Synthesis and characterization of 4,6-O-butylidene-N-(2-hydroxybenzylidene)-beta-D-glucopyranosylamine: crystal structures of 4,6-O-butylidene-alpha-D-glucopyranose, 4,6-O-butylidene-beta-D-glucopyranosylamine and 4,6-O-butylidene-N-(2-hydroxybenzylidene)-beta-D-glucopyranosylamine

4,6-O-Butylidene-N-(2-hydroxybenzylidene)-beta-D-glucopyranosylamine was synthesized and characterized using analytical, spectral and single-crystal X-ray diffraction methods. 1H and 13C NMR studies showed the presence of the beta-anomer, which has also been confirmed by the crystal structure. The molecular structure of this compound showed the presence of the tridentate ONO ligation-core. Both precursors, 4,6-O-butylidene-alpha-D-glucopyranose and 4,6-O-butylidene-beta-D-glucopyranosylamine were characterized using single crystal X-ray diffraction. The alpha-anomeric nature of the former and beta-anomeric nature of the latter were proposed based on 1H NMR studies and were confirmed by determining the crystal structures. In addition, the crystal structure of 4,6-O-butylidene-beta-D-glucopyranosylamine revealed the C-1-N-glycosylation. In all the three molecules, the saccharide unit exhibits a 4C(1) chair conformation. In the lattice, the molecules are connected by hydrogen-bond interactions. The conformation of 4,6-O-butylidene-N-(2-hydroxybenzylidene)-beta-D-glucopyranosylamine is stabilized via an O-H...N intramolecular interaction, and each molecule in the lattice interacts with three neighboring molecules through hydrogen bonds of the type O-H...O and C-H...O.     

Glycosylamines of 4,6-O-butylidene-alpha-D-glucopyranose: synthesis and characterization of glycosylamines, and the crystal structure of 4,6-O-butylidene-N-(o-chlorophenyl)-beta-D-glucopyranosylamine

A total of nine glycosylamines of 4,6-O-butylidene-alpha-D-glucopyranose were synthesized using primary amines having various groups in their ortho- or para-positions. Among these, six are monoglycosylamines, including one primary glycosylamine, and three are bis-glycosylamines. All these compounds were characterized by 1H, 1H-1H COSY, 1H-13C COSY and 13C NMR spectroscopy and FTIR spectra. The FAB mass spectra provided the molecular weights of the products by exhibiting the corresponding molecular ion peaks. The crystal structure of 4,6-O-butylidene-N-(o-chlorophenyl)-beta-D-glucopyranosylamine revealed the C-1 glycosylation, the beta-anomeric nature, and the 4C1 chair conformation of the saccharide unit in the product. In the lattice two types of dimers exist. While one type of dimer is formed through O-H...O type of interactions, the other type is formed via C-H...O type of interactions. In the direction of these C-H...O type of interactions, the dimeric units are connected to form a chain.     

Interaction of D-glucose with alkaline-earth metal ions. Synthesis, spectroscopic, and structural characterization of Mg(II)- and Ca(II)-D-glucose adducts and the effect of metal-ion binding on anomeric configuration of the sugar

The interaction of D-glucose with the hydrated alkaline-earth metal halides has been studied in solution, and adducts of the type Mg(D-glucose)X2.4 H2O, Ca(D-glucose)X2.4 H2O, and Ca(D-glucose)2X2.4 H2O, where X = Cl- and Br-, have been isolated, and characterized by means of F.t.-i.r. and 1H-n.m.r. spectroscopy, X-ray powder diffraction, and molar conductivity measurements. Spectroscopic and other evidence suggested that the Mg(II) ion in the Mg(D-glucose)X2.4 H2O adducts six-coordinate, binding to a D-glucose molecule (possibly via O-1 and O-2 atoms) and to four H2O molecules, whereas, in the corresponding 1:1 Ca-D-glucose adduct, the Ca(II) ion is possibly seven-coordinate, binding to a sugar moiety (through the O-1, O-2, and other sugar donor atoms) and to four H2O molecules. In 1:2 Ca(D-glucose)2X2.4 H2O, the calcium ion may be eight-coordinate, binding to two D-glucose molecules (possibly via the O-1 and O-2 atoms of each sugar moiety) and to four H2O molecules. The strong, sugar H-bonding network is rearranged upon D-glucose adduct-formation, and the alpha-anomeric configuration is favored by these metal cation coordinations.     

