Crystal structures of rare disaccharides, α-d-glucopyranosyl β-d-psicofuranoside, and α-d-galactopyranosyl β-d-psicofuranoside

The crystal structures of α-d-glucopyranosyl β-d-psicofuranoside and α-d-galactopyranosyl β-d-psicofuranoside were determined by a single-crystal X-ray diffraction analysis, refined to R₁ = 0.0307 and 0.0438, respectively. Both disaccharides have a similar molecular structure, in which psicofuranose rings adopt an intermediate form between ⁴E and ⁴T₃. Unique molecular packing of the disaccharides was found in crystals, with the molecules forming a layered structure stacked along the y-axis.     

A direct enzymatic synthesis of β-d-galactopyranosyl-d-xylopyranosides and their use to evaluate rat intestinal lactase activity in vivo

By enzymatic β-d-galactosylation of d-xylose a mixture of 4-, 3-, and (1, 4, and 7, respectively) was obtained in 50% isolated yield. Disaccharides 1, 4, and 7 are substrates of intestinal lactase isolated from lamb small intestine with K_m values of 250.0, 4.5, and 14.0 mM, respectively. The mixture was used to monitor the normal decline in lactase activity in rats that takes place after weaning. The data obtained by this method correlated with the levels of intestinal lactase activity in the same animals after being sacrificed.     

The crystal structure of isopenicillin N synthase with δ-(l-α-aminoadipoyl)-l-cysteinyl-d-methionine reveals thioether coordination to iron

Isopenicillin N synthase (IPNS) catalyses cyclization of δ-(l-α-aminoadipoyl)-l-cysteinyl-d-valine (ACV) to isopenicillin N (IPN), the central step in penicillin biosynthesis. Previous studies have shown that IPNS turns over a wide range of substrate analogues in which the valine residue of its natural substrate is replaced with other amino acids. IPNS accepts and oxidizes numerous substrates that bear hydrocarbon sidechains in this position, however the enzyme is less tolerant of analogues presenting polar functionality in place of the valinyl isopropyl group. We report a new ACV analogue δ-(l-α-aminoadipoyl)-l-cysteinyl-d-methionine (ACM), which incorporates a thioether in place of the valinyl sidechain. ACM has been synthesized using solution phase methods and crystallized with IPNS. A crystal structure has been elucidated for the IPNS:Fe(II):ACM complex at 1.40 Å resolution. This structure reveals that ACM binds in the IPNS active site such that the sulfur atom of the methionine thioether binds to iron in the oxygen binding site at a distance of 2.57 Å. The sulfur of the cysteinyl thiolate sits 2.36 Å from the metal.     

Immunostimulatory activities of monoglycosylated α-d-pyranosylceramides

We compared the immunostimulatory effects of chemically synthesized α-galactosylceramides (α-GalCers), α-glucosylceramides (α-GluCers), 6″-monoglycosylated α-GalCer and 6″- or 4″-monoglycosylated α-GluCer and made the following observations: (1) the length of the fatty acid side chain in the ceramide portions greatly affects the immunostimulatory effects of α-GalCers and α-GluCers; (2) the configuration of the 4″-hydroxyl group of the inner pyranose moiety plays an important role in the immunostimulatory effects of monoglycosylated α- -pyranosylceramides; (3) the free 4″-hydroxyl group of the inner pyranose of monoglycosylated α- -pyranosylceramides plays a more important role in their immunostimulatory effects than the free 6″-hydroxyl group.     

Large scale isolation of 1,2:3,4-di-O-isopropylidene-α-d-glucoseptanose and 2,3:4,5-di-O-isopropylidene-β-d-glucoseptanose

Isolation of 1,2:3,4-di-O-isopropylidene-α-d-glucoseptanose and 2,3:4,5-di-O-isopropylidene-β-d-glucoseptanose from the mother-liquors from commercial scale preparation of 1,2:5,6-di-O-isopropylidene-α-d-glucofuranose is described.     

