Dual and multiple stimuli-responsive platonic micelles bearing disaccharides.

We recently identified completely monodisperse micelles whose aggregation number (Nagg) coincides with the vertex number of regular polyhedra, named Platonic micelles. The combination of both the micellar properties and controlling their structures by external stimuli could be promising for producing precisely controlled self-assembled structures. From this perspective, we newly synthesized a calix[4]arene-based amphiphile bearing disaccharides, cellobioses. The crowded and bulky structure in the hydrophilic group could provide a novel stimuli-responsiveness of disaccharides in amphiphiles. The aggregation behavior such as the morphologies and the aggregation number of the calix[4]arene-based micelle was characterized using small angle scattering techniques and analytical ultracentrifugation measurements. Owing to hydrogen bonding among the disaccharide, the head volume became smaller than expected, resulting in the formation of cylindrical ones. However, cleaving the hydrogen bond by controlling temperature or pH induced morphological transition of the micellar structure from cylindrical to spherical. The dual-stimuli (temperature and pH) generated smaller micelles with Nagg of 12. Interestingly, when the amphiphile formed spherical micelles at various conditions, the Nagg matched the Platonic number, and the change of Nagg in response to the external stimuli was non-continuous, which is consistent with the concept of Platonic micelles.

Physicochemical properties of thermotolerant extracellular ß-glucosidase from Talaromyces thermophilus and enzymatic synthesis of cello-oligosaccharides.

A thermophilic fungus, Talaromyces thermophilus that produces a novel thermotolerant extra-cellular ß-glucosidase (Bgl.tls), was isolated from Tunisian soil samples. The enzyme was purified from the culture filtrates of T. thermophilus grown on lactose using gel filtration, ion exchange chromatography and FPLC. The monomeric enzyme had a molecular mass of 116.0 kDa and a high specific activity of 1429 UI/mg. Bgl.tls exhibited optimal activity at pH 5.0 and 65 °C. It was also stable over a wide range of pH (4.0-10.0) and stable at 50 °C for 34 h. Bgl.tls retained about 80% of its initial activity after 1.0 hours of preincubation at 60 °C. The Km and Vmax values recorded for pNPG were 0.25 mM and 228.7 µmol min(-1), respectively. Bgl.tls was activated by Mn(2+), Mg(2+), Ca(2+) and Co(2+) but obviously inhibited by Fe(2+) and Cu(2+). It was able to hydrolyze a variety of aryl / alkyl -ß-glucosides and disaccharides as well as (1 → 6) and (1 → 4)-ß-glucosidic linkages and α-glycosidic substrates, thus providing evidence for its broad substrate specificity. The enzyme also displayed high hydrolytic and transglycosylation activities. Overall, this study is the first report on the purification and physicochemical properties of a ß-glucosidase secreted by T. thermophilus. The cello-oligosaccharides synthesized by this enzyme within 2 h were mainly cellotriose, cellotetraose and cellopentaose identified by HPLC and ESI-MS techniques.

Useful approach to the synthesis of aryl thio- and selenoglycosides in the presence of rongalite.

A simple, mild, and cost effective methodology has been developed for the synthesis of aryl thio-and selenoglycosides from glycosyl halides and diaryl dichalcogenides. Diaryl dichalcogenides undergo reductive cleavage in the presence of rongalite (HOCH2SO2Na) to generate a chalcogenide anion in situ followed by reaction with glycosyl halides to furnish the corresponding aryl thio- and selenoglycosides in excellent yields. Using this protocol, synthesis of 4-methyl-7-thioumbelliferyl-ß-D-cellobioside (MUS-CB), a fluorescent non-hydrolyzable substrate analogue for cellulases has been achieved.

Synthesis of quercetin glycosides and their melanogenesis stimulatory activity in B16 melanoma cells.