Crystal structure of 4,6-O-ethylidene-N-(2-hydroxybenzylidene)-beta-D-glucopyranosylamine.

4,6-O-Ethylidene-N-(2-hydroxybenzylidene)-beta-D-glucopyranosylamine was synthesized and characterized using analytical, spectral and single-crystal X-ray diffraction methods. The anomeric nature of the saccharide moiety was proposed based on 1H NMR studies and was confirmed by the crystal structure. The lattice structure of this compound was compared with that of its analogues.     

Structures and mechanisms of glycosyltransferases

Glycosyltransferases (GTs) catalyze the transfer of a sugar moiety from an activated donor sugar onto saccharide and nonsaccharide acceptors. A sequence-based classification spreads GTs in many families thus reflecting the variety of molecules that can be used as acceptors. In contrast, this enzyme family is characterized by a more conserved three-dimensional architecture. Until recently, only two different folds (GT-A and GT-B) have been identified for solved crystal structures. The recent report of a structure for a bacterial sialyltransferase allows the definition of a new fold family. Progress in the elucidation of the structures and mechanisms of GTs are discussed in this review. To accommodate the growing number of crystal structures, we created the 3D-Glycosyltransferase database to gather structural information concerning this class of enzymes.     

Crystal structure of N-(1-deoxy-beta-D-fructopyranos-1-yl)-L-proline-an Amadori compound

Here we report the crystal structure data on N-(1-deoxy-beta-D-fructopyranos-1-yl)-L-proline (Fru-Pro)-an Amadori compound. X-ray crystal and molecular structures of its two isomorphous crystalline forms, (Fru-Pro)xMeOH, C(11)H(19)NO(7)xCH(4)O (1a) and (Fru-Pro)x2H(2)O, C(11)H(19)NO(7)x2H(2)O (1b) were determined. In 1a and 1b the compound crystallizes as the beta-anomer with the overall geometry of Fru-Pro zwitterions being very similar. Fructose ring adopts the chair (2)C(5) conformation with the proline moiety bonded to equatorial C-1 atom and remaining in a trans-gauche (tg) orientation with respect to the sugar ring. The five-membered pyrrolidine ring adopts an envelope conformation, with the Cbeta atom puckered. Fructosyl and carboxylate groups are in bisectional and axial positions of pyrrolidine ring, respectively. The overall molecular geometry of Fru-Pro zwitterions, especially the relative orientation of sugar and amino acid moieties, is stabilized by intramolecular, three-centred N-H...O(Fru)/O(Pro) hydrogen bonds (with bifurcated acceptor) formed between aminium and hydroxyl/carboxylate groups. The packing diagrams are very similar in both 1a and 1b with the adjacent zwitterions linked to each other by the extensive network of O-H...O and C-H...O hydrogen bonds to form channels along the a-axis, filled up with solvent molecules.     

Crystal structures of ethyl 3-azido-2,3-dideoxy-D-arabino-hexopyranoside anomers

The structures of the title alpha (1) and beta (2) anomers of ethyl 3-azido-2,3-dideoxy-d-arabino-exopyranoside (C(8)H(15)N(3)O(4)) are reported. The single-crystal structures of C(8)H(15)N(3)O(4) were determined by X-ray crystallography at 293K. It has been found that both title compounds crystallize in the orthorhombic space group. In both cases, the unit cell contains four asymmetric molecules. From intensity measurements, it has been shown that each of these molecules adopts a (4)C(1) chair conformation. The packing arrangement in the unit cell displays a stratified structure. Moreover, medium strength O-H...O hydrogen bonds in both crystal lattices can be observed.     

