Role of hydroxyl group on the mesomorphism of alkyl glycosides:synthesis and thermal behavior of alkyl 6-deoxy-beta-D-glucopyranosides

A homologous series of alkyl 6-deoxy-beta-D-glucopyranoside amphiphiles was prepared,in an effort to identify the role of hydroxyl group in the mesomorphic behavior of alkyl glycosides. Synthesis was performed by a chlorination of the sugar moiety in alkyl-beta-D-glucopyranosides with methylsulfonyl chloride in DMF, followed by a metal mediated dehalogenation to secure alkyl 6-deoxy-beta-D-glucopyranosides, wherein the alkyl chain length varied from C-9 to C-16. The mesomorphic behavior of these 6-deoxy alkyl glycosides was assessed using polarizing optical microscopy, differential scanning calorimetry and X-ray diffraction method. Whereas the lower homologues exhibited a monotropic SmA phase till sub-ambient temperatures, the higher homologues formed a plastic phase. A partial interdigitized bilaye structure of SmA phase is inferred from experimental d-spacing and computationally derived lengths of the molecules. The results were compared with those of normal alkyl glucopyranosides, retained with hydroxyl groups at C-2-C-6 carbons, and alkyl 2-deoxy-glucopyranosides, devoid of a hydroxyl group at C-2 and the comparison showed important differences in the mesomorphic behavior.(C)2010 Elsevier Ireland Ltd. All rights reserved.

Expression of hyoscyamine 6 beta-hydroxylase in the root pericycle cells and accumulation of its product scopolamine in leaf and stem tissues of Datura metel L.

Hyoscyamine 60-hydroxylase (H6H: EC 1.14.11.11), a key enzyme at the terminal step of tropane alkaloid biosynthesis, converts hyoscyamine to scopolamine. The accumulation of scopolamine in different organs, in particular the aerial parts for storage, is subject to the expression of hyoscyamine 6-phydroxylase as well as its transport from the site of synthesis. To understand the molecular basis of this regulation, we have analyzed, in parallel, the relative levels of hyoscyamine and scopolamine, and the accumulation of H6H (both protein and transcript) in leaves, stems and roots of D. metel. The root, stem and leaf tissues all contain about 0.51-0.65 mg g(-1) dry weight of scopolamine. Hyoscyamine content was extremely low in leaf and stem tissues and was about 0.28 mg g(-1) dry weight in the root tissue. H6H protein and its transcript were found only in roots but not in the aerial parts viz. stems and leaves. The immunolocalization studies performed on leaf, stem, root as well as hairy root tissues showed that H6H was present only in the pericycle cells of young lateral and hairy roots. These studies suggest that the conversion of hyoscyamine to scopolamine takes place in the root pericycle cells, and the alkaloid biosynthesized in the roots gets translocated to the aerial parts in D. metel. (C) 2009 Elsevier Ireland Ltd. All rights reserved.

Biochemical and structural characterization of recombinant hyoscyamine 6 beta-hydroxylase from Datura metel L.

Hyoscyamine 6 beta-hydroxylase (H6H; EC 1.14.11.11), an important enzyme in the biosynthesis of tropane alkaloids, catalyzes the hydroxylation of hyoscyamine to give 6 beta-hydroxyhyoscyamine and its epoxidation in the biosynthetic pathway leading to scopolamine. Datura metel produces scopolamine as the predominant tropane alkaloid. The cDNA encoding H6H from D. mete! (DmH6H) was cloned, heterologously expressed and biochemically characterized. The purified recombinant His-tagged H6H from D. mete! (DmrH6H) was capable of converting hyoscyamine to scopolamine. The functionally expressed DmrH6H was confirmed by HPLC and ESI-MS verification of the products, 6 beta-hydroxyhyoscyamine and its derivative, scopolamine; the DmrH6H epoxidase activity was low compared to the hydroxylase activity. The K-m values for both the substrates, hyoscyamine and 2-oxoglutarate, were 50 mu M each. The CD (circular dichroism) spectrum of the DmrH6H indicated a preponderance of alpha-helicity in the secondary structure. From the fluorescence studies, Stern-Volmer constants for hyoscyamine and 2-oxoglutarate were found to be 0.14 M-1 and 0.56 M-1, respectively. These data suggested that the binding of the substrates, hyoscyamine and 2-oxoglutarate, to the enzyme induced significant conformational changes. (C) 2010 Elsevier Masson SAS. All rights reserved.

