Anomeric Forms Research Articles

On the electrochemical reactivity of anomers: electrocatalytic oxidation of α- and β- d-glucose on platinum electrodes in acid and basic media

Polarimetric studies of d-glucose solutions were carried out at low temperature (2°C). This procedure allowed us to stabilize the solutions of d-glucose and to differentiate the behaviour of the two main dissolved anomeric forms (α or β). In particular, the dependence of their stability on the pH of the electrolytic solutions was followed and their electrochemical reactivity was tested. Using cyclic voltammetry it was possible to demonstrate that, of the two possible pyranic forms of d-glucose, only β- d-glucose exhibited an appreciable reactivity at platinum electrodes, i.e. α- d-glucose was almost unreactive at low temperature. This difference in reactivity between anomers has never been shown clearly in any of the previous electrochemical studies of glucose. Long-term electrolysis was also performed. Ionic chromatography as well as in situ IR spectroscopic experiments allowed us to conclude that the oxidation of d-glucose in basic media at low temperature leads selectively to formation of the δ-gluconolactone, which later undergoes hydrolysis forming d-gluconate as a final product. Mechanisms are proposed and discussed.

Reaction mechanism of chitosanase from Streptomyces sp. N174.

Chitosanase was produced by the strain of Streptomyces lividans TK24 bearing the csn gene from Streptomyces sp. N174, and purified by S-Sepharose and Bio-Gel A column chromatography. Partially (25-35%) N-acetylated chitosan was digested by the purified chitosanase, and structures of the products were analysed by NMR spectroscopy. The chitosanase produced heterooligosaccharides consisting of D-GlcN and GlcNAc in addition to glucosamine oligosaccharides [(GlcN)n, n = 1, 2 and 3]. The reducing- and non-reducing-end residues of the heterooligosaccharide products were GlcNAc and GlcN respectively, indicating that the chitosanase can split the GlcNAc-GlcN linkage in addition to that of GlcN-GlcN. Time-dependent 1H-NMR spectra showing hydrolysis of (GlcN)6 by the chitosanase were obtained in order to determine the anomeric form of the reaction products. The chitosanase was found to produce only the alpha-form; therefore it is an inverting enzyme. Separation and quantification of (GlcN)n was achieved by HPLC, and the time course of the reaction catalysed by the chitosanase was studied using (GlcN)n (n = 4, 5 and 6) as the substrate. The chitosanase hydrolysed (GlcN)6 in an endo-splitting manner producing (GlcN)2, (GlcN)3 and (GlcN)4, and did not catalyse transglycosylation. Product distribution was (GlcN)3 >> (GlcN)2 > (GlcN)4. Cleavage to (GlcN)3 + (GlcN)3 predominated over that to (GlcN)2 + (GlcN)4. Time courses showed a decrease in rate of substrate degradation from (GlcN)6 to (GlcN)5 to (GlcN)4. It is most likely that the substrate-binding cleft of the chitosanase can accommodate at least six GlcN residues, and that the cleavage point is located at the midpoint of the binding cleft.

Synthesis of cyclic and acyclic nucleoside analogues having a thiophene or dihydrothiophene ring fused to the d side of an uracil

AbstractSynthesis of cyclic and acyclic nucleosides was achieved by alkylation of various new aglycones following the Vorbrüggen and Niedballa's procedure [6]. The analytical results allowed us to conclude that the alkylation occured on N1 site and led to the β anomeric form for the cyclic nucleosides.

Rapid gas-chromatographic assay of lactulose and mannitol for estimating intestinal permeability

We developed a gas-chromatographic method to determine urinary mannitol and lactulose. The procedure for purification of urine by a resin was optimized for purification of analytes and high recovery; the aliquot of resin chosen (500 mg) was kept in contact with the urine for 1 min. The recoveries of mannitol and lactulose were > 85% at concentrations that include both normal and pathological values. Sugars were converted to oximes before the silylation step to avoid multiple peaks for the anomeric forms. The calibration was linear over the range 0.1-1 microgram of sugar injected. Analytical recovery of the sugars ranged from 90% to 95.3% for mannitol and from 90.4% to 95.8% for lactulose. The mean within-day imprecision (CV) was 6.2% for mannitol and 4.7% for lactulose; the between-day CV was 6.7% for mannitol and 5.1% for lactulose. A lactulose/mannitol ratio of 0.035 completely differentiated 28 normal children and 28 children with active gluten-sensitive enteropathy, whose mean ratios were 0.022 (SD 0.007) and 0.084 (SD 0.054), respectively.

