Sugar Structure Research Articles

Unexpected chiral induction from achiral cationic polythiophene aggregates and its application to the sugar pattern recognition

The aggregates of a cationic polythiophene (PT1) formed in poor solvent in the presence of sugars showed the distinct circular dichroism activity, the intensity being well correlated with the specific optical rotation [α] of sugars: therefore, the present system is useful as a novel sugar structure reading-out method.

α,β-D-Constrained Nucleic Acids Are Strong Terminators of Thermostable DNA Polymerases in Polymerase Chain Reaction

(S C5′, RP) α,β-D- Constrained Nucleic Acids (CNA) are dinucleotide building blocks that can feature either B-type torsional angle values or non-canonical values, depending on their 5′C and P absolute stereochemistry. These CNA are modified neither on the nucleobase nor on the sugar structure and therefore represent a new class of nucleotide with specific chemical and structural characteristics. They promote marked bending in a single stranded DNA so as to preorganize it into a loop-like structure, and they have been shown to induce rigidity within oligonucleotides. Following their synthesis, studies performed on CNA have only focused on the constraints that this family of nucleotides introduced into DNA. On the assumption that bending in a DNA template may produce a terminator structure, we investigated whether CNA could be used as a new strong terminator of polymerization in PCR. We therefore assessed the efficiency of CNA as a terminator in PCR, using triethylene glycol phosphate units as a control. Analyses were performed by denaturing gel electrophoresis and several PCR products were further analysed by sequencing. The results showed that the incorporation of only one CNA was always skipped by the polymerases tested. On the other hand, two CNA units always stopped proofreading polymerases, such as Pfu DNA polymerase, as expected for a strong replication terminator. Non-proofreading enzymes, e.g. Taq DNA polymerase, did not recognize this modification as a strong terminator although it was predominantly stopped by this structure. In conclusion, this first functional use of CNA units shows that these modified nucleotides can be used as novel polymerization terminators of proofreading polymerases. Furthermore, our results lead us to propose that CNA and their derivatives could be useful tools for investigating the behaviour of different classes of polymerases.

Synthesis and biological evaluation of RON-neoglycosides as tumor cytotoxins

Cardenolides such as digitoxin have been shown to inhibit cancer cell growth, to reduce cancer metastasis, and to induce apoptosis in tumor cells. Among the most potent digitoxin-based cytotoxins identified to date are MeON-neoglycosides generated via oxyamine neoglycosylation. Here, we report our studies of oxyamine neoglycosylation aimed at facilitating the elucidation of linkage-diversified digitoxin neoglycoside structure–activity relationships. We identified conditions suitable for the convenient synthesis of digitoxin neoglycosides and found that sugar structure, rather than RON-glycosidic linkage, exerts the strongest influence on neoglycoside yield and stereochemistry. We synthesized a library of digitoxin neoglycosides and assessed their cytotoxicity against eight human cancer cell lines. Consistent with previous findings, our data show that the structure of RON-neoglycosidic linkages influences both the potency and selectivity of digitoxin neoglycosides.

Searching and Optimizing Structure Ensembles for Complex Flexible Sugars

NMR restrictions are suitable to specify the geometry of a molecule when a single well-defined global free energy minimum exists that is significantly lower than other local minima. Carbohydrates are quite flexible, and therefore, NMR observables do not always correlate with a single conformer but instead with an ensemble of low free energy conformers that can be accessed by thermal fluctuations. In this communication, we describe a novel procedure to identify and weight the contribution to the ensemble of local minima conformers based on comparison to residual dipolar couplings (RDCs) or other NMR observables, such as scalar couplings. A genetic algorithm is implemented to globally minimize the R factor comparing calculated RDCs to experiment. This is done by optimizing the weights of different conformers derived from the exhaustive local minima conformational search program, fast sugar structure prediction software (FSPS). We apply this framework to six human milk sugars, LND-1, LNF-1, LNF-2, LNF-3, LNnT, and LNT, and are able to determine corresponding population weights for the ensemble of conformers. Interestingly, our results indicate that in all cases the RDCs can be well represented by only a few most important conformers. This confirms that several, but not all of the glycosidic linkages in histo-blood group "epitopes" are quite rigid.

