Methyl ß-lactoside [methyl ß-D-galactopyranosyl-(1→4)-ß-D-glucopyranoside] monohydrate: a solvomorphism study.

Methyl ß-lactoside [methyl ß-D-galactopyranosyl-(1→4)-ß-D-glucopyranoside] monohydrate, C13H24O11·H2O, (I), was obtained via spontaneous transformation of methyl ß-lactoside methanol solvate, (II), during air-drying. Cremer-Pople puckering parameters indicate that the ß-D-Galp (ß-D-galactopyranosyl) and ß-D-Glcp (ß-D-glucopyranosyl) rings in (I) adopt slightly distorted 4C1 chair conformations, with the former distorted towards a boat form (BC1,C4) and the latter towards a twist-boat form (O5SC2). Puckering parameters for (I) and (II) indicate that the conformation of the ßGalp ring is slightly more affected than the ßGlcp ring by the solvomorphism. Conformations of the terminal O-glycosidic linkages in (I) and (II) are virtually identical, whereas those of the internal O-glycosidic linkage show torsion-angle changes of 6° in both C-O bonds. The exocyclic hydroxymethyl group in the ßGalp residue adopts a gt conformation (C4' anti to O6') in both (I) and (II), whereas that in the ßGlcp residue adopts a gg (gauche-gauche) conformation (H5 anti to O6) in (II) and a gt (gauche-trans) conformation (C4 anti to O6) in (I). The latter conformational change is critical to the solvomorphism in that it allows water to participate in three hydrogen bonds in (I) as opposed to only two hydrogen bonds in (II), potentially producing a more energetically stable structure for (I) than for (II). Visual inspection of the crystalline lattice of (II) reveals channels in which methanol solvent resides and through which solvent might exchange during solvomorphism. These channels are less apparent in the crystalline lattice of (I).

One-pot lipase-catalyzed esterification of ε-caprolactone with methyl-d-glucopyranoside and its elongation with free 6-hydroxyhexanoate monomer units.

One-pot synthesis of sugar-functionalized oligomeric caprolactone was carried out by lipase-catalyzed esterification of ε-caprolactone (ECL) with methyl-d-glucopyranoside (MGP) followed by the elongation of functionalized oligomer chain. Functionalization was performed in a custom-fabricated glass reactor equipped with Rushton turbine impeller and controlled temperature at 60 °C using tert-butanol as reaction medium. The overall reaction steps include MGP esterification of ECL monomer and its subsequent elongation by free 6-hydroxyhexanoate monomer units. A ping-pong bi-bi mechanism without ternary complex was proposed for esterification of ECL and MGP with apparent values of kinetic constant, namely maximal velocity (Vmax ), Michaelis constant for MGP (KmMGP ), and Michaelis constant for ECL (KmECL ) at 3.848 × 10-3  M H-1 , 8.189 × 10-2  M, and 6.050 M, respectively. Chain propagation step of MGP-functionalized ECL oligomer exhibits the properties of living polymerization mechanism. Linear relationship between conversion (%) and number average molecular weight, Mn (g mol-1 ), of functionalized oligomer was observed. Synthesized functionalized oligomer showed narrow range of molecular weight from 1,400 to 1,600 g mol-1 with more than 90% conversion achieved. Structural analysis confirmed the presence of covalent bond between the hydroxyl group in MGP with carboxyl end group of ECL oligomer.

A Galactoside-Binding Protein Tricked into Binding Unnatural Pyranose Derivatives: 3-Deoxy-3-Methyl-Gulosides Selectively Inhibit Galectin-1.

Galectins are a family of galactoside-recognizing proteins involved in different galectin-subtype-specific inflammatory and tumor-promoting processes, which motivates the development of inhibitors that are more selective galectin inhibitors than natural ligand fragments. Here, we describe the synthesis and evaluation of 3-C-methyl-gulopyranoside derivatives and their evaluation as galectin inhibitors. Methyl 3-deoxy-3-C-(hydroxymethyl)-ß-d-gulopyranoside showed 7-fold better affinity for galectin-1 than the natural monosaccharide fragment analog methyl ß-d-galactopyranoside, as well as a high selectivity over galectin-2, 3, 4, 7, 8, and 9. Derivatization of the 3-C-hydroxymethyl into amides gave gulosides with improved selectivities and affinities; methyl 3-deoxy-3-C-(methyl-2,3,4,5,6-pentafluorobenzamide)-ß-d-gulopyranoside had Kd 700 µM for galectin-1, while not binding any other galectin.