Structural studies of five novel bile acid-4-aminopyridine conjugates

Synthesis and solid-state structural characterization of five bile acid amides of 4-aminopyridine (4-AP) are reported. Systematic crystallization experiments revealed a number of structural modifications and/or solvate/hydrate systems for these conjugates. Particularly, cholic acid conjugate exhibited five distinct structure modifications, including one anhydrous form, mono- and dihydrates, as well as ethanol and 2-butanol solvates. The obtained crystal forms were examined extensively with various analytical methods, including solid-state NMR, Raman, and IR spectroscopies, powder and single crystal X-ray diffraction methods, thermogravimetry, and differential scanning calorimetry. After releasing their crystal solvent molecules, the resulted non-solvated structure forms showed 50-75°C higher melting points than corresponding bile acids, and thermal degradation occurred for all conjugates at about 300-330°C. Moreover, the single crystal X-ray structure of the ursodeoxycholic acid-4-aminopyridine conjugate is reported.     

An orthorhombic crystal form of cyclohexaicosaose, CA26.32.59 H(2)O: comparison with the triclinic form.

Cycloamylose containing 26 glucose residues (cyclohexaicosaose, CA26) crystallized from water and 30% (v/v) polyethyleneglycol 400 in the orthorhombic space group P2(1)2(1)2(1) in the highly hydrated form CA26.32.59 H(2)O. X-ray analysis of the crystals at 0.85 A resolution shows that the macrocycle of CA26 is folded into two short left-handed V-amylose helices in antiparallel arrangement and related by a twofold rotational pseudosymmetry as reported recently for the (CA26)(2).76.75 H(2)O triclinic crystal form [Gessler, K. et al. Proc. Natl. Acad. Sci. USA 1999, 96, 4246-4251]. In the orthorhombic crystal form, CA26 molecules are packed in motifs reminiscent of V-amylose in hydrated and anhydrous forms. The intramolecular interface between the V-helices in CA26 is dictated by formation of an extended network of interhelical C-H...O hydrogen bonds; a comparable molecular arrangement is also evident for the intermolecular packing, suggesting that it is a characteristic feature of V-amylose interaction. The hydrophobic channels of CA26 are filled with disordered water molecules arranged in chains and held in position by multiple C-H...O hydrogen bonds. In the orthorhombic and triclinic crystal forms, the structures of CA26 molecules are equivalent but the positions of the individual water molecules are different, suggesting that the patterns of water chains are perturbed even by small structural changes associated with differences in packing arrangements in the two crystal lattices rather than with differences in the CA26 geometry.     

Crystal structures of (2R,4R)-2-(polyhydroxyalkyl)-1,3-thiazolidine-4-carboxylic acids: condensation products of l-cysteine with d-hexoses

We report herein the first crystal structures of (4-carboxy-1,3-thiazolidin-2-yl)pentitols [2-(polyhydroxyalkyl)thiazolidine-4-carboxylic acids], condensation products of l-cysteine with d-galactose and d-mannose: 2-(d-galacto-pentahydroxypentyl)thiazolidine-4-carboxylic acid hydrate, Gal-Cys·H(2)O (1), and 2-(d-manno-pentahydroxypentyl)thiazolidine-4-carboxylic acid hydrate, Man-Cys·H(2)O (2). In 1 and 2 the compounds crystallize as zwitterions, with the carboxylic groups deprotonated and the thiazolidine N atoms protonated. The sugar moiety and carboxylate group are in a cis configuration relative to the thiazolidinium ring, which adopts different conformation: twisted (T) on C(β)-S in 1, and S-puckered envelope (E) in 2. The carbon chain of the galactosyl/mannosyl moiety remains in an extended zig-zag conformation. The orientation of the sugar O2 atom with respect to the thiazolidinium S and N atoms is trans-gauche in 1 and gauche-gauche in 2. The molecular conformation is stabilized by the intramolecular N-H?O(Cys) contacts in both 1 and 2 and by the additional N-H?O(Man) interaction in 2. The crystal packing of orthorhombic 1 and monoclinic 2 is determined mainly by N/O/C-H?O hydrogen bonds forming ribbons linked to each other by direct and water-mediated O/C-H?O/S contacts.     