A New Monoclonal Antibody (A78-G/A7) to the Thomsen—Friedenreich Pan-Tumor Antigen

A new monoclonal antibody to the Thomsen-Friedenreich (TF) antigen (or, more precisely, epitope; Gal beta 1-3GalNAc-) has been developed that is specific for both anomeric forms of this disaccharide (TF alpha and TF beta, including related structures on glycolipids), and not assay restricted. We demonstrate that this avid antibody (A78-G/A7) is well suited for immunohistochemistry on paraffin-embedded and cryosectioned tissues, immunoblotting, ELISA techniques, and hemagglutination. Immunohistochemistry on paraffin sections does not require proteolytic or microwave pretreatment. The binding characteristics of this antibody are largely independent of variations in pH (6.0-8.2) and temperature (4-37 degrees C). Immunoblotting with KG-1 (human acute myelogenous leukemia) cells revealed a series of TF-active glycoproteins with a main band at about 155 kDa. Immunoprecipitation was performed using a new technique applicable to IgM-type antibodies.

Comparative Biochemistry of Chitinases—Anomeric Form of the Reaction Products

Anomeric form of the products from chitinase-catalyzed hydrolysis was analyzed by 1H-NMR spectroscopy. When the chitinase from Streptomyces griseus was added to the solution of substrate, N,N,N,N,N-pentaacetylchitopentaitol, β-anomer was first produced by the enzymatic hydrolysis, and then transformed to α-anomer by mutarotation. In contrast, the chitinase from Dioscorea opposita (yam) produced only α-anomer, which was transformed to β-anomer by mutarotation.

Anomeric Dependence of Fluorodeoxyglucose Transport in Human Erythrocytes

The transport of several n-fluoro-n-deoxy-D-glucose derivatives across the human erythrocyte membrane has been studied under equilibrium exchange conditions using one- and two-dimensional nuclear magnetic resonance (NMR) techniques. This approach is based on the intracellular 19F shift, which was found to depend on the anomeric form and on the F/OH substitution position. Since the transport behavior of both glucose anomers can be followed simultaneously, this approach is particularly sensitive to differences in anomeric permeability. For 2-, 3-, 4-, and 6-fluorodeoxyglucose analogs, the alpha anomers permeate more rapidly, and the P alpha/P beta ratio is dependent on the position of fluorination, with values of 1.1, 1.3, 2.5, and 1.6, respectively, obtained at 37 degrees C. These results have been analyzed in terms of a simple alternating conformation model for the glucose transporter. Although mutarotase activity has been reported for red cells, mutarotation behavior for all anomers was found to be completely negligible on the transport and spin-lattice relaxation time scales. Metabolic transformation of the fluorinated glucose analogs, primarily to fluorinated gluconate and sorbitol analogs, is very slow and does not significantly interfere with the transport measurements. A mean ratio of 2.6 was found for the extracellular/intracellular fluorine spin-lattice relaxation rates.

A Method for Obtaining 13C Isotopomer Populations in 13C-Enriched Glucose

13C NMR analysis of 13C-enriched glucose containing multiple isotopomers is hampered by chemical shift similarities of several carbon resonances and by the presence of two anomeric forms. A convenient and quantitative method of enzymatically oxidizing glucose to gluconate in tissue and perfusate extracts is presented. The six carbon resonances of the resulting 13C-enriched gluconate are fully resolved at high pH, thereby allowing a determination of the fractional population of each 13C isotopomer by 13C NMR. The utility of this method is demonstrated using the effluent from an isolated perfused liver containing 13C-enriched glucose produced by hepatic metabolism of sodium [1,2,3-13C3]propionate via the citric acid cycle and gluconeogenesis. An analysis of the gluconate C2 and C5 resonances in this sample showed that pentose phosphate activity was insignificant during this perfusion protocol. As demonstrated, this method provides a means of fully describing 13C isotopomer populations in enriched glucose samples where isotope may be derived from multiple metabolic pathways, thus expanding the scope of experimental design and enrichment strategies.

