Biosynthèse de cellulose bactérienne à pertir de glycérol sélectivement deutérié en position 1, 2, ou 3: Etude par résonance magnétique nucléaire

Glycerol specifically deuterated at C-1, C-2, or C-3 was prepared and used for the biosynthesis of bacterial cellulose with Acetobacter xylinum.The material obtained were converted into glucitol hexaacetate and analyzed by 250-MHz nuclear magnetic resonance and mass spectrometry. The spectra indicated that the protons of the C-3 position of the starting glycerol were incorporated as substituents of the C-6 and C-1 positions of the cellulose. Similarly, protons of the C-2 and C-5 positions of the cellulose came essentially from water and the protons bonded at the C-3 and C-4 positions of the cellulose from protons bonded to C-1 of the starting glycerol.     

Mode of action on deacetylation of acetylated methyl glycoside by cellulose acetate esterase from Neisseria sicca SB

The regioselective deacetylation of purified cellulose acetate esterase from Neisseria sicca SB was investigated on methyl 2,3,4,6-tetra-O-acetyl-beta-D-glucopyranoside and 2,3,4,6-tetra-O-acetyl-beta-D-galactopyranoside. The substrates were used as model compounds of cellulose acetate in order to estimate the mechanism for deacetylation of cellulose acetate by the enzyme. The enzyme rapidly deacetylated at position C-3 of methyl 2,3,4,6-tetra-O-acetyl-beta-D-glucopyranoside to accumulate 2,4,6-triacetate as the main initial reaction product in about 70% yield. Deacetylation was followed at position C-2, and generated 4,6-diacetate in 50% yield. The enzyme deacetylated the product at positions C-4 and C-6 at slower rates, and generated 4- and 6-monoacetates at a later reaction stage. Finally, it gave a completely deacetylated product. For 2,3,4,6-tetra-O-acetyl-beta-D-galactopyranoside, CA esterase deacetylated at positions C-3 and C-6 to give 2,4,6- and 2,3,4-triacetate. Deacetylation proceeded sequentially at positions C-3 and C-6 to accumulate 2,4-diacetate in 55% yield. The enzyme exhibited regioselectivity for the deacetylation of the acetylglycoside.     

Chemical and enzymatic synthesis of fructose analogues as probes for import studies by the hexose transporter in parasites

Various D-fructose analogues modified at C-1 or C-6 positions were synthesized from D-glucose by taking advantage of the Amadori rearrangement or using the aldol condensation between dihydroxyacetone phosphate and appropriate aldehyde catalyzed by fructose 1,6-diphosphate aldolase from rabbit muscle. The affinities of the analogues for the glucose transporter expressed in the mammalian form of Trypanosoma brucei were determined by inhibition of radiolabelled 2-deoxy-D-glucose (2-DOG) transport using zero-trans kinetic analysis. Interestingly, the analogues bearing an aromatic group (i.e. a fluorescence marker) at C-1 or C-6 positions present comparable apparent affinities to D-fructose for the transporter. This result could find applications for hexose transport studies and also provides criteria for the design of glucose import inhibitors.     

Etude de quelques propriétés de la phosphofructokinase érythrocytaire de l'homme normal

Human erythrocyte phosphofructokinase was purified 150 fold by DEAE cellulose adsorption and ammonium sulfate precipitation.At pH 7,5 the enzyme exhibits allosteric kinetics with respect to ATP, fructose 6 phosphate, and Mg²⁺.ATP at high concentration acted as an inhibitor and ADP, 5′AMP, 3′,5′, AMP, acted as activators. Both effectors seemed to decrease the homotropic interactions beetween the fructose 6 phosphate molecules.The activators increased the affinity of phosphofructokinase for the substrate (F6P), the inhibitor decreased it.These ligands had no effect on the maximum velocity of the reaction except in the case of ADP.Interactions between the substrates and the effector ligands on the enzyme were considered in terms of the Monod - Changeux - Wyman model for allosteric proteins.With GTP and ITP, no inhibition was observed. At saturing concentration of GTP, ATP still inhibited phosphofructokinase.Both 3′5′ AMP and fructose 6 phosphate increased the concentration of ATP required to produce an inhibition of 50 %.Citrate, like ATP, inhibited phosphofructokinase by binding most likely at the same allosteric site. Erythrocyte phosphofructokinase is inhibited by 2–3 DPG.The study of the relation log V max = f (pH) suggested, that the active center contains at least one imidazole and one sulfhydryl group.     

