Ab Initio studies of receptor interactions with AMPA ((S)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl) propionic acid ) and Kainic acid (2S-(2α,3β,4β))-2-carboxy-4-(1-methylethenyl)-3-pyrrolidineacetic acid

The optimum geometries and binding energies of the complexes formed by AMPA and Kainic acid, as well as their anions with tyrosine, proline and some tripeptides are investigated with quantum chemical calculations (HF/6-31G**). It was found that receptors featuring the Tyr-Ala-Pro sequence exhibit stronger binding energies to the substrates than the Tyr-Ser-Pro and Tyr-Ser-Ser. As expected, the anions are more bound than the neutral species. This work can lead to investigations on the effect of AMPA receptors mutations on the brain functions, possibly related to criminal tendencies.     

Synthesis and stability assay of 4-methylumbelliferyl (1→3)-β-D-pentaglucoside

The synthesis and stability of 4-methylumbelliferyl (1 → 3)-β-D-pentaglucoside 3 are described. The (1 → 3)-β-D-glucan isolated from the cell walls of Saccharomyces cerevisiae was recovered from the aqueous medium as water-insoluble particles by the spray drying (GS) method. The acid-solubilized (1 → 3)-β-D-oligoglucosides were prepared by partial acid hydrolysis of glucan. The peracetylated (1 → 3)-β-D-pentaglucoside 1 was obtained by isolation of peracetylated (1 → 3)-β-D-oligoglucoside mixture. The peracetylated 4-methylumbelliferyl (1 → 3)-β-D-pentaglucoside 2 was synthesized by treating compound 1 with the 4-methylumbelliferone and a Lewis acid (SnCl₄) catalyst. NaOMe in dry methanol was used for the deacetylation of the blocked derivative, to give the target compound 3 in an overall yield of 35%. Activity assays with β-glucosidase indicated that compound 3 was much more stable than the corresponding pentasaccharide.     

Synthesis of methyl O-(3-deoxy-3-fluoro-β-d-galactopyranosyl)-(1→6)-β-d-galactopyranoside and methyl O-(3-deoxy-3-fluoro-β-d-galactopyranosyl)-(1→6)-O-β-d-galactopyranosyl-(1→6)-β-d-galactopyranoside

Condensation of 2,4,6-tri-O-acetyl-3-deoxy-3-fluoro-α-d-galactopyranosyl bromide (3) with methyl 2,3,4-tri-O-acetyl-β-d-galactopyranoside (4) gave a fully acetylated (1→6)-β-d-galactobiose fluorinated at the 3′-position which was deacetylated to give the title disaccharide. The corresponding trisaccharide was obtained by reaction of 4 with 2,3,4-tri-O-acetyl-6-O-chloroacetyl-α-d-galactopyranosyl bromide (5), dechloroacetylation of the formed methyl O-(2,3,4-tri-O-acetyl-6-O-chloroacetyl-β-d-galactopyranosyl)-(1→6)- 2,3,4-tri-O-acetyl-β-d-galactopyranoside to give methyl O-(2,3,4-tri-O-acetyl-β-d-galactopyranosyl)-(1→6)-2,3,4-tri-O-acetyl-β-d-galactopyranoside (14), condensation with 3, and deacetylation. Dechloroacetylation of methyl O-(2,3,4-tri-O-acetyl-6-O-chloroacetyl-β-d-galactopyranosyl)-(1→6)-O-(2,3,4-tri-O-acetyl- β-d-galactopyranosyl)-(1→6)-2,3,4-tri-O-acetyl-β-d-galactopyranoside, obtained by condensation of disaccharide 14 with bromide 5, was accompanied by extensive acetyl migration giving a mixture of products. These were deacetylated to give, crystalline for the first time, the methyl β-glycoside of (1→6)-β-d-galactotriose in high yield. The structures of the target compounds were confirmed by 500-MHz, 2D, ¹H- and conventional ¹³C- and ¹⁹F-n.m.r. spectroscopy.     