Fluorescence-polarization studies on binding of 4-methylumbelliferyl beta-D-galactopyranoside to Ricinus communis (castor-bean) agglutinin

The binding of Ricinus communis (castor-bean) agglutinin 1 to saccharides was studied by equilibrium dialysis and fluorescence polarization by using the fluorescently labelled sugar 4-methylumbelliferyl beta-D-galactopyranoside. No appreciable change in ligand fluorescence of 4-methylumbelliferyl beta-D-galactopyranoside was considerably polarized on its binding to the lectin. The association constants obtained by Scatchard analysis of equilibrium-dialysis and fluorescence-polarization data do not differ much from each other, and at 25 degrees C, Ka = 2.4 (+/- 0.2) X 10(4)M-1. These values agree reasonably well with that reported in the literature for Ricinus agglutinin 1. The number of binding sites obtained by the different experimental procedures is 1.94 +/- 0.1 per molecule of 120 000 daltons and is equal to the reported value of 2. The consistency in the values of Ka and number of binding sites indicate the absence of additional subsites on Ricinus agglutinin 1 for its specific sugars. In addition, the excellent agreement between the binding parameters obtained by equilibrium dialysis and fluorescence polarization indicate the potential of ligand-fluorescence-polarization measurements in the investigation of lectin-sugar interactions.

Structure of 7-methyl-1(2)a,1(6)a,3(4)a-trihomocubane-1(6)a,3(4)a-dione,star-c12h12o2- a case of enantiomeric and rotational disorder

M r = 188.22, monoclinic, P21/n, a = 6.219 (2), b= 10.508 (2), c=7.339 (1)A, t= 107.64 (2) °, V= 457 ,/k 3, Z = 2, D m - - 1.360 (3), D x = 1.366 (2)Mgm -3, ~,(MoKa) = 0.7107/~, #= 0.053 mm -I, F(000) = 200, T= 293 K. Final R = 5.8% for 614 significant reflections. The molecule, which does not possess a centre of symmetry, occupies a crystallographic centre of symmetry because of the statistical enantiomeric and rotational disorder. Latticeenergy calculations, based on van der Waals attractive and repulsive potentials, clearly show minima at the observed disordered positions.

Theoretical studies on the conformation of beta-D-N-acetyl neuraminic acid (sialic acid)

The possible conformations of sialic acid were analysed using semi-empirical potential functions. The solid state conformation has approx. 0.2 kcal/mol higher energy than the minimum energy conformation. These studies suggest that in solution sialic acid may exist preponderantly in two different conformations which differ in the orientation of the terminal hydroxymethyl group of glycerol side-chain. The present model is consistent with 1H- and 13C-NMR data, but differs from the earlier models.

The modes of binding methyl-alpha (and beta)-D-glucopyranosides and some of their derivatives to concanavalin A--a theoretical approach

The probable modes of binding of Methyl--alpha (and beta)-D-glucopyranosides and some of their derivatives to concanavalin A have been proposed from theoretical studies. Theory predicts that beta-MeGlcP can bind to ConA in three different modes whereas alpha-MeGlcP can bind only in one mode. beta-MeGlcP in its most favourable mode of binding differs from alpha-MeGlcP in its alignment in the active-site of the lectin where it binds in a flipped or inverted orientation. Methyl substitution at the C-2 atom of the alpha-MeGlcP does not significantly affect the possible orientations of the sugar in the active-site of the lectin. Methyl substitution at C-3 or C-4, however, affects the allowed orientations drastically leading to the poor inhibiting power of Methyl-3-O-methyl-alpha-D-glucopyranoside and the inactivity of Methyl-4-O-methyl-alpha-D-glycopyranoside. These studies suggest that the increased activity of the alpha-MeGlcP over beta-MeGlcP may be due to the possibility of formation of better hydrogen bonds and to hydrophobic interactions rather than to steric factors as suggested by earlier workers. These models explain the available NMR and other binding studies.

Stereospecific photodimerization of coumarins in crystalline inclusion complexes. Molecular and crystal structure of 1:2 complex of (S,S)-(-)-1,6-bis(o-chlorophenyl)-1,6-diphenyl-hexa-2,4-diyne-1,6-diol and coumarin

Proximity of molecules is a crucial factor in many solid- state photochemical processes.'S2 The biomolecular photodimerization reactions in the solid state depend on the relative geometry of reactant molecules in the crystal lattice with center-to-center distance of nearest neighbor double bonds of the order of ca. 4 A. This fact emanates from the incisive studies of Schmidt and Cohen.2 One of the two approaches to achieve this distance requirement is the so-called "Crystal-Engineering" of structures, which essentially involves the introduction of certain functional groups that display in-plane interstacking interactions (Cl...Cl, C-He-0, etc.) in the crystal The chloro group is by far the most successful in promoting the /3- packing m ~ d e ,th~o,u~gh recent studies have shown its limitations? Another approach involves the use of constrained media in which the reactants could hopefully be aligned.