Studies on the stereoselective synthesis of a protected α-d-Gal-(1→2)-d-Glc fragment

A novel 1,2-cis stereoselective synthesis of protected α-d-Gal-(1→2)-d-Glc fragments was developed. Methyl 2-O-acetyl-3-O-allyl-4,6-O-benzylidene-α-d-galactopyranosyl-(1→2)-3-O-benzoyl-4,6-O-benzylidene-α-d-glucopyranoside (13), methyl 2-O-acetyl-3-O-allyl-4,6-O-benzylidene-α-d-galactopyranosyl-(1→2)-3,4,6-tri-O-benzoyl-α-d-glucopyranoside (15), methyl 2-O-acetyl-3-O-allyl-4,6-O-benzylidene-α-d-galactopyranosyl-(1→2)-3-O-benzoyl-4,6-O-benzylidene-β-d-glucopyranoside (17), and methyl 2-O-acetyl-3-O-allyl-4,6-O-benzylidene-α-d-galactopyranosyl-(1→2)-3,4,6-tri-O-benzoyl-β-d-glucopyranoside (19) were favorably obtained by coupling a new donor, isopropyl 2-O-acetyl-3-O-allyl-4,6-O-benzylidene-1-thio-β-d-galactopyranoside (2), with acceptors, methyl 3-O-benzoyl-4,6-O-benzylidene-α-d-glucopyranoside (4), methyl 3,4,6-tri-O-benzoyl-α-d-glucopyranoside (5), methyl 3-O-benzoyl-4,6-O-benzylidene-β-d-glucopyranoside (8), and methyl 3,4,6-tri-O-benzoyl-β-d-glucopyranoside (12), respectively. By virtue of the concerted 1,2-cis α-directing action induced by the 3-O-allyl and 4,6-O-benzylidene groups in donor 2 with a C-2 acetyl group capable of neighboring-group participation, the couplings were achieved with a high degree of α selectivity. In particular, higher α/β stereoselective galactosylation (5.0:1.0) was noted in the case of the coupling of donor 2 with acceptor 12 having a β-CH₃ at C-1 and benzoyl groups at C-4 and C-6.     

Synthesis and conformational analysis of 1,2-cis fused bicyclic α-d-galactofuranosyl thiocarbamate from per-O-tert-butyldimethylsilyl-β-d-galactofuranosyl isothiocyanate

Per-O-tert-butyldimethylsilyl-α,β-d-galactofuranosyl isothiocyanate (4) was synthesized by the reaction of per-O-TBS-β-d-galactofuranose (1) with KSCN, promoted by TMSI. Upon O-desilylation (1,2-dideoxy-α-d-galactofuranoso)[1,2d]-1,3-oxazolidine-2-thione (6), the cis-fused bicyclic thiocarbamate was obtained as the only product. Conformational analysis, aided by molecular modelling, showed two stable twist forms (³T₄ and ⁴T_O) for the five-membered sugar ring in 6. In aqueous solution, the equilibrium favours the first conformation (3:1 ratio). On the other hand, this ratio decreases for less polar solvents.     

The Crystal Structure of the Complexes of Concanavalin A with 4′-Nitrophenyl-α-d-mannopyranoside and 4′-Nitrophenyl-α-d-glucopyranoside

Concanavalin A (Con A) is the best-known plant lectin and has importantin vitrobiological activities arising from its specific saccharide-binding ability. Its exact biological role still remains unknown. The complexes of Con A with 4′-nitro-phenyl-α-d-mannopyranoside (α-PNM) and 4′-nitrophenyl-α-d-glucopyranoside (α-PNG) have been crystallized in space group P2₁2₁2 with cell dimensionsa= 135.19 Å,b= 155.38 Å,c= 71.25 Å anda= 134.66 Å,b= 155.67 Å, andc= 71.42 Å, respectively. X-ray diffraction intensities to 2.75 Å for the α-PNM and to 3.0 Å resolution for the α-PNG complex have been collected. The structures of the complexes were solved by molecular replacement and refined by simulated annealing methods to crystallographic R-factor values of 0.185/0.186 and free-R-factor values of 0.260/0.274, respectively. In both structures, the asymmetric unit contains four molecules arranged as a tetramer, with approximate 222 symmetry. A saccharide molecule is bound in the sugar-binding site near the surface of each monomer. The nonsugar (aglycon) portion of the compounds used helps to identify the exact orientation of the saccharide in the sugar-binding pocket and is involved in major interactions between tetramers. The hydrogen bonding network in the region of the binding site has been analyzed, and only minor differences with the previously reported Con A–methyl-α-d-mannopyranoside complex structure have been observed. Structural differences that may contribute to the slight preference of the lectin for mannosides over glucosides are discussed. Calculations indicate a negative electrostatic surface potential for the saccharide binding site of Con A, which may be important for its biological activity. It is also shown in detail how a particular class of hydrophobic ligands interact with one of the three so-called characteristic hydrophobic sites of the lectins.     