Hydroxyl distribution in sugar structure and its contributory role in the inhibition of Stachybotrys microspora β-glucosidase (bglG)

Stachybotrys microspora is a filamentous fungus that produces various β-glucosidases, of which two have already been characterized. The present study reports on the production of a third one, named bglG, in the presence of d-glucose used as a sole carbon source, and on its subsequent purification and characterization. Although efficiently produced in the presence of d-glucose, bglG continues to be highly inhibited by this sugar. In fact, the addition of d-glucose significantly decreases the glucose formation rates during the hydrolysis of pNPG. This work reports on the effect of various carbohydrates on bglG activity in order to understand the mechanisms adopted by d-glucose to inhibit this enzyme. The findings indicate that bglG is strongly inhibited by d-glucose (44% of the relative activity at 5mM), d-glucitol (96% of the relative activity), d-mannose (56% of the relative activity), cellobiose and maltose (72% and 71% of the relative activity, respectively). On the other hand, d-galactose, d-fructose, lactose, and sucrose have no effect on bglG activity. Similarly, several isomers, such as 2-acetamido-2-deoxy-d-glucose and 2-deoxy-d-arabino-hexose (2-deoxy-d-glucose) were noted to bring no change on the relative activity of bglG. d-xylose and xylitol, on the other hand, enhanced bglG activity up to 123% and 120% of relative activity, respectively. Accordingly, the configuration, epimerisation, isomerisation, and substitutions played key roles in bglG inhibition. The effect of the combination of iron (the best activator of bglG, 161%) with some of those additives was also investigated. The findings revealed that, while a combination of iron at a concentration of 10mM with d-glucose resulted in a two-fold decrease in bglG inhibition (84% at 5mM), iron maintained the same effect with the remainder of the additives being tested.

Enzymatic synthesis of novel branched sugar alcohols mediated by the transglycosylation reaction of pullulan-hydrolyzing amylase II (TVA II) cloned from Thermoactinomyces vulgaris R-47

Transglycosylation reactions are useful for preserving a specific sugar structure during the synthesis of branched oligosaccharides. We have previously reported a panosyl unit transglycosylation reaction by pullulan-hydrolyzing amylase II (TVA II) cloned from Thermoactinomyces vulgaris R-47 (Tonozuka et al., Carbohydr. Res., 1994, 261, 157–162). The acceptor specificity of the TVA II transglycosylation reaction was investigated using pullulan as the donor and sugar alcohols as the acceptor. TVA II transferred the α-panosyl unit to the C-1 hydroxyl group of meso-erythritol, C-1 and C-2 of xylitol, and C-1 and C-6 of d-sorbitol. TVA II differentiated between the sugar alcohols’ hydroxyl groups to produce five novel non-reducing branched oligosaccharides, 1-O-α-panosylerythritol, 1-O-α-panosylxylitol, 2-O-α-panosylxylitol, 1-O-α-panosylsorbitol, and 6-O-α-panosylsorbitol. The Trp356→Ala mutant showed similar transglycosylation reactions; however, panose production by the mutant was 4.0–4.5-fold higher than that of the wild type. This suggests that Trp356 is important for recognizing both water and the acceptor molecules in the transglycosylation and the hydrolysis reaction.

Glycosphingolipid receptors for pathogenic vibrios in intestines of mariculture fish

Vibrios are highly motile, facultatively anaerobic bacteria that are ubiquitous in aquatic environments and part of the normal intestinal microflora of healthy fish, but some species can cause vibriosis. The adherence of vibrios to host fish intestines is a significant event not only for their survival and growth, but also in terms of pathogenicity. However, the molecular mechanism underlying the adhesion of vibrios to the intestinal tract of fish is not fully understood. We report here the identification of intestinal glycosphingolipid (GSL) receptors to which pathogenic vibrios attach in typical mariculture fish. Thin-layer chromatography overlay assays using five species of 35S-labeled vibrios and intestinal glycosphingolipids of seven species of mariculture fish revealed that all of the fish tested possessed GM3 (NeuAcα2-3Galβ1-4Glcβ1-1′Cer) and/or GM4 (NeuAcα2-3Galβ1-1′Cer) as major acidic intestinal GSLs and that all of the vibrios tested specifically adhered to GM3 and/or GM4. Our results demonstrate that these GSLs were able to function as a receptor for the various vibrios tested. Analysis of the relationship between sugar structure and receptor activity for vibrios revealed that ‘NeuAcα2-3Galβ1-’ is required at the non-reducing end of glycosphingolipids for the bacteria to attach.

