The conformation of tetrafluorinated methyl galactoside anomers: crystallographic and NMR studies

The first single-crystal X-ray diffraction study of tetrafluorinated monosaccharide derivatives is presented. Both α- and β-methyl 2,3-dideoxy-2,2,3,3-tetrafluoro-d-galactopyranoside anomers adopt the ⁴C₁ conformation. The values for the C1–O1 and C1–O5 bond lengths and the O5–C1–O1–CH₃ dihedral angles are in line with what can be expected from the anomeric and exo-anomeric effects. The chair conformations are slightly distorted, presumably due to repulsion between 1,3-diaxial C–O and C–F bonds. The asymmetric unit of both compounds contains up to three independent molecules, which differ in the conformation of the hydroxymethyl group (including in one case a ‘forbidden’ gg rotamer). The molecular packing of the β-anomer shows a clear segregation between fluorinated and hydrophilic domains, while for the α-anomer the regions of fluorine segregation are broken by interleafing of OMe groups. There is one close OH?F contact, which is likely to arise from the crystal packing. NMR studies show that the two anomers also adopt a ⁴C₁ conformation in solution (D₂O, CDCl₃).     

O-Aryl α,β-d-ribofuranosides: Synthesis & highly efficient biocatalytic separation of anomers and evaluation of their Src kinase inhibitory activity

A series of peracetylated O-aryl α,β-d-ribofuranosides have been synthesized and an efficient biocatalytic methodology has been developed for the separation of their anomers which was otherwise almost impossible by column chromatographic or other techniques. The incubation of 2,3,5-tri-O-acetyl-1-O-aryl-α,β-d-ribofuranoside with Lipozyme® TL IM immobilized on silica led to the selective deacetylation of only one acetoxy group, viz the C-5′-O-acetoxy group of the α-anomer over the other acetoxy groups derived from the two secondary hydroxyl groups present in the molecule and also over three acetoxy groups (derived from one primary and two secondary hydroxyls of the β-anomer). This methodology led to the easy synthesis of both, α- and β-anomers of O-aryl d-ribofuranosides. All the arylribofuranosides were screened for inhibition of Src kinase. 1-O-(3-Methoxyphenyl)-β-d-ribofuranoside exhibited the highest activity for inhibition of Src kinase (IC₅₀ = 95.0 μM).     

Application of ab initio molecular-orbital calculations to the structural moieties of carbohydrates. Part VI

Ab initio RHF/4–31G molecular-orbital calculations have been conducted on methoxymethyl formate and methoxymethyl acetate as models for examining the anomeric effect and stereochemistry of 1-O-acetylglycopyranoses. The results indicate that, as with the methyl glycopyranosides, the α-⁴C₁(D) configurations are more stable than the β-⁴C₁(D), except that the energy difference is more dependent on the disposition about the glycosidic bond. The lowest-energy conformations occur with glycosidic torsion-angles of ?  180°, where the anomeric energy is about 4 kcal/mol. There is a secondary energy-minimum at ?  90°, for which the anomeric energy is less, about 2 kcal/mol. This orientation corresponds to the conformation most commonly observed in the crystal structures of peracetylated glycopyranoses. Small differences in the CO single-bond lengths, which are observed experimentally in both the α and β anomers, are reproduced by the theoretical calculations.     

Inverting character of α-glucuronidase A from Aspergillus tubingensis

α-Glucuronidase A from Aspergillus tubingensis was found to be capable of liberating 4-O-methyl-D-glucuronic acid (MeGlcA) only from those beechwood glucuronoxylan fragments in which the acid is attached to the non-reducing terminal xylopyranosyl residue. Reduced aldotetrauronic acid, 4-O-methyl-D-glucuronosyl-α-1,2-D-xylopyranosyl-β-1,4-xylopyranosyl-β-1,4-xylitol, was found to be a suitable substrate to follow the stereochemical course of the hydrolytic reaction catalyzed by the purified enzyme. The configuration of the liberated MeGlcA was followed in a D₂O reaction mixture by ¹H-NMR spectroscopy. It was unambiguously established that MeGlcA was released from the substrate as its β-anomer from which the α-anomer was formed on mutarotation. This result represents the first experimental evidence for the inverting character of a microbial α-glucuronidase, a member of glycosyl hydrolase family 67 (EC 3.1.1.139).     