Evidence for two asymmetric conformational states in the human erythrocyte sugar-transport system.

6-O-methyl-, 6-O-propyl-, 6-O-pentyl- and 6-O-benzyl-D-galactose, and 6-O-methyl-, 6-O-propyl- and 6-O-pentyl-D-glucose inhibit the glucose-transport system of the human erythrocyte when added to the external medium. Penetration of 6-O-methyl-D-galactose is inhibited by D-glucose, suggesting that it is transported by the glucose-transport system, but the longer-chain 6-O-alkyl-D-galactoses penetrate by a slower D-glucose-insensitive route at rates proportional to their olive oil/water partition coefficients. 6-O-n-Propyl-D-glucose and 6-O-n-propyl-D-galactose do not significantly inhibit L-sorbose entry or D-glucose exit when present only on the inside of the cells whereas propyl-beta-D-glucopyranoside, which also penetrates the membrane slowly by a glucose-insensitive route, only inhibits L-sorbose entry or D-glucose exit when present inside the cells, and not when on the outside. The 6-O-alkyl-D-galactoses, like the other nontransported C-4 and C-6 derivatives, maltose and 4,6-O-ethylidene-D-glucose, protect against fluorodinitrobenzene inactivation, whereas propyl beta-D-glucopyranoside stimulates the inactivation. Of the transported sugars tested, those modified at C-1, C-2 and C-3 enhance fluorodinitrobenzene inactivation, where those modified at C-4 and C-6 do not, but are inert or protect against inactivation. An asymmetric mechanism is proposed with two conformational states in which the sugar binds to the transport system so that C-4 and C-6 are in contact with the solvent on the outside and C-1 is in contact with the solvent on the inside of the cell. It is suggested that fluorodinitrobenzene reacts with the form of the transport system that binds sugars at the inner side of the membrane. An Appendix describes the theoretical basis of the experimental methods used for the determination of kinetic constants for non-permeating inhibitors.     

Specificity of acceptor binding to Leuconostoc mesenteroides B-512F dextransucrase: binding and acceptor-product structure of alpha-methyl-D-glucopyranoside analogs modified at C-2, C-3, and C-4 by inversion of the hydroxyl and by replacement of the hydroxyl with hydrogen

The specificity of acceptor binding to the active site of dextransucrase was studied by using alpha-methyl-D-glucopyranoside analogs modified at C-2, C-3, and C-4 positions by (a) inversion of the hydroxyl group and (b) replacement of the hydroxyl group with hydrogen. 2-Deoxy-alpha-methyl-D-glucopyranoside was synthesized from 2-deoxyglucose; 3- and 4-deoxy-alpha-methyl-D-glucopyranosides were synthesized from alpha-methyl-D-glucopyranoside; and alpha-methyl-D-allopyranoside was synthesized from D-glucose. The analogs were incubated with [14C]sucrose and dextransucrase, and the products were separated by thin-layer chromatography and quantitated by liquid scintillation spectrometry. Structures of the acceptor products were determined by methylation analyses and optical rotation. The relative effectiveness of the acceptor analogs in decreasing order were 2-deoxy, 2-inverted, 3-deoxy, 3-inverted, 4-inverted, and 4-deoxy. The enzyme transfers D-glucopyranose to the C-6 hydroxyl of analogs modified at C-2 and C-3, to the C-4 hydroxyl of 4-inverted, and to the C-3 hydroxyl of 4-deoxy analogs of alpha-methyl-D-glucopyranoside. The data indicate that the hydroxyl group at C-2 is not as important for acceptor binding as the hydroxyl groups at C-3 and C-4. The hydroxyl group at C-4 is particularly important as it determines the binding orientation of the alpha-methyl-D-glucopyranoside ring.     