Synthesis of nortropane alkaloid calystegine B2 from methyl α-d-xylopyranoside.

A new synthetic route for formation of a central cycloheptanone intermediate leading to the nortropane alkaloid calystegine B2 is described. The approach installs the desired ketone functionality directly in a ring-closing metathesis step. The target compound was prepared over 10 steps from commercially available methyl α-d-xylopyranoside.

Vibrational normal modes calculation in the crystalline state of methylated monosaccharides: Anomers of the methyl-D-glucopyranoside and methyl-D-xylopyranoside molecules.

A structural investigation of the organic molecules is being carried out using vibrational spectroscopy. In this study, normal co-ordinate calculations of anomers of the methyl-D-glucopyranoside and methyl-ß-D-xylopyranoside in the crystalline state have been performed using the modified Urey-Bradley-Shimanouchi force field (mUBSFF) combined with an intermolecular potential energy function. The latter includes Van der Waals interactions, electrostatic terms, and explicit hydrogen bond functions. The vibrational spectra of the compounds recorded in the crystalline state, in the 4000-500 cm(-1) spectral region for the IR spectra, and in the 4000-20 cm(-1) spectral range for the Raman spectra are presented. After their careful examination, several differences in the intensities and frequency shifts have been observed. The theoretical spectra have been obtained after a tedious refinement of the force constants. Thus, on the basis of the obtained potential distribution, each observed band in IR and in Raman has been assigned to a vibrational mode. The obtained results are indeed in agreement with those observed experimentally and thus confirm the previous assignments made for the methyl-α and ß-D-glucopyranoside, as well as for the methyl-ß-D-xylopyranoside.

Direct assay of iduronate-2-sulfatase for Hunter disease using UPLC-tandem mass spectrometry and fluorogenic substrate.

OBJECTIVE: We devised iduronate-2-sulfatase (IDS) enzyme activity assays by combining fluorometric substrate and LC-MS/MS based detection. DESIGN AND METHODS: 4-Methylumbelliferyl α-L-idopyranosiduronic acid 2-sulfate (IDS-S) was used as a substrate for IDS. Its enzymatic product, 4-methylumbelliferyl α-L-idopyranosiduronic acid (IDS-P) and internal standard, 4-methylumbelliferyl α-L-idopyranoside (IDS-IS), were directly measured by UPLC-MS/MS. We determined the precision of our enzyme assay and the effects of sample amounts and incubation time based on the results. Dried blood spots (DBSs) of 110 normal newborns and three patients with Hunter disease were analyzed. RESULTS: IDS-IS, IDS-P and IDS-S were fully separated using UPLC without any ion suppressions. The intra- and inter-assay precisions were 8.5-10.5% and 11.9-15.3%, respectively. The amount of product obtained was proportional to the number of DBSs and increased linearly with the incubation period from 0 to 15 h. The enzyme activities in DBSs from three patients with MPS II were markedly lower than those in the DBSs of 110 normal newborns. CONCLUSION: To the best of our knowledge, this is the first report describing the use of LC-MS/MS for the diagnosis of Hunter disease with a commercially available substrate. Our method would be a rapid and effective screening tool for the diagnosis of Hunter disease with further study.

Conformational interchange of a carbohydrate by mechanical compression at the air-water interface.