Synthesis of β-l-fucopyranosyl phosphate andl-fucofuranosyl phosphates by the MacDonald procedure

Fusion or β-l-fucopyranose tetraacetate with phosphoric acid for 1 min at 50° gives a 9:1 anomeric mixture of the α-and β-pyranosyl phosphates. Longer fusion times give the α-anomer exclusively. The l-fucofuranose tetraacetates were synthesized for the first time by acetolysis or methyl-2,3,5-tri-O-acetyl-β-l-fucofuranoside. Fusion or the furanose tetraacetates with phosphoric acid gave a mixture or the fucofuranosyl phosphates in which the β-anomer predominated (β/α = 2.4). Anomeric pairs in the fucofuranose series appear to be distinguishable by the chemical shift of the C-6 methyl protons, as already shown by Sinclair and Sleeter in the pyranose series.     

A NMR method to determine the anomeric specificity of glucose phosphorylation

A NMR method related to 2D CH correlation with an additional double quantum filter for ³¹P spin coupling was employed to follow the reaction kinetics of the two anomers of glucose during phosphorylation catalyzed by the enzyme yeast hexokinase. The kinetic parameters according to Michaelis–Menten for these reactions have been determined and it is shown that the β-anomer of glucose is phosphorylated faster by a factor of 1.4 versus the α-anomer. Use of human liver glucokinase as an enzyme yields more complex kinetics.     

Tautomeric composition of D-fructose phosphates in solution by fourier transform carbon-13 nuclear magnetic resonance

The Fourier transform ¹³C magnetic resonance spectra of D-fructose 6-phosphate (F6P) and D-fructose 1,6-diphosphate (FDP) were obtained. The signal assignments made on the basis of ¹³C chemical shifts and ¹³C-³¹P spin-spin couplings indicate that the earlier assignments of the C-4 and C-5 resonances of α- and β-fructofuranose in oligosaccharides and D-fructose [Allerhand, A. and Doddrell, D., J. Amer. Chem. Soc., $\text{93}$, 2777, 2779 (1971)] should be reversed. Integration of signal intensities yields the following equilibrium composition at 35°C: F6P, α-anomer 19±2% and β-anomer 81±2%, FDP, α-anomer 23±4% and β-anomer 77±4%. Less than 1.5% keto or hydrated keto form is present in solutions of either fructose phosphate. The bearing of these findings on the tautomeric specificity of phosphofructokinase is discussed.     

The crystal and molecular structure of O-α-D-mannopyranosyl-(1→3)-O-β-D-mannopyranosyl-(1→4)-2-acetamido-2-deoxy-α-D-glucopyranose

The crystal structure of α-D-Manp-(1→3)-β-D-Manp-(1→4)-α-D-GlcNAcp has been determined by the direct method using the multi-solution, tangent formula, and “magic integer” procedures. The space group is P2₂, and 2 molecules are in the unit cell with a  9.894 (5), b  10.372 (6), c  11.816 (6) Å, and β  95.03° (6). The structure was refined to R 0.059 for 2099 reflections measured with Mo Kα radiation. Difference synthesis showed all the hydrogen atoms, and indicated a partial (～30%) substitution of the α-anomer molecules by the β-anomer molecules. The D-mannopyranose and the D-glucopyranose have the normal ⁴C₁ conformation; an intramolecular hydrogen-bond O-3″-H.....O-5′ (2.703 Å) stabilises the GlcNAc in relation to β-D-mannopyranose.     

Synthesis of potassium (2R)-2-O-α-d-glucopyranosyl-(1→6)-α-d-glucopyranosyl-2,3-dihydroxypropanoate a natural compatible solute

Ethyl 6-O-acetyl-2,3,4-tribenzyl-1-thio-d-glucopyranoside, as a mixture of anomers, was employed for the stereoselective synthesis of the potassium salt of (2R)-2-O-α-d-glucopyranosyl-(1→6)-α-d-glucopyranosyl-2,3-dihydroxypropanoic acid (α-d-glucosyl-(1→6)-α-d-glucosyl-(1→2)-d-glyceric acid, GGG), a recently isolated compatible solute. The α-anomer was by far the major product of both glycosylation reactions using NIS/TfOH as activator.     