The structure and (local) stability constants of borate esters of mono- and di-saccharides as studied by 11B and 13C NMR spectroscopy

The formation of borate esters of various mono- and di-saccharides in aqueous solution was studied by 11B and 13C NMR spectroscopy. Association constants K(B −L) at a carbohydrate-borate molar ratio of 1:1, pH 7, and 25°C were determined and compared with literature values obtained from potentiometry. The association constants K(B −L) were converted into local association constants K loc(B −L) by using the distribution of the various anomeric forms in D 2O. In this way, values of K loc(B −L) were obtained, which appear to be characteristic of the configuration concerned. They explain the favourable effect of borate in the alkaline isomerisation of lactose into lactulose. At a low molar ratio (1:3) of carbohydrate-borate, predominantly diborate esters (B −) 2L were formed.

Global and internal molecular dynamics of poly(acrylamide-co-allyl 2-acetamido-2-deoxy-D-glucopyranoside) glycopolymers from 13C NMR relaxation studies

13C spin-lattice and spin–spin relaxation times and nuclear Overhauser enhancements have been used to examine the molecular dynamics of the α- (1) and β- (2) anomeric forms of poly(acrylamide-co-allyl 2-acetamido-2-deoxy-D-glucopyranoside) glycopolymers. The timescale of motions and the spatial restriction of these motions were determined by using various forms of the "model-free" approach. It is shown that the motions of the C—H vectors of the polymer backbone may be described by a scaled Lorentzian spectral density function, giving rise to an effective correlation time for overall tumbling. The temperature dependence of this correlation time suggests that the overall motion is dependent on viscosity. The overall motion of the polymer molecules is shown to be anisotropic in nature by including the spin–spin relaxation data in the analysis. The N-acetyl methyl and sugar hydroxymethyl (C6) groups exhibit internal motions. The activation energies associated with these internal motions are derived. The difference in relaxation rates between the α and β anomeric forms, though small, suggests that the motions of the sugar ring may be different for the two systems. This conclusion is supported by potential energy contour map calculations, which indicate that the β anomer (2) has almost twice the conformational flexibility of the α anomer (1).

Nucleic‐Acid Analogues with Constraint Conformational Flexibility in the Sugar‐Phosphate Backbone (‘Bicyclo‐DNA’). Part 1. Preparation of (3S,5′R)‐2′‐Deoxy‐3′,5′‐ethano‐αβ‐D‐ribonucleosides (‘Bicyclonucleosides’)

AbstractWe describe the synthesis of 2′‐deoxy‐3′,5′‐ethano‐D‐ribonucleosides 1–8 (= (5′,8′‐dihydroxy‐2′‐oxabicyclo‐[3.3.0]oct‐3′‐yl)purines or ‐pyrimidines) of the nucleobases adenine, thymine, cytosine, and guanine. They differ from natural 2′‐deoxyribonucleosides only by an additional ethylene bridge between the centers C(3′) and C(5′). The configuration at these centers (3S,5′R) was chosen as to match the geometry of a repeating nucleoside unit in duplex DNA as close as possible. These nucleosides were designed to confer, as constituents of an oligonucleotide chain, a higher degree of preorganization of a single strand for duplex formation with respect to natural DNA, thus leading to an entropic advantage for the pairing process. The synthesis of these ‘bicyclonucleosides’ was achieved by construction of an enantiomerically pure carbohydrate precursor 18/19 (Schemes 1), which was then converted to the corresponding nucleosides by known methods in nucleoside synthesis (Schemes 2 and 3). In all cases, both anomeric forms of the nucleosides were obtained in pure crystalline form, the relative configuration of which was established by 1H‐NMR‐NOE spectroscopy. A conformational analysis of the nucleosides with β‐configuration at the anomeric center by means of X‐ray and 1H‐NMR (including NOE) spectroscopy show the furanose part of the molecules to adopt uniformly a 1′exo‐conformation with the base substituents preferentially in the anti‐range in the pyrimidine nucleosides (anti/syn ca. 2:1) distribution in the purine nucleosides (in solution).

Gas Chromatographic determination of sugars and polyalcohols in apple liqueurs

This paper proposes a gas-chromatographic method to determine sugars and polyalcohols in apple liqueurs. The samples were prepared previously and the sugars determined as trimethylsilyl derivates. This technique enables isolation of theα andβ anomeric forms of fructose, glucose and maltose. The precision and accuracy of the method has been verified. The liqueurs analysed are sweet liqueurs, as can be deduced from their high sugar content (over 100 g/L). Reducing sugars (glucose and fructose) were predominant.