Distribution of tritium in D-glucose and starch labeled by tritium-atom bombardment

Samples of D-glucose and starch were labeled by tritium-atom bombardment. Up to 51% incorporation into D-glucose as non-labile tritium was achieved for crystalline, anhydrous D-glucose and 41% for the amylose-butyl alcohol complex. Distribution of tritium in the carbon skeleton of D-glucose was calculated by comparing the specific molar activity of D-glucose with that of its derivatives. Derivatives prepared were D-gluconic acid, D-arabino-hexulose phenylosotriazole, 4-formyl-2-phenyltriazole, 2-phenyltriazole-4-carboxylic acid, D-arabino-hexulose phenylflavazole, 3-formyl-1-phenylflavazole, and formaldehyde dimedone. The tritium distribution showed definite structural effects. Generally, the products from films of D-glucose and the amylose-butyl alcohol complex had nearly uniform distribution of tritium in D-glucose. The product from crystalline α-D-glucose monohydrate had zero tritium at C-2 and twice the expected amount of tritium at C-5, and that from starch granules had zero or near zero tritium at C-3 and close to twice the expected amount of tritium at C-2.     

Préparation et dosage immunonéphélémétrique du fibrinogène de lapin

Pure rabbit fibrinogen was prepared by a method involving two ammonium sulfate precipitations, one 2 M phosphate buffer precipitation, one DEAE cellulose chromatography and lastly one Sepharose 6 B chromatography. The aminoacid composition was determined and an immunonephelemetric assay was proposed. This assay allowed an accurate determination of fibrinogen concentration in a rabbit with inflammatory reaction.     

Biosynthesis of the 2-(aminomethyl)-4-(hydroxymethyl)furan subunit of methanofuran

2H- and 13C-labeled precursors were used to establish the pathway for the biosynthesis of the 2-(aminomethyl)-4-(hydroxymethyl)furan (F1) component of methanofuran in methanogenic archaebacteria. The extent and position of the label incorporated into F1 were measured from the mass spectrum of the diacetyl derivative of F1. [1,2-13C2]Acetate was found to be incorporated into two separate positions of the F1 molecule as a unit. The extent of incorporation of 13C2 into each of these positions was the same as that observed for the incorporation of acetate into the alanine and proline produced by the cells. From [2,2,2-2H3]acetate, deuterium was incorporated into two separate sites of the F1 molecule, one containing up to two deuteriums and the other only one. On the basis of the fragmentation pattern of the F1 diacetyl derivative, it was determined that two deuteriums were incorporated into the hydroxymethyl group at C-4 and one was incorporated at C-3 of the furan ring. The extent and distribution of the incorporated deuterium at the C-4 methylene were the same as that observed for C-6 of the glucose produced by the cells. On the basis of this and additional information presented in this paper, it is concluded that F1 is generated by the condensation of dihydroxyacetone phosphate with pyruvate. The resulting dihydroxy-substituted tetrahydrofuran after elimination of 2 mol of water would produce the phosphate ester of 2-carboxy-4-(hydroxymethyl)furan. Reduction of the carboxylic acid to an aldehyde and subsequent transamination would produce the phosphate ester of F1.     