Catalytic properties of endo-1,3-β-D-glucanase from the Vietnamese edible mussel Perna viridis

A β-1,3-glucanase with a molecular mass of 33 kDa was isolated in the homogeneous state from a crystalline stalk of the commercially available Vietnamese edible mussel Perna viridis. It hydrolyzes β-1,3-bonds in glucans and is capable of catalyzing the transglycosylation reaction. The β-1,3-glucanase has a K _m value of 0.3 mg/ml for the hydrolysis of laminaran and shows a maximum activity in the pH range from 4 to 6.5 and at 45°C. Its half-inactivation time is 180 min at 45°C and 20 min at 50°C. The enzyme was ascribed to glucan-endo-(1 → 3)-β-D-glucosidases (EC 3.2.1.39). The enzyme could be used in the structure determination of β-1,3-glucans and enzymatic synthesis of new carbohydrate-containing compounds.     

Theoretical studies on the conformations of methyl glycopyranosides

The σ-charges on various atoms of methyl glycosides have been computed by using the MO-LCAO method of Del Re. The potential and free energies of methyl aldohexopyranosides and methyl aldopentopyranosides in their C1(d) and 1C(d) conformations have been calculated. Minimization of the energies of these conformations has been studied by suitably tilting the axial C-C and C-O bonds. Considerable release of strain is achieved when tilts of 4.5 and 3° are given to the axial hydroxymethyl and hydroxyl groups, respectively, that are involved in Hassel-Ottar effect. A tilt of 3° is also found necessary for the axial OMe group involved in the Hassel-Ottar effect. The calculated free-energy values are in accord with experimental ones, after adding a value of 0.8 kcal.mole^?1 for the anomeric effect of -OMe group. These studies predict that all of the methyl aldohexopyranosides, except methyl α-d- and methyl β-d-idopyranosides, favour the C1 conformation. On the other hand, the energy calculations also predict that, of the eight methyl aldopentopyranosides studied, only methyl α-d- and methyl β-d-xylopyranosides and methyl α-d -ribopyranoside favour the C1(d) conformation; for the other pentopyranosides, considerable amounts of both C1(d) and 1C(d) conformations are present in the equilibrium mixture. The calculated values of the percentage of α-anomer present in the equilibrium mixture agree fairly well with those obtained experimentally.     

Convenient approaches to synthesis of furanoid sugar-aza-crown ethers from C-ribosyl azido aldehyde via a reductive amination/amidation

A short and highly efficient route to the α-anomer of a furanoid sugar-aza-crown ether was developed by a one-pot reductive amination of an α-anomer C-ribosyl azido aldehyde. In addition, the β-anomer furanoid sugar-aza-crown ether was synthesized from a linear disaccharide precursor via amidation and then followed by microwave-assisted amide reduction.     

Specificity for the glucose-6-P inhibition site of hexokinase

Inhibition of the three animal hexokinase isozymes by the following glucose-6-P analogs has been determined: α-glucose-1,6-P₂, α- and β-methyl glucose-6-P, α- and β-glucose-6-P, 2-Cl- and 4F-glucose-6-P, 5-deoxyglucose-6-P, glucose-6-sulfate, and δ-gluconolactone-6-P. Although both anomers of glucose-6-P were about equally active inhibitors, the β-methyl derivative was inactive. Generally the α-methyl and α-PO₃^? derivatives were good inhibitors though weaker than glucose-6-P except in the case of hexokinase II for which α-glucose-1,6-P₂ was an excellent inhibitor.     

Purification and biochemical characterization of a novel α-glucosidase from Aspergillus niveus