Further glucosides of lichens' acids from Central Asian lichens

Eight compounds isolated from an extract of Central Asian lichens comprised new glucosides having murolic, protoconstipatic and allo-murolic acids as the aglycones and a saccharide moiety linked at C-18 made up of four glucoses. The structures were elucidated using extensive spectroscopic analysis (1D and 2D NMR, MS, IR, UV and CD) and by chemical methods.     

Peptide design: influence of a guest Aib-Pro segment on the stereochemistry of an oligo-val sequence--solution conformations and crystal structure of Boc-(Val)2-Aib-Pro-(Val)3-OMe

The peptide Boc-Val-Val-Aib-Pro-Val-Val-Val-OMe has been synthesized to investigate the effect of introduction of a strong beta-turn promoting guest segment into an oligopeptide with a tendency to form extended structures. 1H-nmr studies in solution using analysis of NH group solvent accessibility and nuclear Overhauser effects suggest an appreciable solvent dependence of conformations. In chloroform a 3(10)-helical structure is favored, while in dimethylsulfoxide an Aib-Pro beta-turn with extended arms on either side is suggested. In the crystal, the backbone forms a somewhat distorted 3(10)-helix despite the presence of a Pro residue in the middle. Among the four possible intrahelical hydrogen bonds three are of the 4----1 type and one 5----1. Head-to-tail NH...O = C hydrogen bonds link the helical molecules into continuous columns. The space group is P2(1)2(1)2(1) a = 11.320(2), b = 19.889(3), and c = 21.247(3) A.     

Effect of asymmetric modification on the conformation of ascidiacyclamide analogs.

Ascidiacyclamide (ASC), cyclo(-Ile1-Oxz2-d-Val3-Thz4-)2 (Oxz=oxazoline and Thz=thiazole) has a C2-symmetric sequence, and the relationships between its conformation and symmetry have been studied. In a previous study, we performed asymmetric modifications in which an Ile residue was replaced by Gly, Leu or Phe to disturb the symmetry [Doi et al. (1999) Biopolymers49, 459-469]. In this study, the modifications were extended. The Ile1 residue was replaced by Gly, Ala, aminoisobutyric acid (Aib), Val, Leu, Phe or d-Ile, and the d-Val3 residue was replaced by Val. The structures of these analogs were analyzed by X-ray diffraction, 1H NMR and CD techniques. X-Ray diffraction analyses revealed that the [Ala1], [Aib1] and [Phe1]ASC analogs are folded, whereas [Val1]ASC has a square form. These structures are the first examples of folded structures for ASC analogs in the crystal state and are similar to the previously reported structures of [Gly1] and [Phe1]ASC in solution. The resonances of amide NH and Thz CH protons linearly shift with temperature changes; in particular, those of [Aib1], [d-Ile1] and [Val3]ASCs exhibited a large temperature dependence. DMSO titration caused nonlinear shifts of proton resonances for all analogs and largely affected [d-Ile1] and [Val3]ASCs. A similar tendency was observed upon the addition of acetone to peptide solutions. Regarding peptide concentration changes, amide NH and Thz CH protons of [Gly1]ASC showed a relatively large dependence. CD spectra of these analogs indicated approximately two patterns in MeCN solution, which were related to the crystal structures. However, all spectra showed a similar positive Cotton effect in TFE solution, except that of [Val3]ASC. In the cytotoxicity test using P388 cells, [Val1]ASC exhibited the strongest activity, whereas the epimers of ASC ([d-Ile1] and [Val3]ASCs), showed fairly moderate activities.     