Ligand recognition by the lactose permease of Escherichia coli: specificity and affinity are defined by distinct structural elements of galactopyranosides

Specificity of substrate recognition in lactose permease is directed toward the galactosyl moiety of lactose. In this study, binding of 31 structural analogues of D-galactose was examined by site-directed N-[(14)C]ethylmaleimide-labeling of the substrate-protectable Cys148 in the binding site. Alkylation of Cys148 is blocked by D-galactose with an apparent affinity of approximately 30 mM. Epimers of D-galactose at C-3 (D-gulose) and C-4 (D-glucose) or deoxy derivatives at these positions exhibit no binding whatsoever, indicating that these OH groups participate in essential interactions. Interestingly, the C-2 epimer alpha-D-talose binds almost as well as D-galactose, while 2-deoxy-D-galactose affords no substrate protection, indicating that nonstereospecific H-bonding at C-2 is required for stable binding. No substrate protection is detected with D-fucose, L-arabinose, 6-deoxy-6-fluoro-D-galactose, 6-O-methyl-D-galactose, or D-galacturonic acid, suggesting that the C-6 OH is an essential H-bond donor. Both alpha- and beta-methyl D-galactopyranosides bind more strongly than galactose, supporting the notion that the cyclic pyranose conformation is the bound form and that the anomeric configuration at C-1 does not contribute to substrate specificity. However, methyl or allyl alpha-D-galactopyranosides exhibit 60-fold lower apparent K(d)'s than D-galactose, demonstrating that binding affinity is significantly influenced by the functional group at C-1 and its orientation. Taken together, the observations confirm and extend the current binding site model [Venkatesan, P., and Kaback, H. R. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 9802-9807] and indicate that specificity toward galactopyranosides is governed by H-bonding interactions at C-2, C-3, C-4, and C-6 OH groups, while binding affinity can be increased dramatically by hydrophobic interactions with the nongalactosyl moiety.     

Preparation of 6-deoxy-6-fluorocellulose

6-Deoxy-6-fluorocellulose was prepared from cellulose 2,3-diacetate (1) or cellulose 2,3-dibenzoate (2) in various solvents, and was characterized by ¹⁹F and ¹³C NMR measurements. The best product, having ds of 0.95 at C-6 and 0.04 at C-3, was prepared from cellulose 2,3-dibenzoate in nitrobenzene. Other combinations of starting material and solvent gave a lower (≈ 0.8) ds of fluorine at C-6 and higher (≈ 0.12) at C-2 or C-3. Substitution at C-2 was observed when the combination of 1 and 1,4-dioxane, or 2 and chloroform was used. The products substituted at C-2 by fluorine were relatively resistant to acid hydrolysis.     

Studies on insulin action. V. Structural requirements of primers for subsequent accumulation of D-glucose at 0 degrees C in insulinized frog muscles.

Systematic analysis was made of structural requirements for possible priming action of D-glucose and 40 related compounds in the presence of insulin to enhance subsequent labeled D-glucose uptake at 0 degrees C. It was concluded that a major portion if not all of the H-bonding groups on the D-glucose molecules are involved although their relative importance varies. Free OH and H on C-1, and upward orientation of OH and downward orientation of H on C-3, are the most important. Orientation of OH, H, and CH2OH on C-2, C-4, and C-5 are also important, but their perturbations are not nearly as detrimental to the priming action as those on C-1 and C-3.     

Homogeneous sulfation of bagasse cellulose in an ionic liquid and anticoagulation activity

Homogeneous sulfation of bagasse cellulose (BC) with chlorosulfonic acid-dimethylformamide was accomplished in an ionic liquid 1-butyl-3-methylimidazolium chloride ([C(4)mim]Cl). The BCS products from the sulfation had degrees of substitution (DS) in the range of 0.52-2.95 and a simultaneous substitution pattern at C-6, C-2 and C-3 positions. The sulfated BCS attained significant anticoagulation activity, causing a dose-dependent prolongation of coagulation time and inhibition of FIIa and FXa activities in human plasma. The anticoagulation activity of BCS showed a positive correlation with DS, and some of the activity indexes exceeded those of heparin.     