Regioselective monoacylation of 2-O-α-d-glucopyranosyl-l-ascorbic acid by a polymer catalyst in N,N-dimethylformamide

6-O-Dodecanoyl-2-O-α-d-glucopyranosyl-l-ascorbic acid (6-sDode-AA-2G) was synthesized from 2-O-α-d-glucopyranosyl-l-ascorbic acid and lauric anhydride with a polymer catalyst, poly(4-vinylpyridine), in N,N-dimethylformamide without the introduction of protecting groups. The optimum reaction conditions enabled 6-sDode-AA-2G to be synthesized in a yield of 49.7%. The yield and the regioselectivity in this method were far superior to those in our previous method by using an enzyme. The polymer catalyst could be recycled more than five times without any significant activity loss.     

Influence of the solvent in low temperature glycosylations with O-(2,3,5,6-tetra-O-benzyl-β-d-galactofuranosyl) trichloroacetimidate for 1,2-cis α-d-galactofuranosylation

Glycosylation studies for the construction of 1,2-cis α-linkages with O-(2,3,5,6-tetra-O-benzyl-β-d-galactofuranosyl) trichloroacetimidate (1) and several acceptors, including d-mannosyl and l-rhamnosyl derivatives were performed. The reactions were conducted at low temperatures using CH₂Cl₂, Et₂O, and acetonitrile as solvents. A non-participating solvent such as CH₂Cl₂ at −78 °C, favored the α-d-configuration. In contrast, acetonitrile strongly favored the β-d-configuration, whereas no selectivities were observed with Et₂O. The use of thiophene as an additive did not enhance the α-d-selectivity as in the pyranose counterpart. Although selectivities strongly depended on the acceptor, trichloroacetimidate 1 constitutes a valuable donor for the synthesis of α-d-Galf-(1→2)-l-Rha and α-d-Galf-(1→6)-d-Man. As these motifs are present in pathogenic microorganisms, these procedures described here are useful for the straightforward synthesis of natural oligosaccharides.     

Synthesis of β-d-glucopyranuronosylamine in aqueous solution: kinetic study and synthetic potential

A systematic study of the synthesis of β-d-glucopyranuronosylamine in water is reported. When sodium d-glucuronate was reacted with ammonia and/or volatile ammonium salts in water a mixture of β-d-glucopyranuronosylamine and ammonium N-β-d-glucopyranuronosyl carbamate was obtained at a rate that strongly depended on the experimental conditions. In general higher ammonia and/or ammonium salt concentrations led to a faster conversion of the starting sugar into intermediate species and of the latter into the final products. Yet, some interesting trends and exceptions were observed. The use of saturated ammonium carbamate led to the fastest rates and the highest final yields of β-d-glucopyranuronosylamine/carbamate. With the exception of 1 M ammonia and 0.6 M ammonium salt, after 24 h of reaction all tested protocols led to higher yields of β-glycosylamine/carbamate than concentrated commercial ammonia alone. The mole fraction of α-d-glucopyranuronosylamine/carbamate at equilibrium was found to be 7–8% in water at 30 °C. Concerning bis(β-d-glucopyranuronosyl)amine, less than 3% of it is formed in all cases, with a minimum value of 0.5% in the case of saturated ammonium carbamate. Surprisingly, the reaction was consistently faster in the case of sodium d-glucuronate than in the case of d-glucose (4–8 times faster). Finally, the synthetic usefulness of our approach was demonstrated by the synthesis of three N-acyl-β-d-glucopyranuronosylamines and one N-alkylcarbamoyl-β-d-glucopyranuronosylamine directly in aqueous–organic solution without resorting to protective group chemistry.     