4'-O-ß-d-Glucopyranosyl-quercetin-3-O-ß-d-glucopyranosyl-(1→4)-ß-d-glucopyra-noside (3) was isolated from Helminthostachys zeylanica root extract as a melanogenesis acceleration compound and was synthesized using rutin as the starting material. Related compounds were also synthesized to understand the structure-activity relationships in melanin biosynthesis. Melanogenesis activities of the glycosides were determined by measuring intracellular melanin content in B16 melanoma cells. Among the synthesized quercetin glycosides, quercetin-3-O-ß-d-glucopyranoside (1), quercetin-3-O-ß-d-glucopyranosyl-(1→4)-ß-d-glucopyranoside (2), and 3 showed more potent intracellular melanogenesis acceleration activities than theophyline used as positive control in a dose-dependent manner with no cytotoxic effect.

Synthesis of bis-cellobiose and bis-glucose derivatives of azacrown macrocycles as hosts in complexes with acetylsalicylic acid and 4-acetamidophenol.

Two new C2 symmetric bis-cellobiose and bis-glucose azacrown derivatives were prepared according to the one-step procedure using azacrown ethers and azidosaccharides. Their complexes with aspirin and paracetamol were studied with the use of proton NMR spectroscopy. It was found that these pseudocryptands bind aspirin and paracetamol but each one in a different manner.

Azido derivatives of cellobiose: oxidation at C1 with cellobiose dehydrogenase from Sclerotium rolfsii.

We report the chemo-enzymatic synthesis of three cellobiono-1,5-lactone azido derivatives, designed as building blocks for biomedical polymer scaffolds. The synthesis is based on regioselective protection of cellobiose or 1,6-O-anhydro-ß-d-cellobiose before azidation and subsequent deprotection. The oxidation to the corresponding cellobiono-1,5-lactones was investigated with 6'-azido-6'-deoxycellobiose (6'N3Clb, 5), 6-azido-6-deoxycellobiose (6N3Clb, 11) and 2-azido-2-deoxycellobiose (2N3Clb, 15) under the catalysis of cellobiose dehydrogenase (CDH) from the plant-pathogenic fungus Sclerotium rolfsii. Substrate binding characteristics and kinetics of CDH for the three cellobiose azido derivatives were studied employing computational docking, steady-state and presteady-state techniques. The process of enzymatic oxidation of the cellobiose azido intermediates was optimized by using the available kinetic information. Whereas the conversion of 15 by CDH is very slow, the conversion of 5 and 11 by a regenerated, bi-enzymatic process (CDH/redox mediator/laccase/O2) is fast, quantitative and produces azido derivatives of cellobiono-1,5-lactone in an environmentally friendly, oxygen-driven process.

Rational design, synthesis, evaluation and enzyme-substrate structures of improved fluorogenic substrates for family 6 glycoside hydrolases.

Methylumbelliferyl-ß-cellobioside (MUF-G2) is a convenient fluorogenic substrate for certain ß-glycoside hydrolases (GH). However, hydrolysis of the aglycone is poor with GH family 6 enzymes (GH6), despite strong binding. Prediction of the orientation of the aglycone of MUF-G2 in the +1 subsite of Hypocrea jecorina Cel6A by automated docking suggested umbelliferyl modifications at C4 and C6 for improved recognition. Four modified umbelliferyl-ß-cellobiosides [6-chloro-4-methyl- (ClMUF); 6-chloro-4-trifluoromethyl- (ClF3MUF); 4-phenyl- (PhUF); 6-chloro-4-phenyl- (ClPhUF)] were synthesized and tested with GH6, GH7, GH9, GH5 and GH45 cellulases. Indeed the rate of aglycone release by H. jecorina Cel6A was 10-150 times higher than with MUF-G2, although it was still three orders of magnitude lower than with H. jecorina Cel7B. The 4-phenyl substitution drastically reduced the fluorescence intensity of the free aglycone, while ClMUF-G2 could be used for determination of k(cat) and K(M) for H. jecorina Cel6A and Thermobifida fusca Cel6A. Crystal structures of H. jecorina Cel6A D221A mutant soaked with the MUF-, ClMUF- and ClPhUF-ß-cellobioside substrates show that the modifications turned the umbelliferyl group 'upside down', with the glycosidic bond better positioned for protonation than with MUF-G2.