Mechanism of Melibiose/Cation Symport of the Melibiose Permease of Salmonella typhimurium

The MelB permease of Salmonella typhimurium (MelB-ST) catalyzes the coupled symport of melibiose and Na(+), Li(+), or H(+). In right-side-out membrane vesicles, melibiose efflux is inhibited by an inwardly directed gradient of Na(+) or Li(+) and stimulated by equimolar concentrations of internal and external Na(+) or Li(+). Melibiose exchange is faster than efflux in the presence of H(+) or Na(+) and stimulated by an inwardly directed Na(+) gradient. Thus, sugar is released from MelB-ST externally prior to the release of cation in agreement with current models proposed for MelB of Escherichia coli (MelB-EC) and LacY. Although Li(+) stimulates efflux, and an outwardly directed Li(+) gradient increases exchange, it is striking that internal and external Li(+) with no gradient inhibits exchange. Furthermore, Trp → dansyl FRET measurements with a fluorescent sugar (2'-(N-dansyl)aminoalkyl-1-thio-β-D-galactopyranoside) demonstrate that MelB-ST, in the presence of Na(+) or Li(+), exhibits (app)K(d) values of ∼1 mM for melibiose. Na(+) and Li(+) compete for a common binding pocket with activation constants for FRET of ∼1 mM, whereas Rb(+) or Cs(+) exhibits little or no effect. Taken together, the findings indicate that MelB-ST utilizes H(+) in addition to Na(+) and Li(+). FRET studies also show symmetrical emission maximum at ∼500 nm with MelB-ST in the presence of 2'-(N-dansyl)aminoalkyl-1-thio-β-D-galactopyranoside and Na(+), Li(+), or H(+), which implies a relatively homogeneous distribution of conformers of MelB-ST ternary complexes in the membrane.

Effects of mono- and disaccharides on the antimicrobial activity of bovine lactoperoxidase system.

The effects of mono- and disaccharides on the antimicrobial activity of the lactoperoxidase (LPO) system against Salmonella Enteritidis were investigated. The results clearly reveal that most of the sugars inhibit the antimicrobial activity of the LPO system. The inhibitory potency varies depending on the structure of sugar. L-Fructose and D-allose were strongly inhibitive to the action of the LPO system, while sucrose was the weakest inhibitor. The decreased antimicrobial activity is due to the reduction of LPO catalytic activity by sugar. An inhibitory kinetic study showed the noncompetitive inhibitor. D-Allose and L-fructose yielded strikingly low K(i) values of 0.36 and 0.42 mM, respectively, while the K(i) values of the other sugars ranged from 1.37 to 3.60 mM. Since LPO activity is inhibited by the saccharides, the sugar content in food should be considered when the LPO system is applied to the preservation of food.

Conformational Energies of DNA Sugar−Phosphate Backbone: Reference QM Calculations and a Comparison with Density Functional Theory and Molecular Mechanics

The study investigates electronic structure and gas-phase energetics of the DNA sugar−phosphate backbone via advanced quantum chemical (QM) methods. The analysis has been carried out on biologicall...

Assessment of Chemoselective Neoglycosylation Methods Using Chlorambucil as a Model

To systematically assess the impact of glycosylation and the corresponding chemoselective linker upon the anticancer activity/selectivity of the drug chlorambucil, herein we report the synthesis and anticancer activities of a 63-member library of chlorambucil-based neoglycosides. A comparison of N-alkoxyamine-, N-acylhydrazine-, and N-hydroxyamine-based chemoselective glycosylation of chlorambucil revealed sugar- and linker-dependent partitioning among open- and closed-ring neoglycosides and corresponding sugar-dependent variant biological activity. Cumulatively, this study represents the first neoglycorandomization of a synthetic drug and expands our understanding of the impact of sugar structure upon product distribution/equilibria in the context of N-alkoxyamino-, N-hydroxyamino-, and N-acylhydrazine-based chemoselective glycosylation. This study also revealed several analogues with increased in vitro anticancer activity, most notably D-threoside 60 (NSC 748747), which displayed much broader tumor specificity and notably increased potency over the parent drug.

Noninvasive Imaging of Dendrimer‐Type N‐Glycan Clusters: In Vivo Dynamics Dependence on Oligosaccharide Structure

Body image: Self-activating Huisgen 1,3-dipolar cycloaddition and 6π azaelectrocyclization of lysine-based dendrimers (see picture) enable the in vivo dynamics and organ-specific accumulation of N-glycans to be visualized. The sugar structure and glycosyl bond linkages of N-glycans control the whole-body trafficking of the clusters in nude mice and a cancer model.