Methyl xylopyranoside containing three 4-(pyrene-1-yl)benzoyl groups (PyXy) undergoes conformational interchange within a Langmuir monolayer upon mechanical compression. This xylose-type molecular machine PyXy was immobilized within two different matrix lipids, methyl stearate and methyl 2,3,4-tri-O-stearoyl-ß-D-xylopyranoside, which respectively form rigid and soft monolayers. Structural properties of the monolayer were characterized by assessing the compressibility, compression modulus, and ideal limiting molecular area of PyXy, all of which were estimated from the π-A isotherm measurements. Only the rigid monolayer exhibited a transition to the condensed phase with a limiting molecular area of PyXy smaller than that of the cross-sectional area of the xylopyranose ring in its C1 chair conformation. This suggests conformational interchange of PyXy from the most stable (4)C1 (C1) form to the metastable (1)C4 (1C) form. Surface-reflective fluorescence spectroscopy of the monolayer was applied to detect excimer emission resulting from the face-to-face dimerization of pyrenes attached at the O-2 and O-4 positions of xylose. Fluorescence intensity of the excimer increased abruptly in the condensed region only when the rigid monolayer was applied. These results indicate that the rigidity of the matrix monolayer is a critical aspect of the precise manipulation of molecular machines at interfaces. Consequently, this study demonstrates that including a molecular machine into a rigid lipid matrix is a promising means for the preparation of a novel nanoassembly with dynamic functionalities variable depending on a mechanical stimulus.

Isolation and characterization of the microflora of nixtamalized corn masa.

Corn tortillas are a staple in the diet among the Mexican population, and are traditionally produced through a process known as nixtamalization. This traditional process involves steeping whole-kernel corn in an alkaline solution overnight and then grinding the corn into dough (masa), which is then baked. While the masa is held before baking, significant microbial change can occur which leads to fermentation and spoilage. The objective of this research was to characterize and identify the microflora of nixtamalized corn masa from six different commercial tortilla mills throughout Guadalajara, Mexico. The identification of samples was conducted using the microbial identification system (MIS), which analyzes cellular fatty acids via gas chromatography to identify bacterial species. Lactic acid bacteria and aerobic mesophiles were the predominant organisms, with both groups having counts ranging from 10(4) to 10(7)cfu/g across all mills. Coliform populations were observed at counts of 10(2) to 10(3)cfu/g, while yeast and mold counts were typically less than 10(1)cfu/g. Some mills showed no presence of coliforms or yeast or mold. Streptococcus bovis and Lactobacillus oris were isolated from all mills, and were the most prevalent organisms representing 43% and 17% of all lactic acid bacteria isolated, respectively. S. bovis was also isolated on the aerobic tryptic soy plates and was the most prevalent species representing 19% of the total organisms from these aerobic plates.

Carbohydrate-lipid interactions: affinities of methylmannose polysaccharides for lipids in aqueous solution.

The interactions between 3-O-methyl-mannose polysaccharides (MMPs), extracted from Mycobacterium smegmatis (consisting of a mixture of MMP-10, -11, -12 and -13) or obtained by chemical synthesis (MMP-5(s), -8(s), -11(s) and -14(s)), and linear saturated and unsaturated fatty acids (FAs), and a commercial mixture of naphthenic acids (NAs) in aqueous solution at 25 °C and pH 8.5 were quantified by electrospray ionization mass spectrometry (ESI-MS). Association constants (K(a)) for MMP binding to four FAs (myristic acid, palmitic acid, stearic acid and trans-parinaric acid) were measured by using an indirect ESI-MS assay, the "proxy protein" method. The K(a) values are in the 10(4)-10(5) M(-1) range and, based on results obtained for the binding of the synthetic MMPs with palmitic acid, increase with the size of the carbohydrate. Notably, the measured affinity of the extracted MMPs for trans-parinaric acid is two orders of magnitude smaller than the reported value, which was determined by using a fluorescence assay. Using a newly developed competitive binding assay, referred to as the "proxy protein/proxy ligand" ESI-MS method, it was shown that MMPs bind specifically to NAs in aqueous solution, with apparent affinities of approximately (5×10(4)) M(-1) for the mixture of NAs tested. This represents the first demonstration that MMPs can bind to hydrophobic species more complex than those containing linear alkyl/alkenyl chains. Moreover, the approach developed here represents a novel method for probing carbohydrate-lipid interactions.