Hybrid immobilization of galactosyl lactose and cellobiose on a gold substrate to modulate biological responses.

Bioactive O-ß-d-galactopyranosyl-(1→4)-O-ß-d-galactopyranosyl-(1→4)-d-glucopyranose (4'-galactosyl lactose) was site-selectively modified at a reducing end with thiosemicarbazide (TSC). As-synthesized 4'-galactosyl lactose-TSC was immobilized on a gold substrate with cellobiose-TSC as a spacer through spontaneous self-assembly chemisorption via SAu bonding. Quartz crystal microbalance analysis suggested the successful formation of self-assembled monolayers (SAMs) of 4'-galactosyl lactose-TSC and/or cellobiose-TSC. Galactose-binding lectin exhibited the highest affinity for hybrid SAMs with an equimolar ratio of the two oligosaccharide-TSCs, while glucose-binding lectin showed decreasing adsorption with a decrease in cellobiose-TSC ratios. Human hepatocellular carcinoma cells, which recognize galactose residues, efficiently adhered to the hybrid SAMs. Higher enzymatic deethoxylation of ethoxyresorufin via cytochrome P450 appeared on hybrid SAMs. These results suggested that clustering of the bioactive sugars was involved in the cellular responses, possibly via biological carbohydrate-protein interactions. This approach to designing carbohydrate-based scaffolds should provide a basis for the functional development of glyco-decorated biointerfaces for cell culture applications.

A study of the acid-catalyzed hydrolysis of cellulose dissolved in ionic liquids and the factors influencing the dehydration of glucose and the formation of humins.

An investigation was carried out into the hydrolysis of cellulose dissolved in 1-ethyl-3-methylimidazolium chloride ([Emim][Cl]) and 1-butyl-3-methylimidazolium chloride ([Bmim][Cl]) catalyzed by mineral acids. Glucose, cellobiose, and 5-hydroxymethylfurfural (5-HMF) were observed as the primary reaction products. The initial rate of glucose formation was determined to be of first order in the concentrations of dissolved glucan and protons and of zero order in the concentration of water. The absence of a dependence on water concentration suggests that cleavage of the ß-1,4-glycosidic linkages near chain ends is irreversible. The apparent activation energy for glucose formation is 96 kJ mol(-1). The absence of oligosaccharides longer than cellobiose suggests that cleavage of interior glycosidic bonds is reversible due to the slow diffusional separation of cleaved chains in the highly viscous glucan/ionic liquid solution. Progressive addition of water during the course of glucan hydrolysis inhibited the rate of glucose dehydration to 5-HMF and the formation of humins. The inhibition of glucose dehydration is attributed to stronger interaction of protons with water than the 2-OH atom of the pyranose ring of glucose, the critical step in the proposed mechanism for the formation of 5-HMF. The reduction in humin formation associated with water addition is ascribed to the lowered concentration of 5-HMF, since the formation of humins is suggested to proceed through the condensation polymerization of 5-HMF with glucose.

New radiosynthesis of 2-deoxy-2-[(18)F]fluoroacetamido-D-glucopyranose and its evaluation as a bacterial infections imaging agent.