Interaction Analyses of Amyloid β Peptide (1–40) with Glycosaminoglycan Model Polymers

Abstract We synthesized a novel glycopolymer library with 6-sulfo-GlcNAc and glucuronic acid (GlcA) based on the structure of glycosaminoglycans. The molecular weights of the polymers were controlled via living radical polymerization. The interactions of Aβ(1–40) with glycopolymers were analyzed by inhibition activity of protein aggregation using ThT fluorescence assay, atomic force microscopy observation, and CD spectra. The inhibition activity of Aβ was much affected by the sugar structure and molecular weight of the polymer. The glycopolymers carrying 6-sulfo-GlcNAc showed inhibition activity toward Aβ aggregate, and those with 6-suflo-GlcNAc and GlcA showed the strong inhibition activity. The glycopolymer libraries yielded valuable information about Aβ aggregate with glycosaminoglycans.

GlycoViewer: a tool for visual summary and comparative analysis of the glycome

The GlycoViewer (http://www.systemsbiology.org.au/glycoviewer) is a web-based tool that can visualize, summarize and compare sets of glycan structures. Its input is a group of glycan structures; these can be entered as a list in IUPAC format or via a sugar structure builder. Its output is a detailed graphic, which summarizes all salient features of the glycans according to the shapes of the core structures, the nature and length of any chains, and the types of terminal epitopes. The tool can summarize up to hundreds of structures in a single figure. This allows unique, high-level views to be generated of glycans from one protein, from a cell, a tissue or a whole organism. Use of the tool is illustrated in the analysis of normal and disease-associated glycans from the human glycoproteome.

Sugar Acetates as CO2-philes: Molecular Interactions and Structure Aspects from Absorption Measurement Using Quartz Crystal Microbalance

Sugar acetates, recognized as attractive CO(2)-philic compounds, have potential uses as pharmaceutical excipients, controlled release agents, and surfactants for microemulsion systems in CO(2)-based processes. This study focuses on the quantitative examination of absorption of high pressure CO(2) into these sugar derivatives using quartz crystal microbalance (QCM) as a detector. In addition to the absorption measurement, the QCM is initially found to be able to detect the CO(2)-induced deliquescence of sugar acetates, and the CO(2) pressure at which the deliquescence happens depends on several influencing factors such as the temperature and thickness of the film. The CO(2) absorption in alpha-D-glucose pentaacetate (Ac-alpha-GLU) is revealed to be of an order of magnitude larger in comparison with its anomer Ac-beta-GLU, whereas alpha-D-galactose pentaacetate (Ac-alpha-GAL) absorbs CO(2) less than Ac-alpha-GLU due to the steric-hindrance between the acetyl groups on the anomeric and C4 carbons, implying the significant importance of the molecular structure and configuration of sugar acetates on the absorption. The effects of molecular size and acetyl number of sugar acetates on the CO(2) absorption are evaluated and the results indicate that the conformation and packing of crystalline sugar acetate as well as the accessibility of the acetyls are also vital for the absorption of CO(2). It is additionally found that a CO(2)-induced change in the structure from a crystalline system to an amorphous system results in an order of magnitude increase in CO(2) absorption. Further investigation illustrates the interaction strength between sugar acetates and CO(2) by calculating the thermodynamic parameters such as Henry's law constant, enthalpy and entropy of dissolution from the determined CO(2) absorption. Experiments and calculations demonstrate that sugar acetates exhibit high CO(2) absorption, as at least comparable to ionic liquids. Since the ionic liquids have potential uses in the separation of acidic gases, it is evident from this study that sugar acetates could be used as possible materials for CO(2) separation.

Impact of sugar pucker on base pair and mispair stability.

The selection of nucleoside triphosphates by a polymerase is controlled by several energetic and structural features, including base pairing geometry as well as sugar structure and conformation. Whereas base pairing has been considered exhaustively, substantially less is known about the role of sugar modifications for both nucleotide incorporation and primer extension. In this study, we synthesized oligonucleotides containing 2'-fluoro-modified nucleosides with constrained sugar pucker in an internucleotide position and, for the first time, at a primer 3'-end. The thermodynamic stability of these duplexes was examined. The nucleoside 2'-deoxy-2'-fluoroarabinofuranosyluracil [U(2'F(ara))] favors the 2'-endo conformation (DNA-like), while 2'-deoxy-2'-fluororibofuranosyluracil [U(2'F(ribo))] favors the 3'-endo conformation (RNA-like). Oligonucleotides containing U(2'F(ara)) have slightly higher melting temperatures (T(m)) than those containing U(2'F(ribo)) when located in internucleotide positions or at the 3'-end and when correctly paired with adenine or mispaired with guanine. However, both modifications decrease the magnitude of DeltaH degrees and DeltaS degrees for duplex formation in all sequence contexts. In examining the thermodynamic properties for this set of oligonucleotides, we find entropy-enthalpy compensation is apparent. Our thermodynamic findings led to a series of experiments with DNA ligase that reveal, contrary to expectation based upon observed T(m) values, that the duplex containing the U(2'F(ribo)) analogue is more easily ligated. The 2'-fluoro-2'-deoxynucleosides examined here are valuable probes of the impact of sugar constraint and are also members of an important class of antitumor and antiviral agents. The data reported here may facilitate an understanding of the biological properties of these agents, as well as the contribution of sugar conformation to replication fidelity.