Evidence for molybdate complexes of ketoses and aldosesin the furanose form: a 13C and 1H NMR study

The formation of dimolybdate complexes of ketoses of the ribo and lyxo series and of aldoses of the lyxo series was investigated by 13C and 1H NMR spectroscopy. All these sugars possess two cis hydroxyl groups adjacent to the anomeric centre and form two different series of complexes involving the furanose forms. The tridentate complexes of 2-ketoses involve the ring HO-3,4 and the cis exocyclic HO-1 of the β ( ribo series) or of the α ( lyxo series) anomer, whereas the trans anomeric HO-2 is not bound to molybdenum. In the lyxo series, the β anomers form tetradentate complexes involving HO-1,2,3,5 (aldoses) or HO-2,3,4,6 (ketoses), that appear similar to a β- d-lyxofuranose complex already characterized in the solid state. A mixture of tridentate and tetradentate species was obtained in the case of d-tagatose. The relative stabilities of the complexes are discussed.

1-N-glycyl beta-oligosaccharide derivatives as stable intermediates for the formation of glycoconjugate probes.

Incubation of reducing sugars in ammonium bicarbonate was found to be a simple procedure for the formation of beta-D-glycosylamines of purified complex oligosaccharides in 70-80% yield. These provide valuable intermediates for the synthesis of a wide range of oligosaccharide probes and derivatives by acylation of the 1-amino function. The 1-amino function showed different rates of reactivity with different reagents. In general, interactions with large ring systems such as the fluorophores dansyl chloride and carboxyfluorescein gave 10-20% yields of products, which consisted of mixtures of both anomeric forms, whereas smaller acylating reagents gave near-quantitative yields of the desired beta-D-derivatives. Steric effects may explain differences in reactivity. N-Chloroacetamido derivatives could be obtained in high yield with retention of the beta-anomeric configuration. Subsequent ammonolysis of the chloroacetamido function afforded the corresponding N-glycyl beta-derivatives. The linker thereby introduced retains the amino function, possesses the useful properties of fixed anomeric configuration, improved stability, and uniform reactivity with a variety of reagents, and is structurally analogous to an asparagine side chain. The potential therefore exists for the generation of oligosaccharide derivatives tailored for different applications.

Specificity mapping of cellulolytic enzymes: classification into families of structurally related proteins confirmed by biochemical analysis.

The specificities of 15 cellulolytic enzymes have been examined using chromophoric glycosides derived from D-glucose, cellobiose, higher cellooligosaccharides, lactose, D-xylose, and beta-(1,4)-xylobiose. Coinciding with a classification based on hydrophobic cluster analysis of amino acid sequences, six families each showing a characteristic specificity pattern were observed. Furthermore, in these cases where the anomeric forms of reaction products were determined, results seem to indicate conservation of intrinsic reaction mechanism (single or double displacement) within each family. On the other hand, the low molecular weight substrates do not discriminate exo- from endocellulases. This functional differentiation is speculated to originate from the presence, in exoenzymes, of a tunnel-shaped active site formed by extra loops in their structure.

Synthesis of methyl alpha- and beta-N-dansyl-D-galactosaminides, probes for the combining sites of N-acetyl-D-galactosamine-specific lectins.

The synthesis of the methyl alpha- and beta-N-dansyl-D-galactosaminides is described using methyl alpha,beta-2-azido-2-deoxy-D-galactopyranoside as starting material. This was reduced to the corresponding methyl alpha,beta-2-amino-2-deoxy-D-galactopyranoside and then treated with dansyl chloride to yield a mixture of methyl alpha,beta-N-dansyl-D-galactosaminides which was separated into individual anomeric forms by flash chromatography on silica gel. Methyl alpha-N-dansyl-D-galactosaminide was used as a fluorescent indicator ligand in continuous substitution titrations to determine the association constants of nonchromophoric carbohydrates with the N-acetyl-D-galactosamine specific lectin from Erythrina corallodendron.