An extracellular α-glucosidase produced by Aspergillus niveus was purified using DEAE-Fractogel ion-exchange chromatography and Sephacryl S-200 gel filtration. The purified protein migrated as a single band in 5% PAGE and 10% SDS–PAGE. The enzyme presented 29% of glycosylation, an isoelectric point of 6.8 and a molecular weight of 56 and 52 kDa as estimated by SDS-PAGE and Bio-Sil-Sec-400 gel filtration column, respectively. The enzyme showed typical α-glucosidase activity, hydrolyzing p-nitrophenyl α-d-glucopyranoside and presented an optimum temperature and pH of 65°C and 6.0, respectively. In the absence of substrate the purified α-glucosidase was stable for 60 min at 60°C, presenting t ₅₀ of 90 min at 65°C. Hydrolysis of polysaccharide substrates by α-glucosidase decreased in the order of glycogen, amylose, starch and amylopectin. Among malto-oligosaccharides the enzyme preferentially hydrolyzed malto-oligosaccharide (G10), maltopentaose, maltotetraose, maltotriose and maltose. Isomaltose, trehalose and β-ciclodextrin were poor substrates, and sucrose and α-ciclodextrin were not hydrolyzed. After 2 h incubation, the products of starch hydrolysis measured by HPLC and thin layer chromatography showed only glucose. Mass spectrometry of tryptic peptides revealed peptide sequences similar to glucan 1,4-alpha-glucosidases from Aspergillus fumigatus, and Hypocrea jecorina. Analysis of the circular dichroism spectrum predicted an α-helical content of 31% and a β-sheet content of 16%, which is in agreement with values derived from analysis of the crystal structure of the H. jecorina enzyme.     

Effects of End Group and Aggregation on Helix Conformation: Crystal Structure of Ac-(Aib-Val-Ala-Leu)2-Aib- OMe

The role of end groups in determining stereochemistry and packing in hydrophobic helical peptides has been investigated using an α-aminosobutyric acid (Aib) containing model nonapeptide sequence. In contrast to the Boc-analogue, Ac-(Aib-Val-Ala-Leu)₂-Aib-OMe crystallizes with two independent molecules in a triclinic cell. The cell parameters are: space group P1, a=10.100(2)Å, b=15.194(4) Å, c=19.948(5) Å, α=63.12(2)°, β=88.03(2)°, γ=88.61(2)°, Z=2, R=7.96% for 5140 data where |F_o|>3σ(F). The two independent molecules alternate in infinite columns formed by head-to-tail hydrogen bonding. The helices in the two independent molecules are quite similar to each other but one molecule is rotated ≈?123° about its helix axis with respect to the other. All the helical columns pack parallel to each other in the crystal. Replacement of the bulky Boc group does not lead to any major changes in conformation. Packing characteristics are also similar to those observed for similar helical peptides.     

X-ray structure and characterization of a thermostable lipase from Geobacillus thermoleovorans

Thermo-alkalophilic bacterium, Geobacillus thermoleovorans secrets many enzymes including a 43?kDa extracellular lipase. Significant thermostability, organic solvent stability and wide substrate preferences for hydrolysis drew our attention to solve its structure by crystallography. The structure was solved by molecular replacement method and refined up to 2.14?Å resolution. Structure of the lipase showed an alpha-beta fold with 19 α-helices and 10 β-sheets. The active site remains covered by a lid. One calcium and one zinc atom was found in the crystal. The structure showed a major difference (rmsd 5.6?Å) from its closest homolog in the amino acid region 191 to 203. Thermal unfolding of the lipase showed that the lipase is highly stable with T_m of 76?°C. ¹³C NMR spectra of products upon triglyceride hydrolysate revealed that the lipase hydrolyses at both sn-1 and sn-2 positions with equal efficiency.     

Sugar ring isomerization in C-arabinosylflavones

6-C-α-l-Arabinopyranosyl- and furanosylacacetins have been synthesized. They are isomerized by short acid treatment to give a mixture of the four anomer/ring size combinations without any Wessely-Moser isomerization. In the same conditions molludistin (8-C-α-l-arabinopyranosylgenkwanin) led only to a mixture of molludistin and 8-C-α-l-arabinofuranosylgenkwanin. This is the first demonstration of ring sugar isomerization in C-glycosylflavones. In usual solvent systems, α-anomers are easily separated from β-anomers, whereas corresponding pyranosyl and furanosyl anomers are not. However, they are easily separated after permethylation and characteristic features are found in the mass spectra of PM 6-C-arabinofuranosyl isomers.     