INTRODUCTION: The diagnosis of infection and the ability to distinguish bacterial infection from nonbacterial inflammation by positron emission tomography (PET) have gained interest in recent years, but still few specific radiopharmaceuticals are available for use. In this study, we developed a new radiosynthesis method of 2-deoxy-2-[(18)F]fluoroacetamido-d-glucopyranose ([(18)F]FAG) by applying microwave irradiation and demonstrated that [(18)F]FAG could be a potential radiopharmaceutical to distinguish bacterial infection from nonbacterial inflammation. METHODS: 1,3,4,6-Tetra-O-acetyl-2-deoxy-2-bromoacetamido-d-glucopyranose was used as precursor, and labeling was performed under microwave irradiation conditions followed by alkaline hydrolysis and high-performance liquid chromatography (HPLC) purification. In vitro uptake of [(18)F]FAG by Escherichia coli was performed. Tissue biodistribution of [(18)F]FAG was performed in mice. Moreover, PET imaging acquisition of E. coli infection and nonbacterial inflammation models was performed in rats. Tissue radiotracer-accumulated sites were analyzed by hematoxylin and eosin staining and anti-E.coli immunostaining. RESULTS: The radiosynthesis of [(18)F]FAG was achieved with microwave irradiation, and the radiochemical yield was 9.7%±2.8% end of bombardment (EOB); the radiochemical purity was more than 98%, and the total synthesis time was 62 min. Compared with control group, in vitro uptake of [(18)F]FAG by E. coli was significantly decrease in inhibition group (P<.05). Biodistribution studies in mice showed rapid clearance of [(18)F]FAG from the animal body. [(18)F]FAG clearly visualized the infection areas but not nonbacterial inflammation areas in PET studies. Quantitative analysis revealed that the uptake of [(18)F]FAG into infection areas was significantly higher than that of [(18)F]FAG into inflammation areas (P<.05). Histological analysis demonstrated the presence of bacterial cells at the sites of accumulation of [(18)F]FAG. CONCLUSIONS: Using 1,3,4,6-tetra-O-acetyl-2-deoxy-2-bromoacetamido-d-glucopyranose as a precursor, the new radiosynthesis method of [(18)F]FAG was achieved in fewer steps and with a shorter synthesis time than previously reported. Furthermore, [(18)F]FAG was able to distinguish bacterial infection from nonbacterial inflammation.

Van der Waals versus hydrogen-bonding forces in a crystalline analog of cellotetraose: cyclohexyl 4'-O-cyclohexyl beta-D-cellobioside cyclohexane solvate.

Hydrogen bonding is important in cellulosic and other carbohydrate structures, but the role of interactions between nonpolar groups is less understood. Therefore, we synthesized cyclohexyl 4'-O cyclohexyl beta-D-cellobioside (8), a molecule that has two glucose rings and two nonpolar cyclohexyl rings. Key to attaching the 4'-Ocyclohexyl group was making the 4'-O,6'-O-cyclohexylidene ketal. After peracetylation, the cyclohexylidene ketal ring was opened regioselectively, providing 65% of 8 after final deacetylation. Comparison of the crystal structure of 8, as the cyclohexane solvate, with those of cellulose and its fragments, especially cellotetraose with four glucose rings, revealed extensive effects from the cyclohexyl groups. Three conformationally unique molecules (A, B, and C) are in the triclinic unit cell of 8, along with two solvent cyclohexanes. When viewed down the crystal's a-axis, the array of C, A, and B looks like the letter N, with A inclined so that its cyclohexyl groups can stack with those of the reducing ends of the B and C molecules. The lower left and upper right points of the N are stacks of cyclohexyl rings on the nonreducing ends of B and C, interspersed with solvent cyclohexanes. Whereas cellotetraose has antiparallel (up-down) packing, A and B in 8 are oriented "down" in the unit cell while C is "up". "Down-down-up" (or, alternatively, "up-up-down") packing is rare for carbohydrates. Other unusual details include 06 in all three staggered orientations: one is tg, two are gg, and three are gt, confirmed with CP/MAS 13C NMR. The tg O6 donates a proton to an intramolecular hydrogen bond to O2', opposite to the major schemes in native cellulose I. A similar but novel O6B-H...O2'B hydrogen bond is based on a slightly distorted gg orientation. The hydrogen bonds between parallel molecules are unique, with linkages between O2'A and O2'B, O3'A and O3'B, and O6A and O6B. Other details, such as the bifurcated O3...O5' and ...O6' hydrogen bonds are similar to those of other cellulosic structures. C-H...O hydrogen bonds are extensive along the [110] line of quarter-staggering. The unusual features described here expand the range of structural motifs to be considered for as-yet undetermined cellulose structures.

Regioselective synthesis of 1I,1II,5I,5II,6I,6I,6II,6II-2H8-cellobiose.

