the 2,3-positions, the latter in both the pyranose and furanose forms. This includes a successful pyranose-into-furanose isomerisation protocol.

Conformational Behavior of d-Lyxose in Gas and Solution Phases by Rotational and NMR Spectroscopies.

Abstract: Understanding the conformational preferences of carbohydrates is crucial to explain the interactions with their biological targets and to improve their use as therapeutic agents. We present experimental data resolving the conformational landscape of the monosaccharide d-lyxose, for which quantum mechanical (QM) calculations offer model-dependent results. This study compares the structural preferences in the gas phase, determined by rotational spectroscopy, with those in solution, resolved by nuclear magnetic resonance (NMR) and molecular dynamics (MD) simulations. In contrast to QM calculations, d-lyxose adopts only pyranose forms in the gas phase, with the α-anomer exhibiting both the 4C1 and 1C4 chairs (60:40). The predominantly populated β-anomer shows the 4C1 form exclusively, as determined experimentally by isotopic substitution. In aqueous solution, the pyranose forms are also dominant. However, in contrast to the gas phase, the α-anomer as 1C4 chair is the most populated, and its solvation is more effective than for the β derivative. Markedly, the main conformers found in the gas phase and solution are characterized by the lack of the stabilizing anomeric effect. From a mechanistic perspective, both rotational spectroscopy and solid-state nuclear magnetic resonance (NMR) corroborate that α ← β or furanose ← pyranose interconversions are prevented in the gas phase. Combining microwave (MW) and NMR results provides a powerful method for unraveling the water role in the conformational preferences of challenging molecules, such as flexible monosaccharides.

Synergetic Depolymerization of Aspen CEL by Pyranose 2-Oxidase and Lignin-degrading Peroxidases

Abstract: Three enzymatic treatments were compared for the depolymerization of cellulolytic enzyme lignin (CEL) from aspen; these systems used pyranose 2-oxidase and lignin-degrading peroxidases. The “P” system was mainly composed of pyranose 2-oxidase, lignin peroxidase (LiP), and manganese peroxidase (MnP). Catalase and vitamin C were added to the P system to decompose H2O2 to control the H2O2 concentration. The system to which catalase was added was called the “C” system. The system to which catalase and vitamin C were added together was called the “V” system. Ultraviolet-visible (UV-Vis) spectra of supernatants after aspen CEL treatment by the P, C, or V system was used to monitor the amount of water-soluble lignin fragments that were generated, which increased with system treatment time. A gel permeation chromatography (GPC) analysis showed that after 12 h of system treatment, the molecular weight (Mw) of CEL was efficiently lowered; the maximum Mw reduction of aspen CEL was 20% when compared to the blank and control runs. The residual enzymatic activity of the supernatant after the CEL treatment by the P, C, or V system indicated that MnP and LiP activity for lignin degradation was dependent upon the H2O2 concentration. Therefore, it is advised that MnP and LiP be applied separately for effective lignin degradation.

Substrate Preference Pattern of Agaricus meleagris Pyranose Dehydrogenase Evaluated through Bioelectrochemical Flow Injection Amperometry

Abstract: Pyranose dehydrogenase (PDH) is a quinone-dependent extracellular flavoglycoprotein mainly produced by litter-decomposing fungi and contributes to the degradation of lignocellulose. PDH in terms of structure and catalytic features pertains to the glucose methanol-choline oxidoreductase family and oxidizes a wide substrate range of aldopyranoses including hexoses, pentoses, disaccharides and oligosaccharides with a high degree of regioselectivity. The purpose of this study was to rationalize the preference of PDH immobilized on an electrode with the structural features of various substrates and thus the kinetic constants were measured for various sugars. PDH was co-immobilized on the electrode with an osmium redox polymer. Response currents for different sugars were measured using flow injection amperometry at +0.3 V vs. Ag|AgCl, KCl (0.1 M). The Michaelis-Menten constants, the turnover numbers and the catalytic efficiency were calculated and revealed that type, orientation and configuration of the substituent play a major role on substrate preference. An OH-group at C-1 and C-6 are not essential and substrate specificities are little affected by the substitution at C-1. The presence and orientation of OH− at C-2 and C-3 are relevant for reactivity. Orientation of OH− at the C-4 position has little effect, and sugars with a substitution below the plane at C-5 are not suitable as substrate. Highest activity for oxidation of glucose, mannose and sucrose was detected at pH 8.5. (Less)

Monosaccharide Isomer Interconversions Become Significant at High Temperatures

Abstract: Quantum-chemical calculations show how low barriers to anomerization and shifting equilibria cause a significant presence of different monosaccharide isomers in high-temperature processes such as pyrolysis. The transition between isomeric forms of monosaccharides is long-studied, but examination has typically been limited to the solution phase and to pyranose isomers. Processes and rates of anomerization by reversible, gas-phase ring-opening and -closing reactions were predicted for the monosaccharides d-glucose, d-mannose, d-galactose, d-xylose, l-arabinose, and d-glucuronic acid. Structures and thermochemistry were computed for stable species and pericyclic transition states using CBS-QB3, and high-pressure-limit Arrhenius reaction parameters were predicted and fitted from 300 to 1000 K. Activation energies for the ring-opening reactions were 162–217 kJ/mol for four-center pericyclic separation of the lactol group but were reduced by catalytic participation of a hydroxyl group within the monosaccharide ...

Copper-mediated anomeric O-arylation with organoboron reagents.