The Crystal Structure of Glutamine-binding Protein fromEscherichia coli

The crystal structure of the glutamine-binding protein (GlnBP) fromEscherichia coliin a ligand-free “open” conformational state has been determined by isomorphous replacement methods and refined to anR-value of 21.4% at 2.3 Å resolution. There are two molecules in the asymmetric unit, related by pseudo 4-fold screw symmetry. The refined model consists of 3587 non-hydrogen atoms from 440 residues (two monomers), and 159 water molecules. The structure has root-mean-square deviations of 0.013 Å from “deal” bond lengths and 1.5° from “ideal” bond angles.The GlnBP molecule has overall dimensions of approximately 60 Å × 40 Å × 35 Å and is made up of two domains (termed large and small), which exhibit a similar supersecondary structure, linked by two antiparallel β-strands. The small domain contains three α-helices and four parallel and one antiparallel β-strands. The large domain is similar to the small domain but contains two additional α-helices and three more short antiparallel β-strands. A comparison of the secondary structural motifs of GlnBP with those of other periplasmic binding proteins is discussed.A model of the “closed form” GlnBP-Gln complex has been proposed based on the crystal structures of the histidine-binding protein-His complex and “open form” GlnBP. This model has been successfully used as a search model in the crystal structure determination of the “closed form” GlnBP-Gln complex by molecular replacement methods. The model agrees remarkably well with the crystal structure of the Gln-GlnBP complex with root-mean-square deviation of 1.29 Å. Our study shows that, at least in our case, it is possible to predict one conformational state of a periplasmic binding protein from another conformational state of the protein. The glutamine-binding pockets of the model and the crystal structure are compared and the modeling technique is described.     

Role of Tryptophan Residues in a Class V Chitinase from Nicotiana tabacum

Tryptophan residues located in the substrate-binding cleft of a class V chitinase from Nicotiana tabacum (NtChiV) were mutated to alanine and phenylalanine (W190F, W326F, W190F/W326F, W190A, W326A, and W190A/W326A), and the mutant enzymes were characterized to define the role of the tryptophans. The mutations of Trp326 lowered thermal stability by 5–7 °C, while the mutations of Trp190 lowered stability only by 2–4 °C. The Trp326 mutations strongly impaired enzymatic activity, while the effects of the Trp190 mutations were moderate. The experimental data were rationalized based on the crystal structure of NtChiV in a complex with (GlcNAc)₄, in which Trp190 is exposed to the solvent and involved in face-to-face stacking interaction with the +2 sugar, while Trp326 is buried inside but interacts with the ?2 sugar through hydrophobicity. HPLC analysis of anomers of the enzymatic products suggested that Trp190 specifically recognizes the β-anomer of the +2 sugar. The strong effects of the Trp326 mutations on activity and stability suggest multiple roles of the residue in stabilizing the protein structure, in sugar residue binding at subsite ?2, and probably in maintaining catalytic efficiency by providing a hydrophobic environment for proton donor Glu115.     

Synthetic C-nucleosides: 3-(alpha- and beta-D-arabinofuranosyl)pyrazolo(4,3-d)pyrimidine-5,7-diones

2,3,5-Tri-O-benzyl-D-arabinofuranosyl bromide (4) was converted into 2,5-anhydro-3,4,6-tri-O-benzyl-D-glucononitrile (5), mixed with 20% of the D-manno epimer 6. The mixture was reduced to the amine 7, which via the N-nitrosoacetamide 10 afforded the 1-deoxy-l-diazo sugar 11. Dipolar addition to dimethyl acetylene-dicarboxylate afforded the C-nucleoside derivative, dimethyl 3-(2,3,5-tri-O-benzyl-α-β-D-arabinofuranosyl)pyrazole-4,5-dicarboxylate (20). Selective ammonolysis afforded the 4-ester-5-carboxamide 21, which was separated chromatographically into the α-(minor) and β-(major) anomers. Hydrazinolysis and Curtius reaction of the pair of 4-acid hydrazides (α-22 and β-22) afforded the anomeric 3-glycosyl-1H-pyrazolo-[4,3-d]pyrimidine-5,7-diones (α-24 and β-24). Hydrogenolytic debenzylation yielded the β-D)-arabino epimer (1) of oxoformycin B, and the α-D-arabino form 2. These anomeric C-nucleosides were distinguished by circular dichroism spectra that showed the same relationship as α- and β-D-arabino anomers of normal purine nucleosides.