Partially deuteriated 1,5,6,6-(2)H(4)-d-glucose and 1(I),1(II),5(I),5(II),6(I),6(I),6(II),6(II)-(2)H(8)-d-cellobiose were synthesized in high yields and on a large scale from d-glucose. (2)H enrichment at C-5 and C-6 of each glucopyranosyl unit in excess of 85% and 90%, respectively, was realized by (1)H-(2)H exchange in (2)H(2)O containing deuteriated Raney Ni. Nucleophilic addition of LiAlD(4) to 5,6,6-(2)H(3)-2,3,4,6-tetra-O-benzyl-d-gluconolactone led to a 98% (2)H enrichment at C-1. Deuteriated cellobiose is of interest as building block for the synthesis of a model compound of cellulose I.

Synthesis of methyl 4'-O-methyl-beta-D-cellobioside-13C12 from D-glucose-13C6. Part 2: solid-state NMR studies.

Double Quantum (DQ) NMR, which utilizes the magnetic dipole interaction between the (13)C atoms, was used for the complete assignment of the (13)C NMR resonances to the corresponding carbon ring positions for the monoclinic and triclinic allomorphs of methyl 4'-O-methyl-beta-D-cellobioside-(13)C(12)(1-(13)C(12)), a cellodextrin model compound of cellulose (13)C-perlabeled at the cellobiose core. The through-space interactions were used to identify the direct chemical bonds between adjacent carbon atoms in the rings. More importantly, the (13)C NMR signals of the carbon sites C1' and C4 involved in the glycosidic bond were identified. This allowed for the complete (13)C chemical shift assignment, that when combined with the X-ray crystallography data provides a complete characterization.

Reaction on D-glucal by an inverting phosphorylase to synthesize derivatives of 2-deoxy-beta-D-arabino-hexopyranosyl-(1-->4)-D-glucose (2II-deoxycellobiose).

Four derivatives of 2(II)-deoxycellobiose were synthesized from d-glucal and acceptor sugars (d-glucose, d-xylose, d-mannose, and 2-deoxy-d-arabino-hexose) using a cellobiose phosphorylase from Cellvibrio gilvus. The enzyme was found to be an effective catalyst to synthesize the beta-(1-->4) linkage of 2-deoxy-d-arabino-hexopyranoside. The acceptor specificity for the d-glucal reaction was identical to that for the alpha-d-glucose 1-phosphate reaction, but the activity of d-glucal was approximately 500 times less than that of alpha-d-glucose 1-phosphate, using 10mM substrates.

Synthesis of urea-tethered disaccharides in water.

A new method for the synthesis of urea-linked disaccharides in aqueous media has been developed. The key feature of our approach is two strained Steyermark-type gluco- and galactopyranosyl oxazolidinones. Each oxazolidinone is attached to a pyranose ring in a di-equatorial trans-annulation framework. Reaction of these oxazolidinones with 4-aminohexopyranose in water proceeded smoothly to afford the urea-tethered cellobiose and lactose analogues. The galactose-type oxazolidinone proved to be more reactive than the glucose-type, which is explained by the presence of an axial hydroxy group at C4 in the former.

Synthesis of methyl 4'-O-methyl-13C12-beta-D-cellobioside from 13C6-D-glucose. Part 1: reaction optimization and synthesis.

A high yielding synthetic route for methyl 4'-O-methyl-beta-D-cellobioside starting from d-glucose was established. The reaction conditions optimized with nonlabeled materials were used for the synthesis of methyl 4'-O-methyl-13C12-beta-D-cellobioside, a compound having more than 99% 13C enrichment at each of the twelve pyranose carbon atoms. The labeled compound is required to study the hydrogen bond network of cellodextrins and cellulose by CPMAS NMR experiments.

The chemical synthesis of 2-deoxy-2-fluorodisaccharide probes of the hen egg white lysozyme mechanism.