Conformational analysis of the anomeric forms of sophorose, laminarabiose, and cellobiose using MM3

Relaxed-residue energy maps based on the MM3 force-field were computed for relative orientations of the pyranosyl rings of sophorose, laminarabiose, and cellobiose, respectively the (1 → 2)-β-; (1 → 3)-β-; and (1 → 4)-β-linked- d-glucosyl disaccharides. Sixteen starting conformations of the rotatable exocyclic side-groups were considered for each molecule. All of the energy surfaces have two intersecting low-energy troughs and illustrate the importance of exo-anomeric effects in determining disaccharide conformation. Local minima were found by relaxed minimization without restriction. The energy surfaces of these disaccharides are very similar to the energy surfaces of their corresponding 6-methyl-tetrahydropyran analogues. There is good agreement between disaccharide structures having minimal MM3 energy and those found by crystallography.

Biological activity of synthetic phosphonooxyethyl analogs of lipid A and lipid A partial structures.

We investigated the biological activity of four new synthetic analogs of lipid A, termed PE-1, PE-2, PE-3, and PE-4. All compounds contain an alpha-oxyethyl-linked (-O-CH2-CH2-) phosphoryl group in position 1 of the reducing glucosaminyl residue (GlcN I) of lipid A. PE-1 is a hexaacylated analog of Escherichia coli lipid A (compound 506). PE-2 differs from PE-1 in carrying two myristic acid residues at GlcN I. PE-3 has the same acylation pattern as PE-2, but GlcN I is present in the beta anomeric form. Finally, PE-4 represents an analog of tetraacyl precursor Ia (compound 406). Structure-activity relationships of these compounds were determined by measuring their capacity to induce tumor necrosis factor alpha, interleukin 1, and interleukin 6 release by human mononuclear cells and to cause mitogenicity of murine spleen cells. The results show that replacement of the glycosidic phosphoryl residue by a phosphonooxyethyl group had no substantial effect on the biological activity of compounds. However, the anomeric configuration of GlcN I was found to be of great biological relevance, as, in general, the alpha anomer (PE-2) expressed high activity, and the beta anomer (PE-3) expressed low mediator-inducing and mitogenic activity. The absence of the 3-hydroxyl groups within the acyl residues at GlcN I in PE-2 was found to only slightly affect the induction of monokines in human mononuclear cells compared with that of PE-1 or lipid A (506). These stable 1-phosphonooxyethyl analogs of lipid A may be candidates in the development of immunomodulators for the treatment of systemic endotoxicosis.

Conformational analysis of the anomeric forms of kojibiose, nigerose, and maltose using MM3

Energy surfaces were computed for relative orientations of the relaxed pyranosyl rings of the two anomeric forms of kojibiose, nigerose, and maltose, the (1 → 2)-α, (1 → 3)-α- and (1 → 4)-α-linked d-glucosyl disaccharides, respectively. Twenty-four combinations of starting conformations of the rotatable side-groups were considered for each disaccharide. Optimized structures were calculated using MM3 on a 20° grid spacing of the torsional angles about the glycosidic bonds. The energy surfaces of the six disaccharides were similar in many respects but differed in detail within the low-energy regions. The maps also illustrate the importance of the exo-anomeric effect and linkage type in determining the conformational flexibility of disaccharides. Torsional conformations of known crystal structures of maltosyl-containing molecules lie in a lower MM3 energy range than previously reported.

Simultaneous Determination of Lactulose and Mannitol in Urine of Burn Patients by Gas-Liquid Chromatography

Ratios of lactulose/mannitol excretion in urine have been used to assess the extent of intestinal permeability in various disease and trauma conditions. Reported studies have used this technique to correlate altered gastrointestinal mucosal permeability to translocation of bacteria and endotoxin, leading to occult sepsis in burn patients. Enzymatic methods of analysis for urine concentrations of mannitol and lactulose were used in these studies. We have found that urine from patients with severe burns frequently contains compounds that interfere with the enzymatic methods. We describe using gas-liquid chromatography to determine mannitol and lactulose simultaneously in the urine of burn patients. To avoid the multiple peaks for the anomeric forms of the reducing sugars during precolumn trimethylsilyl derivatization, we converted the sugars to oximes before the silylation step. The method gave good recoveries of mannitol and lactulose added to burn patients' urine samples. Unlike the enzymatic methods, gas-liquid chromatography eliminates the effect of interfering compounds and allows for the simultaneous determination of both sugars in urine samples.