Too low to kill: concentration of the secondary metabolite ranunculin in buttercup pollen does not affect bee larval survival.

Growing evidence suggests that the freely accessible pollen of some plants is chemically protected against pollen-feeding flower visitors. For example, a diet of pollen from buttercup plants (Ranunculus) recently was shown to have a deleterious effect on developing larvae of several bee species not specialized on Ranunculus. Numerous Ranunculus species contain ranunculin, the glucosyl hydrate form of the highly reactive and toxic lactone protoanemonin, that causes the toxicity of these plants. We tested whether the presence of ranunculin is responsible for the lethal effects of R. acris pollen on the larvae of two bee species that are not Ranunculus specialists. To investigate the effect on bee larval development, we added ranunculin to the pollen provisions of the Campanula specialist bee Chelostoma rapunculi and the Asteraceae specialist bee Heriades truncorum, and allowed the larvae to feed on these provisions. We quantified ranunculin in pollen of R. acris and in brood cell provisions collected by the Ranunculus specialist bee Chelostoma florisomne. We demonstrated that although ranunculin was lethal to both tested bee species in high concentrations, the concentration in the pollen of R. acris was at least fourfold lower than that tolerated by the larvae of C. rapunculi and H. truncorum in the feeding experiments. Ranunculin concentration in the brood cells of C. florisomne was on average even twentyfold lower than that in Ranunculus pollen, suggesting that a mechanism different from ranunculin intoxication accounts for the larval mortality reported for bees not specialized on Ranunculus pollen.

Tetraisopropyldisiloxane-1,3-diyl as a versatile protecting group for pentopyranosides.

The protecting group tetraisopropyldisiloxane-1,3-yl has been investigated for simultaneous protection of two hydroxyls on pentopyranosides. Methyl α-D-xylopyranoside is protected in excellent regioselectivity and high yield to form the 2,3-protected xylopyranoside whereas methyl ß-D-xylopyranoside gives the 3,4-protected product also with excellent regioselectivity.

Methyl beta-D-fructopyranoside.

In methyl beta-D-fructopyranoside, C(7)H(14)O(6), the thermodynamically most stable methyl glycoside of the ketose D-fructose, the pyranose ring is close to being an ideal (2)C(5) chair. The compound forms bilayers involving a complex hydrogen-bonding pattern of five independent hydrogen bonds. Graph-set analysis was applied to distinguish the hydrogen-bond patterns at unary and higher level graph sets.

Methyl 4-O-beta-D-galactopyranosyl alpha-D-mannopyranoside methanol 0.375-solvate.

Methyl beta-D-galactopyranosyl-(1-->4)-alpha-D-mannopyranoside methanol 0.375-solvate, C(13)H(24)O(11).0.375CH(3)OH, (I), was crystallized from a methanol-ethanol solvent system in a glycosidic linkage conformation, with varphi' (O5(Gal)-C1(Gal)-O1(Gal)-C4(Man)) = -68.2 (3) degrees and psi' (C1(Gal)-O1(Gal)-C4(Man)-C5(Man)) = -123.9 (2) degrees , where the ring is defined by atoms O5/C1-C5 (monosaccharide numbering); C1 denotes the anomeric C atom and C6 the exocyclic hydroxymethyl C atom in the betaGalp and alphaManp residues, respectively. The linkage conformation in (I) differs from that in crystalline methyl alpha-lactoside [methyl beta-D-galactopyranosyl-(1-->4)-alpha-D-glucopyranoside], (II) [Pan, Noll & Serianni (2005). Acta Cryst. C61, o674-o677], where varphi' is -93.6 degrees and psi' is -144.8 degrees . An intermolecular hydrogen bond exists between O3(Man) and O5(Gal) in (I), similar to that between O3(Glc) and O5(Gal) in (II). The structures of (I) and (II) are also compared with those of their constituent residues, viz. methyl alpha-D-mannopyranoside, methyl alpha-D-glucopyranoside and methyl beta-D-galactopyranoside, revealing significant differences in the Cremer-Pople puckering parameters, exocyclic hydroxymethyl group conformations and intermolecular hydrogen-bonding patterns.