2,4-Dinitrophenyl 2-acetamido-2-deoxy-beta-d-glucopyranosyl-(1-->4)-2-deoxy-2-fluoro-beta-d-glucopyranoside (GN2FG-DNP) and 2-acetamido-2-deoxy-beta-d-glucopyranosyl-(1-->4)-2-deoxy-2-fluoro-beta-d-glucopyranosyl fluoride (GN2FG-F) were prepared using a divergent synthetic approach involving 10 steps. The key steps involved the preparation of 1-O-acetyl-3,6-di-O-benzyl-2-deoxy-2-fluoro-alpha/beta-d-glucopyranose using Selectfluor(trade mark) in the presence of acetic acid and the subsequent glycosylation of this acceptor to generate the core 2-fluorodisaccharide. After further elaboration, the target molecules were obtained and tested as probes of the mechanism of hen egg white lysozyme (HEWL). Compound GN2FG-DNP is not a substrate for the enzyme while compound GN2FG-F is cleaved slowly with an apparent K(m) greater than 5mM and a second-order rate constant of k(cat)/K(m)=9.6s(-1)M(-1). Comparison of this value to that estimated for the hydrolysis of beta-chitobiosyl fluoride by HEWL (1200s(-1)M(-1)) [Ballardie, F. W.; Capon, B.; Cuthbert, M. W.; Dearie, W. M. Bioorg. Chem.1977, 6, 483-509] revealed a 126-fold rate decrease upon substitution of a fluorine group for the 2-acetamido group of beta-chitobiosyl fluoride. This decrease resulted in the steady-state accumulation of an intermediate as visualized by mass spectrometry and the ultimate crystallographic determination of its structure [Vocadlo, D. J.; Davies, G. J.; Laine, R.; Withers, S. G. Nature2001, 412, 835-838].

Incremented alkyl derivatives enhance collision induced glycosidic bond cleavage in mass spectrometry of disaccharides.

Electrospray ionization and collision induced dissociation on a triple quadrupole mass spectrometer were used to determine the effect of spatial crowding of incremented alkyl groups of two anomeric pairs of peralkylated (methyl to pentyl) disaccharides (maltose/cellobiose and isomaltose/gentiobiose). Protonated molecules were generated which underwent extensive fragmentation under low energy conditions. For both the 1 --> 4 and 1 --> 6 alpha and beta isomers, at comparable collision energies the methyl derivative exhibited the least fragmentation followed by ethyl, propyl, butyl, and pentyl. Collision energy is converted to rotational-vibrational modes in competition with bond cleavage, as represented by the slope of product/parent ion (D/P) ratio versus offset energy. Variable rotational freedom at the glycosidic linkage with incremented alkyl groups is hypothesized to be responsible for this effect. Discrimination of anomeric configuration was also assessed for these stereoiosmeric disaccharides. A systematic study showed that an increasing discrimination was attained for the 1 --> 4 isomeric pair as the size of the derivative increased from methyl to pentyl. No anomeric discrimination was attained for the 1 --> 6 isomeric pair. Parent and product ion scans confirmed the consistency of fragmentation pathways among derivatives. Chem-X and MM3 molecular modeling programs were used to obtain minimum energy structures and freedom of motion volumes for the permethylated disaccharides. The modeling results correlated with the fragmentation ratios obtained in the mass spectrometer giving strong indication that the collision induced spectra are dependent on the freedom of rotational motion around the glycosidic bond.

Commercial synthesis of alpha-D-cellobiosyl bromide heptaacetate.

A detailed commercial process for the synthesis and purification of the activated disaccharide, alpha-D-cellobiosyl bromide heptaacetate (1) was developed. Reaction of alpha-D-cellobiose octaacetate (CBO) with HBr in glacial acetic acid or in glacial acetic acid/methylene chloride combination affords alpha-D-cellobiosyl bromide heptaacetate in high yield and excellent quality. Process variables such as reaction solvent, reaction time, reaction temperature, HBr stoichiometry, isolation methods and product purification options were optimized for large-scale synthesis. alpha-D-Cellobiosyl bromide heptaacetate was successfully prepared in a commercial manufacturing plant.