Specificity and kinetics of hexose transport in Trypanosoma brucei

Transport of 6-deoxy-D-glucose was studied in Trypanosoma brucei in order to characterise the kinetics of hexose transport in this organism using a nonphosphorylated sugar. Kinetic parameters for efflux and entry, measured using zero-trans and equilibrium exchange protocols, indicate that the transporter is probably kinetically symmetrical. Comparison of the kinetic constants of D-glucose metabolism with those for 6-deoxy-D-glucose transport shows that transport across the plasma membrane is likely to be the rate-limiting step of glucose utilisation. The transport rate is nevertheless very fast and 6-deoxy-D-glucose, at concentrations below Km, enters the cells with a half filling time of less than 2 s at 20 degrees C. Thus the high metabolic capacity of these organisms is matched by a high transport rate. The structural requirements for the trypanosome hexose transporter were explored by measuring inhibition constants (Ki) for a range of D-glucose analogues including fluoro and deoxy sugars as well as epimeric hexoses. The relative affinities shown by these analogues indicated H-bonds from the carrier to the C-3, C-4 and C-5 hydroxyl oxygens and from the C-1 and C-3 hydroxyl hydrogens to the binding site. Hydrophobic interactions are likely at the C-2 and C-6 regions of the glucose molecule. Spatial constraints appear to occur around C-4 indicating that the transport site at this position is not freely open to the external solution as is the case with the mammalian hexose transporter. However, the trypanosome transporter appears to accept D-fructose but the common mammalian (erythrocyte type) hexose transporter does not.     

Exclusive and complete introduction of amino groups and their N-sulfo and N-carboxymethyl groups into the 6-position of cellulose without the use of protecting groups

A new regioselective synthesis of 6-amino-6-deoxycellulose with a DS 1.0 (degree of substitution) at C-6, and its 6-N-sulfonated and its 6-N-carboxymethylated derivatives, without using protecting groups is described in this paper. The reaction conditions were optimized for preparing cellulose tosylate with full tosylation at C-6 and partial tosylation at C-2 and C-3. The nucleophilic substitution (S(N)) reaction of the tosyl group by NaN(3) at low temperature of 50 degrees C in Me(2)SO was achieved completely at C-6, whereas the tosyl groups at C-2 and C-3 were not displaced. In contrast to this, at 100 degrees C the tosyl groups at C-6, and also those at C-2 and C-3, were replaced by azido groups. This regioselective reaction that depends on temperature makes it possible to reach a selective and quantitative S(N) reaction at C-6 at low temperatures. In the subsequent reduction step with LiAlH(4), the azido group at C-6 was reduced to the amino group, and the tosyl groups at C-2 and C-3 were simultaneously completely removed. Also reported is a temperature-dependent, regioselective and complete iodination by nucleophilic substitution of the tosyl group at C-6 at 60 degrees C. At higher temperatures from 75 to 130 degrees C, substitution is also observed to occur at C-2. The selective iodination at 60 degrees C was employed to confirm the complete tosylation at C-6 of cellulose. The reaction products were identified by four different independent quantitative methods, namely 13C NMR, elemental analysis, ESCA, and fluorescence spectroscopy. 6-N-Sulfonated and 6-N-carboxymethylated cellulose derivatives were also synthesized. The new derivatives are potent candidates for structure-function studies, e.g., studies in relation to regioselectively 2-N-sulfonated and 2-N-carboxymethylated chitosan derivatives.     

Acétolyse d'une diazocétone derivée d'octose: synthése d'une N-acétyllincosamine protégée, de ses epiméres en c-6 et c-7, et d'une 3-oxétanone á chane latérale glucidique

The acetolysis with anhydrous acetic acid of the diazoketone 1, prepared from potassium 1,2:3,4-di-O-isopropylidene-α-D-galacturonate, gave ketoacetates having the D-glycero (3)and L-glycero (5)configuration at C-7, together with the 3-oxetanone 7, a product of pyranose-ring opening. Starting from the ketoacetates 3 and 5, the lincosamine derivative, 6-acetamido-6,8-dideoxy-1,2:,4-di-O-isopropylidene -α-D-erythro-D-galacto-octopyranose (8), and its three diastereoisomers at C-6 and C-7 (9, 10, and 11) were obtained by oximation, reduction, and N-acetylation.