Abstract: Copper-mediated couplings of arylboroxines with glycosyl hemiacetals furnish O-aryl glycosides via Csp2–O bond formation. The method enables the anomeric O-arylation of protected pyranose and furanose derivatives, and is tolerant of functionalized arylboroxine partners. Whereas mixtures of anomers are formed from glucopyranose, galactopyranose and arabinofuranose hemiacetals, the α-anomer is generated selectively from mannopyranose and mannofuranose-derived substrates.

Synthesis of Mono- and Disaccharide 4-[(ω-Sulfanylalkyl)oxy]benzoylhydrazones as Potential Glycoligands for Noble Metal Nanoparticles

Abstract: A procedure has been developed for the synthesis of previously unknown aldose 4-[(ω-sulfanylalkyl) oxy]benzoylhydrazones (where alkyl is hexyl or decyl and aldoses are D-glucose, D-galactose, D-maltose, and D-lactose) that a repromising glycoligands for noble metal nanoparticles. According to the 1H and 13C NMR data, 4-[(ω-sulfanylalkyl)oxy]benzoylhydrazones derived from D-glucose, D-maltose, and D-lactose in crystal and in DMSO-d6 solution have exclusively the cyclic pyranose structure (α- and β-anomers). D-Galactose 4-[(ω-sulfanylalkyl)oxy]benzoylhydrazones in DMSO-d6 solution exist as tautomeric mixtures of cyclic pyranose and open-chain acylhydrazone structures.

Carbohydrate Spiro-heterocycles via Radical Chemistry

Abstract: This review summarizes the current status of the preparation of spiro-heterocycles fused with a pyranose or furanose carbohydrate skeleton, using free radical chemistry. A variety of heterospiro[m.n]alkane bicyclic structures (m = 3–5, n = 4–6) possessing one, two, or three heteroatoms (N, O, Si, S) have been collected, in addition to three different 1,6,8-trioxadispiro-tetradecane and 1,6,8-trioxadispiro-pentadecane tricyclic systems. C(sp3)–H bond functionalization by 1,5- or 1,6-hydrogen atom transfer (HAT) initiated by C(sp3)-, C(sp2)-, O-, or N-radicals and 5-exo-trig or 6-exo-trig cyclization reactions are the most useful strategies employed for the construction of the heterocyclic rings. The intramolecular HAT promoted by photoexcited monoketones, α-diketones, furanones, and succinimides via a Norrish type II–Yang cyclization process has also been successfully applied.

Anomeric effect in pyranose-ring derivatives containing carbon, silicon, and germanium as anomeric centers: an ab initio systematic study

Abstract: We have performed a systematic conformational analysis focused on the evaluation of the anomeric effect (AE) in a series of pyranose derivatives containing carbon, silicon, and germanium as anomeric centers (c*) using the MP2/aug-cc-pVDZ level of theory together with natural bond orbital (NBO) electronic structure calculations. Although, both endo- and exo-anomeric effects operate within all the systems studied; the conformational preference towards the axial (α) form can be explained in terms of the incidence of the endo-anomeric effect. The magnitude calculated for the AE is considerably higher for compounds containing carbon as c*. On the other hand, the lower magnitude of the hyperconjugative delocalizations towards antibonding exocyclic anomeric orbitals in Si- and Ge-containing compounds can be justified by the availability of energetically accessible vacant d-type orbitals in these atoms. While the conformational preference in the carbon group is purely related to a higher anomeric hyperconjugation in the α conformers, steric and electrostatic factors dictate the conformational α arrangement in the Si- and Ge-containing compounds. Implicit consideration of the solvent (water) produces a notable increase in the population of the β anomers in some of the systems into study. However, the results of NBO energy partition study performed reveal that the merely observation of a change in the α/β ratio for a given system upon solvation should not be taken as an indication of a predominant role of electrostatic effects as the origin for their anomeric preference.

Comparative Study on the Mechanism of Photoinduced Electron Transfer from Tryptophan 168 to Excited Flavin in T169S and Wild-type Pyranose 2-Oxidase

Abstract: The rates of photoinduced electron transfer (ET) from Trp168 to the excited isoalloxazine (Iso*) in the pyranose 2-oxidase (P2O) with T169S mutation were studied using ET theory by fitting structural properties calculated from molecular dynamics simulation to the reported fluorescence lifetimes. The obtained ET rates and related physical quantities were compared to those reported for the wild-type (WT) P2O. Experimental fluorescence lifetimes of Iso in T169S have two components of 92–240 fs (depending on the emission wavelength) and 15 ps (independent of the emission wavelength). One of the four T169S subunits (Sub C) displayed a low rate, and the other three (Sub A, B, and D) had high rates. Mean ET rates of the fast components were ca. 10 ps–1 at 480 nm, 7 ps–1 at 500 nm, and 4.2 ps–1 at 530, 555, and 580 nm, which were lower than the reported rates in the WT P2O of ca. 17 ps–1 at 480 nm, 14 ps–1 at 500 nm, 9 ps–1 at 530 and 555 nm, and 11 ps–1 at 580 nm, while the rate for the slow component in T169S (...

Salt of pyranose substituted heterocyclic compound, and preparation method and use thereof

Abstract: The invention belongs to the field of pharmaceutical chemistry, relates to a salt of a pyranose substituted heterocyclic compound, and a preparation method and a use thereof, and in particular, relates to an acid addition salt of the compound represented by the formula (I) or a prodrug thereof. In addition, the invention relates to D-glucuronate of the compound represented by the formula (I) or the prodrug thereof in a crystalline form. The D-glucuronate of a compound represented by the formula (II) has obvious advantages in crystallinity, subsequent purification, stability, preparation druggability or quality control, and is most suitable for improving the preparation druggability, purity and quality control of the drugs, and for improving the large-scale process development of the drugs.