β-d-(1→4), β-d-(1→3) ‘mixed linkage’ xylans from red seaweeds of the order Nemaliales and Palmariales

Xylans from five seaweeds belonging to the order Nemaliales (Galaxaura marginata, Galaxaura obtusata, Tricleocarpacylindrica, Tricleocarpa fragilis, and Scinaia halliae) and one of the order Palmariales (Palmaria palmata) collected on the Brazilian coasts were extracted with hot water and purified from acid xylomannans and/or xylogalactans through Cetavlon precipitation of the acid polysaccharides. The β-d-(1→4), β-d-(1→3) ‘mixed linkage’ structures were determined using methylation analysis and 1D and 2D NMR spectroscopy. The presence of large sequences of β-(1→4)-linked units suggests transient aggregates of ribbon- or helical-ordered structures that would explain the low optical rotations.     

An improved route to 1,2-dideoxy-β-1-phenyl-d-ribofuranose

An efficient synthesis of the aryl nucleoside analogue 1,2-dideoxy-β-1-phenyl- -ribofuranose (1) is described. This route utilizes the addition of phenyllithium to a protected 2-deoxyribonolactone followed by reduction with triethylsilane/boron trifluoride etherate to selectively produce the β-anomer. Deprotection yields the desired aryl C-nucleoside in 27% overall yield from 2-deoxy- -ribose.     

Solvent effect on enzyme-catalyzed synthesis of β-d-glucosides using the reverse hydrolysis method: Application to the preparative-scale synthesis of 2-hydroxybenzyl and octyl β-d-glucopyranosides

Almond β-d-glucosidase was used to catalyze alkyl-β-d-glucoside synthesis by reacting glucose and the alcohol in organic media. The influence of five different solvents and the thermodynamic water activity on the reaction have been studied. The best yields were obtained in 80 or 90% (v/v) tert-butanol, acetone, or acetonitrile where the enzyme is very stable. In this enzymatic synthesis under thermodynamic control, the yield increases as the water activity of the reaction medium decreases. Enzymatic preparative-scale syntheses were performed in a tert-butanol-water mixture which was found to be the most appropriate medium. 2-Hydroxybenzyl β-d-glucopyranoside was obtained in 17% yield using a 90:10 (v/v) tert-butanol-water mixture. Octyl-β-glucopyranoside was obtained in 8% yield using a 60:30:10 (v/v) tert-butanol-octanol-water mixture.     

Kinetic studies on the loss of water from α-d-glucose monohydrate

Although the dehydration of α-d-glucose monohydrate is an important aspect of several industrial processes, there is uncertainty with regard to the applicable rate law and other factors that affect dehydration. Therefore, the dehydration of three glucose monohydrate samples has been studied using isothermal gravimetric analysis. Dehydration follows a one-dimensional contraction (R1) rate law for the majority of kinetic runs, and an activation energy of 65.0 ± 3.9 kJ mol⁻¹ results when the rate constants are fitted to the Arrhenius equation. Fitting the rate constants to the Eyring equation results in values of 62.1 ± 3.7 kJ mol⁻¹ and −77.8 ± 4.7 J mol⁻¹ K⁻¹ for ΔH^‡ and ΔS^‡, respectively. The impedance effect on the loss of water vapor has also been investigated to determine the values for activation energy, enthalpy, and entropy for diffusion of water. The results obtained for the activation parameters are interpreted in terms of the absolute entropies of anhydrous glucose and the monohydrate.     

Transport of 3-O-methyl d-glucose and β-methyl d-glucoside by rabbit ileum

The intestinal transport of three actively transported sugars has been studied in order to determine mechanistic features that, (a) can be attributed to stereospecific affinity and (b) are common.The apparent affinity constants at the brush-border indicate that sugars are selected in the order, $\text{β-methyl glucose >}\text{d}\text{-galactose}\text{> 3-O-}\text{methyl}$ glucose, (the K_m values are 1.23, 5.0 and 18.1 mM, respectively.) At low substrate concentrations the K_t values for Na⁺ activation of sugar entry across the brush-border are: 27.25, and 140 mequiv. for β-methyl glucose, galactose and 3-O-methyl glucose, respectively. These kinetic parameters suggest that Na⁺, water, sugar and membrane-binding groups are all factors which determine selective affinity.In spite of these differences in operational affinity, all three sugars show a reciprocal change in brush-border entry and exit permeability as Ringer [Na] or [sugar] is increased. Estimates of the changes in convective velocity and in the diffusive velocity when the sugar concentration in the Ringer is raised reveal that with all three sugars, the fractional reduction in convective velocity is approximately equal to the (reduction of diffusive velocity)². This is consistent with the view that the sugars move via pores in the brush-border by convective diffusion.Theophylline reduces the serosal border permeability to β-methyl glucose and to 3-O-methyl glucose relatively by the same extent and consequently, increases the intracellular accumulation of these sugars.The permeability of the serosal border to β-methyl glucose entry is lower than permeability of the serosal border to β-methyl glucose exit, which suggests that β-methyl glucose may be convected out of the cell across the lateral serosal border.     