Enzymatic polymerization to novel polysaccharides having a glucose-N-acetylglucosamine repeating unit, a cellulose-chitin hybrid polysaccharide

A cellulose-chitin hybrid polysaccharide having alternatingly beta(1-->4)-linked D-glucose (Glc) and N-acetyl-d-glucosamine (GlcNAc) was synthesized via two modes of enzymatic polymerization. First, a sugar oxazoline monomer of Glcbeta(1-->4)GlcNAc (1) was designed as a transition-state analogue substrate (TSAS) monomer for chitinase catalysis. Monomer 1 was recognized by chitinase from Bacillus sp., giving rise to a cellulose-chitin hybrid polysaccharide (2) via ring-opening polyaddition with perfect regioselectivity and stereochemistry. Molecular weight (M(n)) of 2 reached 4030, which corresponds to 22 saccharide units. Second, a sugar fluoride monomer of GlcNAcbeta(1-->4)Glc (3) was synthesized for the catalysis of cellulase from Trichoderma viride. The enzyme catalyzed polycondensation of 3, providing a cellulose-chitin hybrid polysaccharide (4) in regio- and stereoselective manner. M(n) of 4 reached 2840, which corresponds to 16 saccharide units. X-ray diffraction measurements revealed that these hybrid polysaccharides did not form any characteristic crystalline structures. Furthermore, these unnatural hybrids of 2 and 4 were successfully digested by lysozyme from human neutrophils.     

Sugar adducts with alkaline earth metal ions. Interaction of L-arabinose with Sr(II) and Ba(II) ions and the effects of metal ion binding on the sugar anomeric configurations

The reaction between L-arabinose and hydrated Sr(II) or Ba(II) halide salts has been studied in H2O solution and adducts of the type M(L-arabinose)X(2).4H(2)O, where M = Sr(II) or Ba(II) and X = Cl- or Br- have been isolated and characterized by means of Fourier transform infrared spectroscopy, 1H-NMR spectroscopy, molar conductivity and X-ray powder diffraction measurements. Due to the marked spectral similarities with those of the structurally known Ca(L-arabinose)X2 . 4H2O (X= Cl- or Br-) compounds, the Sr(II) and the BA(II) ions are eight-coordinated, binding to two l-arabinose molecules via O1, O5 of the first and O3, O4 of the second sugar moiety and to four H2O molecules. 1H-NMR spectroscopy indicated that the free L-arabinose has the beta-anomer configuration in aqueous solution, whereas the alpha-anomer isomer is preferred by Mg(II), Ca(II), Sr(II) and Ba(II) ions, on complexation.     

Purification, characterization, and molecular cloning of a pyranose oxidase from the fruit body of the basidiomycete, Tricholoma matsutake

A new H(2)O(2)-generating pyranose oxidase was purified as a strong antifungal protein from an arbuscular mycorrhizal fungus, Tricholoma matsutake. The protein showed a molecular mass of 250 kDa in gel filtration, and probably consisted of four identical 62 kDa subunits. The protein contained flavin moiety and it oxidized D-glucose at position C-2. H(2)O(2) and D-glucosone produced by the pyranose oxidase reaction showed antifungal activity, suggesting these compounds were the molecular basis of the antifungal property. The V(max), K(m), and k(cat) for D-glucose were calculated to be 26.6 U/mg protein, 1.28 mM, and 111/s, respectively. The enzyme was optimally active at pH 7.5 to 8.0 and at 50 degrees C. The preferred substrate was D-glucose, but 1,5-anhydro-D-glucitol, L-sorbose, and D-xylose were also oxidized at a moderate level. The cDNA encodes a protein consisting of 564 amino acids, showing 35.1% identity to Coriolus versicolor pyranose oxidase. The recombinant protein was used for raising the antibody.     

Conformation stability and organization of mefloquine molecules in different environments

The crystal structures of mefloquine base, [C17H16F6N2O], and two salts of mefloquine: hydrochloride [(C17H17F6N2O)+]3[Cl-]3.3H2O and hydrochloride tetrachlorocobaltate [(C17H17F6N2O)+]3Cl-[CoCl4]2-.C2H6O.H2O, were determined by X-ray diffraction measurements. A comparison of the crystal structures of mefloquine in three different crystalline environments shows that their conformations are stable regardless of mefloquine being a base or a salt. In addition, the conformation of mefloquine is similar to those of crystalline Cinchona alkaloids. The CF3 substituents in the quinoline moiety affect the packing of molecules.     