Résumé

L'acétolyse par l'acide acétique anhydre de la diazocétone 1 dérivée du 1,2:3,4-di-O-isopropylidéne-α-D-galacturonate de potassium, donne les céto-acétates deconfiguration D-glycéro (3) et L-glycéro (5) sur C-7, ainsi que la 3-oxétanone 7 qui résulte de l'ouverture du cycle pyranosique. A partir des céto-acétates 3 et 5, on a obtenu, par oximation, réduction et N-acétylation, le dérivé de la lincosamine, 6-acétamido-6,8-didésoxy-1,2:3,4-di-O-isopropylidéne-α-D-érythro-D-galacto-octo-pyranose (8), et ses trois diastéréoisoméres en C-6 et C-7 (9, 10 et 11).     

Biosynthesis of vitamin B-12 in anaerobic bacteria. Experiments with Eubacterium limosum and D-erythrose 14C-labeled in different positions

In anaerobic microorganisms the origin of C atoms 2 and 4-7 of the 5,6-dimethylbenzimidazole moiety of vitamin B-12 is still unknown. In order to tackle this problem we added several 14C-labeled putative precursors to Eubacterium limosum fermentations. The degradation of the isolated vitamin B-12 revealed that only D-erythrose, 14C-labeled in different positions, was efficiently incorporated into the 5,6-dimethylbenzimidazole part. The 5,6-dimethylbenzimidazole obtained from an experiment with D-[U-14C]erythrose was further degraded. It was found that C-2 was unlabeled, whereas half of the label was located in C-5 plus C-6, and the other half in C-4 plus C-7. These results demonstrate that in E. limosum D-erythrose is a precursor of C-atoms 4, 5, 6 and 7 of the 5,6-dimethylbenzimidazole part of vitamin B-12.     

Synthesis and SAR study of tricyclic sulfones as γ-secretase inhibitors: C-6 and C-8 positions

SAR exploration at C-6 and C-8 positions of the tricyclic sulfone series was carried out. Several functional groups were found to be well tolerated at C-6 and C-8 positions. Selective combination of C-6 and C-8 modification resulted in new tricyclic sulfone analogs with efficacy in in vivo mouse Aβ₄₀ lowering model.     

Transglycosylic reactions of deoxysugars. Biosynthesis of deoxy analogues of starch.

The biosynthesis of starch was investigated in the reaction catalyzed by plant alpha(1 leads to 4)-glucan phosphorylase using alpha-D-glucopyranosyl phosphate and its deoxy analogues as substrates. It was found that the hydroxyl groups at the positions C-2, C-3, C-4 and C-6 in the glucose moiety of the molecule of alpha-D-glucopyranosyl phosphate are not essential for its substrate properties in the transglycosylic reaction. The affinity of plant (alpha(1 leads to 4)-glucan phosphorylase and the rate of hexose incorporation into alpha(1 leads to 4)-glucan decreases in the following sequence: alpha-D-glucopyranosyl phos-phosphate, 2-deoxy-, 6-deoxy, 4-deoxy, and 3-deoxy-alpha-D-glucopyranosyl phosphate. The deoxyglucosyl analogues of alpha-D-glucosylpyranosyl phosphate act as competitive inhibitors on the elongation reaction of the alpha(1 leads to 4) chains of starch. It was found that more than one residue of 2-deoxy-D-glucose or 6-deoxy-D-glucose can be incorporated into the nonreducing terminus of alpha(1 leads to 4)-glucan chains of starch.