Fluorescence polarization as a tool to study lectin-sugar interaction. An investigation of the binding of 4-methylumbelliferyl beta-D-galactopyranoside to Abrus precatorious agglutinin

Polarization of ligand fluorescence was used to study the binding of 4-methylumbelliferyl beta-D-galactopyranoside (MeUmb-Galp) to Abrus precatorious agglutinin. The binding of the fluorescent sugar to the lectin led to considerable polarization of the MeUmb-Galp fluorescence, which was also quenched by about 30% on binding to the lectin. The binding of the fluorescent sugar was carbohydrate-specific, as evidenced by inhibition of both fluorescence polarization and quenching when lectin was preincubated with lactose. The association constant as determined by fluorescence polarization is 1.42 x 10(4) M-1 at 25 degrees C and is in excellent agreement with those determined by fluorescence quenching (Ka = 1.51 x 10(4) M-1) and equilibrium dialysis (Ka = 1.62 x 10(4) M-1) at 25 degrees C. The numbers of binding sites as determined by fluorescence polarization, quenching and equilibrium dialysis agree very well with one another, n being equal to 2.0 +/- 0.05. The consistency between the association constant value determined by fluorescence polarization, quenching and equilibrium dialysis shows the validity of this approach to study lectin-sugar interaction.

Solid state structure of p-bromo phenyl 4,5,7-tri-O-benzyl-beta-D-glycero-D-talo-septanoside and an analysis of non-covalent interactions

The solid state structure of a new seven-membered sugar oxepane derivative, namely, p-bromo phenyl 4,5,7-tri-O-benzyl-beta-D-glycero-D-talo-septanoside is discussed, as determined through single crystal X-ray structural determination and in relation to their conformational features. The molecule adopts twist-chair as the preferred conformation, with conformational descriptor (TC2,3)-T-0,1. The solid state packing of molecules is governed by a rich network of non-covalent bonding originating from O-H center dot center dot center dot O, C-H center dot center dot center dot pi, C-H center dot center dot center dot Br and aromatic pi center dot center dot center dot pi interactions that stabilize the packing of molecules in the crystal. (C) 2015 Elsevier Ltd. All rights reserved.

Purification and characterization of a thermostable glucoamylase from the thermophilic fungus Thermomyces lanuginosus.

Glucoamylase (1,4-alpha-D-glucan glucohydrolase, EC 3.2.1.3) was purified from the culture filtrates of the thermophilic fungus Thermomyces lanuginosus and was established to be homogeneous by a number of criteria. The enzyme was a glycoprotein with an average molecular weight of about 57 000 and a carbohydrate content of 10-12%. The enzyme hydrolysed successive glucose residues from the non-reducing ends of the starch molecule. It did not exhibit any glucosyltransferase activity. The enzyme appeared to hydrolyse maltotriose by the multi-chain mechanism. The enzyme was unable to hydrolyse 1,6-alpha-D-glucosidic linkages of isomaltose and dextran. It was optimally active at 70 degrees C. The enzyme exhibited increase in the Vmax. and decreased in Km values with increasing chain length of the substrate molecule. The enzyme was inhibited by the substrate analogue D-glucono-delta-lactone in a non-competitive manner. The enzyme inhibited remarkable resistance towards chemical and thermal denaturation.

Synthesis of two designed hairpin peptides on a newly developed 1,6-hexanediol diacrylate (HDODA)-crosslinked polystyrene resin

Synthesis of two designed hairpin peptides on 1,6-hexanediol diacrylate crosslinked polystyrene support using the standard solid phase methodology is described. Both the peptides are obtained in high yield and purity. The new polymeric system is an ideal support for the synthesis of hairpin peptides, which is a very difficult task by the solid phase method.

Luteinizing hormone regulates inhibin-alpha subunit expression through multiple signaling pathways involving steroidogenic factor-1 and beta-catenin in the macaque corpus luteum

We employed different experimental model systems to define the role of GATA4, beta-catenin, and steroidogenic factor (SF-1) transcriptional factors in the regulation of monkey luteal inhibin secretion. Reverse transcription polymerase chain reactions and western blotting analyses show high expression of inhibin-alpha, GATA4, and beta-catenin in corpus luteum (CL) of the mid-luteal phase. Gonadotropin-releasing hormone receptor antagonist-induced luteolysis model suggested the significance of luteinizing hormone (LH) in regulating these transcriptional factors. Inducible cyclic AMP early repressor mRNA expression was detected in the CL and no change was observed in different stages of CL. Following amino acid sequence analysis, interaction between SF-1 and beta-catenin in mid-stage CL was verified by reciprocal co-immunoprecipitation experiments coupled to immunoblot analysis. Electrophoretic mobility shift analysis support the role of SF-1 in regulating luteal inhibin-alpha expression. Our results suggest a possible multiple crosstalk of Wnt, cAMP, and SF-1 in the regulation of luteal inhibin secretion.