Synthesis of a small library of bivalent α-d-mannopyranosides for lectin cross-linking

A small library of bivalent α-d-mannopyranosides having rigid linkers was constructed in order to evaluate the effects of inter-saccharide distances upon multivalent binding interactions with plant and bacterial lectins. To this end, iodoaryl and propargyl α-d-mannopyranosides were synthesized and the former treated with TMS-acetylene under palladium chemistry to provide their corresponding ethynylaryl derivatives. A library of 15 dimeric members was then obtained using Lewis acid catalyzed glycosidation, aryl–aryl homocoupling, transition metal catalyzed Sonogashira cross-coupling reactions, and oxidative Glaser homocoupling.     

Activity of Debaryomyces hansenii UFV-1 α-galactosidases against α-d-galactopyranoside derivatives

α-d-Galactopyranosides were synthesized and their inhibitory activities toward the Debaryomyces hansenii UFV-1 extracellular and intracellular α-galactosidases were evaluated. Methyl α-d-galactopyranoside was the most potent inhibitor compared to the others tested, with values of 0.82 and 1.12 mmol L⁻¹, for extracellular and intracellular enzymes, respectively. These results indicate that the presence of a hydroxyl group in the C-6 position of α-d-galactopyranoside derivatives is important for the recognition by D. hansenii UFV-1 α-galactosidases.     

Selective synthesis of 1,6-anhydro-β-d-mannopyranose and -mannofuranose using microwave-assisted heating

The dehydration of d-mannose and the demethanolization of methyl-α-d-mannopyranoside (MαMP) or methyl-α-d-mannofuranoside (MαMF) were examined using microwave-assisted heating for a 3-min irradiation at temperature from 120 to 280 °C in ordinary or dry sulfolane without any catalyst. The microwave-assisted heating of MαMP and MαMF smoothly proceeded to selectively afford the anhydromannoses, 1,6-anhydro-β-d-mannopyranose (AMP) and 1,6-anhydro-β-d-mannofuranose (AMF), respectively, in high yields. For MαMP in ordinary sulfolane at 240 °C, AMP was selectively obtained in the AMF:AMP ratio of 4:96, whereas AMF was the major product at the AMF:AMP ratio of 97:3 from MαMF in dry sulfolane at 220 °C.     

d-Ribose glycates β2-microglobulin to form aggregates with high cytotoxicity through a ROS-mediated pathway

β₂-Microglobulin (β₂M) modified with advanced glycation end products (AGEs) is a major component of the amyloid deposits in hemodialysis-associated amyloidosis (HAA). However, the effect of glycation on the misfolding and aggregation of β₂M has not been studied so far. Here we examine the molecular mechanism of aggregate formation of HAA-related ribosylated β₂M in vitro. We find that the glycating agent d-ribose interacts with human β₂M to generate AGEs that form aggregates in a time-dependent manner. Ribosylated β₂M molecules are highly oligomerized compared with unglycated β₂M, and have granular morphology. Furthermore, such ribosylated β₂M aggregates show significant cytotoxicity to both human SH-SY5Y neuroblastoma and human foreskin fibroblast FS2 cells and induce intracellular reactive oxygen species (ROS). Presence of the antioxidant N-acetylcysteine (1.0 mM) attenuated intracellular ROS and prevented cell death induction in both SH-SY5Y and FS2 cells, indicating that the cytotoxicity of ribosylated β₂M aggregates depends on a ROS-mediated pathway in both cell lines. In other words, d-ribose reacts with β₂M and induces the ribosylated protein to form granular aggregates with high cytotoxicity through a ROS-mediated pathway. These findings suggest that ribosylated β₂M aggregates could contribute to the dysfunction and death of cells and could play an important role in the pathogenesis of β₂M-associated diseases such as HAA.