Methyl beta-allolactoside [methyl beta-D-galactopyranosyl-(1-->6)-beta-D-glucopyranoside] monohydrate.

Methyl beta-allolactoside [methyl beta-D-galactopyranosyl-(1-->6)-beta-D-glucopyranoside], (II), was crystallized from water as a monohydrate, C(13)H(24)O(11).H(2)O. The betaGalp and betaGlcp residues in (II) assume distorted (4)C(1) chair conformations, with the former more distorted than the latter. Linkage conformation is characterized by phi' (C2(Gal)-C1(Gal)-O1(Gal)-C6(Glc)), psi' (C1(Gal)-O1(Gal)-C6(Glc)-C5(Glc)) and omega (C4(Glc)-C5(Glc)-C6(Glc)-O1(Gal)) torsion angles of 172.9 (2), -117.9 (3) and -176.2 (2) degrees , respectively. The psi' and omega values differ significantly from those found in the crystal structure of beta-gentiobiose, (III) [Rohrer et al. (1980). Acta Cryst. B36, 650-654]. Structural comparisons of (II) with related disaccharides bound to a mutant beta-galactosidase reveal significant differences in hydroxymethyl conformation and in the degree of ring distortion of the betaGlcp residue. Structural comparisons of (II) with a DFT-optimized structure, (II(C)), suggest a link between hydrogen bonding, pyranosyl ring deformation and linkage conformation.

The major surface carbohydrates of the Echinococcus granulosus cyst: mucin-type O-glycans decorated by novel galactose-based structures.

The cestodes constitute important but understudied human and veterinary parasites. Their surfaces are rich in carbohydrates, on which very little structural information is available. The tissue-dwelling larva (hydatid cyst) of the cestode Echinococcus granulosus is outwardly protected by a massive layer of carbohydrate-rich extracellular matrix, termed the laminated layer. The monosaccharide composition of this layer suggests that its major carbohydrate components are exclusively mucin-type O-glycans. We have purified these glycans after their release from the crude laminated layer and obtained by MS and NMR the complete structure of 10 of the most abundant components. The structures, between two and six residues in length, encompass a limited number of biosynthetic motifs. The mucin cores 1 and 2 are either nondecorated or elongated by a chain of Galpbeta1-3 residues. This chain can be capped by a single Galpalpha1-4 residue, such capping becoming more dominant with increasing chain size. In addition, the core 2 N-acetylglucosamine residue is in cases substituted with the disaccharide Galpalpha1-4Galpbeta1-4, giving rise to the blood P(1)-antigen motif. Larger, also related, glycans exist, reaching at least 18 residues in size. The glycans described are related but larger than those previously described from an Echinococcus multilocularis mucin [Hulsmeier, A. J., et al. (2002) J. Biol. Chem. 277, 5742-5748]. Our results reveal that the E. granulosus cyst exposes to the host only a few different major carbohydrate motifs. These motifs are composed essentially of galactose units and include the elongation by (Galpbeta1-3)(n) and the capping by Galpalpha1-4, novel in animal mucin-type O-glycans.

The role of sugar configuration in the acetolysis of 6-deoxyhexose methyl glycosides.

The acetolysis of several perbenzylated 6-deoxyhexose methyl glycosides under two mild conditions (10 equiv ZnCl(2) in 2:1 v/v Ac(2)O-AcOH at 5 degrees C; 10:10:1 v/v/v Ac(2)O-AcOH-TFA at 70 degrees C) was studied. We focused on the effect of sugar configuration on the competition between mechanisms with activation at exocyclic or endocyclic oxygen site. No effect was detected in acetolysis using the TFA protocol promoting an exo-activation mechanism, which affords 1-O-Ac-pyranosides regardless of sugar configuration. On the contrary, it has a primary role in determining the endo- versus exo-product distribution on ZnCl(2)-promoted acetolysis.

Efficient synthesis of D-xylo and D-ribo-phytosphingosines from methyl 2-amino-2-deoxy-beta-D-hexopyranosides.