Computational Study of the Conformational Structures of Saccharides in Solution Based on J Couplings and the “Fast Sugar Structure Prediction Software”

This article reports on the implementation of J coupling calculations in our recently developed Fast Sugar Structure Prediction Software (FSPS). The FSPS combines a smart and exhaustive algorithm to search through conformational space with the calculation of different experimental nuclear magnetic resonance observables to establish the conformation of saccharides in solution. Using our algorithm in combination with NMR data, we investigate the solution structure of three simple disaccharides (methyl alpha-sophoroside, methyl alpha-laminarabioside, and methyl alpha-cellobioside) and one complex bacterial polysaccharide (Shigella flexneri 5a).

Striking Structural Transformation from Cyclic Oligosaccharide to Aromatic Series by Means of the Effect of Lithium Carbonate Based on Gas Chromatography Coupled to Time-of-Flight Mass Spectrometry

An interesting paradigm in which the conformation of glucopyranose of beta-CD in the presence of Li(2)CO(3) is transformed into both an aromatic structure, such as the heterocyclic compound C(6)H(6)O(+) with a prominent relative abundance (RA) and the tropylium ions C(3)H(3)(+), C(5)H(5)(+), and C(7)H(7)(+) at comparatively low temperatures, and a linear structure such as C(18)H(30)O(+) and C(4)H(10)O(3)(+) is reported in the present work. The efficient transformation (higher RA, lower temperature) from a sugar structure to an aromatic structure is ascribed to the contribution of the molecule-ion interaction between beta-CD and Li(2)CO(3). Such a transformation is significant because it provides new insight into the link between supramolecular chemistry and other fields such as organic synthesis, biomineralization, environmental protection, and energy utilization.

Thermal Degradation Kinetics and Geometrical Stability of D-Sucrose

Thermal degradation kinetics of solid-state D-sucrose and D-glucose were studied using differential thermal analysis (DTA) and thermogravimetric analysis (TGA) techniques. The melting and decomposition process of the two sugars were displayed as endothermic peaks on the DTA curve under nonisothermal conditions. It was noticed that the melting peak temperature of D-glucose was lower than that of the other sugar. For the decomposition processes, the opposite occurred. The decomposition kinetic parameters, such as the decomposition activation energy (Ed) and the frequency factor (Ko), were calculated by employing different approximations. The relative stability of the studied sugars was determined. Molecular mechanics (MM) calculations showed that the optimal geometric structure (OMG) of D-sucrose was at least three orders of magnitude more stable than the molecular geometric (MG) structure. These calculations indicate that the potential energy (PE (kcal mol−1)) of the OMG is about three (1.142 × 103) orders of magnitude lower than the MG structure of the same sugar. So, optimizing the geometry of the molecular structure (OMG) is very important for understanding the stability of this sugar.

Multiple Anharmonic Vibrational Probes of Sugar Structure and Dynamics

Quantum mechanical computations and molecular dynamics simulations are carried out for the simplest sugar glycolaldehyde to gain insight into the underlying force fields that determine the vibrational spectroscopic parameters relevant to structure and dynamics. The harmonic and anharmonic vibrational frequencies of the 3N--6 modes and their diagonal and off-diagonal anharmonicities are evaluated using the hybrid B3LYP functional in comparison with other high-level theories. Very good performance of B3LYP/6-31+G** is found in predicting the anharmonic frequencies by statistical analysis and by comparison to gas-phase experiments. Full cubic and semidiagonal quartic anharmonic force constants, the origin of the anharmonicities, the isotope dependence of the anharmonicities, and the polarizable continuum solvent effect on the anharmonicities are examined, in particular, for the CO, C-H, and O-H stretching modes. Site-dependent dynamical interactions between glycolaldehyde and water molecules in the hydration shells are examined by molecular dynamics simulations employing a set of molecular mechanical force fields developed on the basis of quantum mechanical computations. The statistical distributions and correlations of the fundamental transition frequencies and transition dipoles are obtained through instantaneous normal-mode analysis. The simultaneous assessment of multiple parameters of multiple vibrational probes shall prove useful in understanding the characteristics of sugar structure and dynamics expressed in two-dimensional infrared correlation spectra.