Two interconverting pentaiodide forms in the cyclomaltohexaose (alpha-cyclodextrin) polyiodide inclusion complex with sodium ion: dielectric and Raman spectroscopy studies

The polycrystalline inclusion complex of cyclomaltohexaose, (alpha-CD)(2) x NaI(5) x 8H(2)O, has been investigated via dielectric spectroscopy over a frequency range of 0-100 kHz and the temperature range of 125-450 K. Additionally, a Raman spectroscopy study was accomplished in the temperature ranges of (i) 153-298 K and (ii) 303-413 K. The ln sigma versus 1/T variation revealed the order-disorder transition of some normal hydrogen bonds to those of a flip-flop type at 200.9 K. From 278.3 up to 357.1K, the progressive transformation (H(2)O)(tightly bound)-->(H(2)O)(easily movable) takes place resulting in an Arrhenius linear increment of the ac-conductivity with activation energy E(a)=0.32 eV. In the range of 357.1-386.1K a second linear part with E(a)=0.55 eV is observed, indicating the contribution of sodium ions via the water-net.The rapid decrease of the ac-conductivity at T>386.1K is due to the removal of the water molecules from the crystal lattice, whereas the abrupt increase at T>414.9 K is caused by the sublimation of iodine.The Raman bands at 160 and 169 cm(-1) indicate the coexistence of (I(2) x I(-) x I(2)) and (I3(-) x I(2)<-->I(2) x I3(-)) units, respectively.The (I3(-) x I(2)<-->I(2) x I3(-)) units are presented as form (I), and their central I(-) ion is disordered in occupancy ratio different from 50/50 (e.g., ...60/40...70/30...).The(I(2) x I(-) x I(2)) units are displayed by the 2 equiv forms (IIa) and (IIb). In (IIa) the central I(-) ion is twofold disordered in an occupancy ratio of 50:50, whereas in (IIb) the central I(-) ion is well-ordered and equidistant from the two I(2) molecules. At low temperatures the transformation (I)-->(IIa) takes place, whereas at high temperatures the inverse one (IIa)-->(I) happens. X-ray powder diffraction and Rietveld analysis revealed a triclinic crystal form with space group P1 and lattice parameters that are in good agreement with the theoretical values.     

Contributions of Ionic Interactions and Protein Dynamics to Cytochrome P450 2D6 (CYP2D6) Substrate and Inhibitor Binding

P450 2D6 contributes significantly to the metabolism of >15% of the 200 most marketed drugs. Open and closed crystal structures of P450 2D6 thioridazine complexes were obtained using different crystallization conditions. The protonated piperidine moiety of thioridazine forms a charge-stabilized hydrogen bond with Asp-301 in the active sites of both complexes. The more open conformation exhibits a second molecule of thioridazine bound in an expanded substrate access channel antechamber with its piperidine moiety forming a charge-stabilized hydrogen bond with Glu-222. Incubation of the crystalline open thioridazine complex with alternative ligands, prinomastat, quinidine, quinine, or ajmalicine, displaced both thioridazines. Quinine and ajmalicine formed charge-stabilized hydrogen bonds with Glu-216, whereas the protonated nitrogen of quinidine is equidistant from Asp-301 and Glu-216 with protonated nitrogen H-bonded to a water molecule in the access channel. Prinomastat is not ionized. Adaptations of active site side-chain rotamers and polypeptide conformations were evident between the complexes, with the binding of ajmalicine eliciting a closure of the open structure reflecting in part the inward movement of Glu-216 to form a hydrogen bond with ajmalicine as well as sparse lattice restraints that would hinder adaptations. These results indicate that P450 2D6 exhibits sufficient elasticity within the crystal lattice to allow the passage of compounds between the active site and bulk solvent and to adopt a more closed form that adapts for binding alternative ligands with different degrees of closure. These crystals provide a means to characterize substrate and inhibitor binding to the enzyme after replacement of thioridazine with alternative compounds.