A general and flexible synthetic approach to biologically important 5,6-unsaturated C(18)-phytosphingosines was developed via olefin cross-metathesis employing truncated C(6)-phytosphingosines as the key intermediates. These were efficiently prepared in high yields by zinc-mediated reductive opening of methyl 2-amino-2-deoxy-beta-hexopyranosides. [structure: see text]

Synthesis of and molecular dynamics simulations on a tetrasaccharide corresponding to the repeating unit of the capsular polysaccharide from Salmonella enteritidis.

Syntheses of two oligosaccharides as methyl glycosides related to the repeating unit of S. enteritidis capsular polysaccharide (CPS) are presented. The trisaccharide corresponds to the backbone of the CPS whereas the tetrasaccharide is a model for the repeating unit which has a branched structure. Molecular dynamics simulations investigating their flexibility and dynamics revealed that the oligosaccharides populate several conformational states and indicate that conformational averaging should be used in describing the accessible conformational space.

The effects of ascorbic acid on homolytic processes involving alpha-hydroxyl-containing carbon-centered radicals.

Effects of ascorbic acid and 5,6-O-isopropylidene-2,3-O-dimethylascorbic acid on final product formation in radiolysis of ethanol, aqueous solutions of ethanol, ethylene glycol, alpha-methylglycoside, maltose, alpha-glycerophosphate, and alpha-glucose phosphate were studied. It was found that ascorbic acid is able to suppress reactions involving various alpha-hydroxyl-containing carbon-centered radicals and depending on the experimental conditions can either oxidize or reduce alpha-hydroxyethyl radicals.

DFT and NMR studies of 2JCOH, 3JHCOH, and 3JCCOH spin-couplings in saccharides: C-O torsional bias and H-bonding in aqueous solution.

Density functional theory (DFT) has been used to investigate the structural dependencies of NMR spin-coupling constants (J-couplings) involving the exchangeable hydroxyl protons of saccharides. 3JHCOH, 3JCCOH, and 2JCOH values were calculated at different positions in model aldopyranosyl rings as a function of one or more torsion angles, and results support the use of a generalized Karplus equation to treat 3JHCOH involving the non-anomeric OH groups. The presence of O5 appended to the H1-C1-O1-H coupling pathway introduces asymmetry in 3JH1,O1H Karplus curves due to internal electronegative substituent effects on the gauche couplings, thus requiring separate equations to treat this coupling. 3JCCOH values depend not only on the C-C-O-H torsion angle but also on the orientation of terminal substituents on the coupled carbon, similar to 3JCOCC studied previously (Bose et al., J. Am. Chem. Soc. 1998, 120, 11158-11173). "In-plane" oxygen increased 3JCCOH by approximately 3-4 Hz, whereas "in-plane" carbon gave more modest enhancements ( approximately 1 Hz). Three Karplus equations were derived for non-anomeric 3JCCOH based on the nature and orientation of substituents on the coupled carbon. Like 3JH1,O1H, 3JC2,O1H is also subject to internal electronegative substituent effects on the gauche couplings, thus necessitating separate equations to treat this coupling. 2JCOH values were found not to be useful probes of C-O torsions as a result of their nonsystematic dependence on these torsions. Experimental measurements of 3JHCOH and 3JCCOH in doubly 13C-labeled methyl beta-lactoside 20 and its constituent 13C-labeled methyl aldopyranosides in H2O/acetone-d6 at -20 degrees C showed that some C-O torsion angles are influenced by molecular context and do not experience complete rotational averaging in solution. A strong bias in the H3-C3-O3-H torsion angle in the Glc residue of 20 favoring a gauche conformation suggests the presence of inter-residue H-bonding between O3HGlc and O5Gal. Quantitative analysis of 3JHCOH and 3JCCOH values in 20 indicates that approximately 85% of the forms in solution have geometries consistent with H-bonding. These results suggest that H-bonding between adjacent and/or remote residues may play a role in dictating preferred glycosidic bond conformation in simple and complex oligosaccharides in aqueous solution.