Behavioral effects of the novel tropane analog, 2β-propanoyl-3β-(4-toluyl)-tropane (PTT)

In vitro studies have demonstrated that a novel tropane analog, PTT, in which both of the esters of cocaine have been removed is 20 times more potent than cocaine and more selective than cocaine in binding to dopamine transporters. The present studies compared the ability of PTT and cocaine to stimulate locomotor activity in rats. The intraperitoneal administration of PTT and cocaine to male Fisher-344 rats produced dose-dependent increases in spontaneous locomotor activity and stereotypic behaviors. PTT was 10–20 times more potent than cocaine in this behavioral assay, closely paralleling its potency relative to cocaine in dopamine transporter binding and uptake assays in vitro. PTT, however, elicited a qualitatively different profile of stereotypic behaviors, and PTT had a longer duration of action than cocaine. These results show how changes in kinetics and selectivity of tropanes can affect stimulant-elicited behaviors.     

New 4-deoxy-(1→3)-β-D-glucan-based oligosaccharides and their immunostimulating potential

(1→3)-β-D-Glucans are well-established natural biological immunomodulators. However, problems inherited with the natural origin of these polysaccharides bring about significant setbacks, including batch-to-batch heterogeneity and significant differences based on the source and isolation techniques. In this study, we tried to overcome these problems by preparation of a quantitatively new set of oligo-(1→3)-β-D-glucan-based synthetic immunomodulators. Some of these non-natural oligosaccharides showed biological activities, such as stimulation of phagocytosis, modulation of gene expression, and anti-cancer activity, which were superior to natural glucans.     

Synthesis and screening of (E)-1-(β-D-galactopyranosyl)-4-(aryl)but-3-ene-2-one against Mycobacterium tuberculosis

A series of galactose-derived aryl enones were synthesised and screened against Mycobacterium tuberculosis H₃₇Rv. Preliminary results were promising with MIC values in the range 1.56-12.5 μg/mL.     

Evolution of mixed-linkage (1 -> 3, 1 -> 4)-β-D-glucan (MLG) and xyloglucan in Equisetum (horsetails) and other monilophytes

Background and Aims

Horsetails (Equisetopsida) diverged from other extant eusporangiate monilophytes in the Upper Palaeozoic. They are the only monilophytes known to contain the hemicellulose mixed-linkage (1 → 3, 1 → 4)-β-d-glucan (MLG), whereas all land plants possess xyloglucan. It has been reported that changes in cell-wall chemistry often accompanied major evolutionary steps. We explored changes in hemicelluloses occurring during Equisetum evolution.

Methods

Hemicellulose from numerous monilophytes was treated with lichenase and xyloglucan endoglucanase. Lichenase digests MLG to di-, tri- and tetrasaccharide repeat-units, resolvable by thin-layer chromatography.

Key Results

Among monilophytes, MLG was confined to horsetails. Our analyses support a basal trichotomy of extant horsetails: MLG was more abundant in subgenus Equisetum than in subgenus Hippochaete, and uniquely the sister group E. bogotense yielded almost solely the tetrasaccharide repeat-unit (G4G4G3G). Other species also gave the disaccharide, whereas the trisaccharide was consistently very scarce. Tetrasaccharide : disaccharide ratios varied interspecifically, but with no consistent difference between subgenera. Xyloglucan was scarce in Psilotum and subgenus Equisetum, but abundant in subgenus Hippochaete and in the eusporangiate ferns Marattia and Angiopteris; leptosporangiate ferns varied widely. All monilophytes shared a core pattern of xyloglucan repeat-units, major XEG products co-chromatographing on thin-layer chromatography with non-fucosylated hepta-, octa- and nonasaccharides and fucose-containing nona- and decasaccharides.

Conclusions

G4G4G3G is the ancestral repeat-unit of horsetail MLG. Horsetail evolution was accompanied by quantitative and qualitative modification of MLG; variation within subgenus Hippochaete suggests that the structure and biosynthesis of MLG is evolutionarily plastic. Xyloglucan quantity correlates negatively with abundance of other hemicelluloses; but qualitatively, all monilophyte xyloglucans conform to a core pattern of repeat-unit sizes.     

Synthetic mucin fragments. Methyl 6-O-(2-acetamido-2-deoxy-β-D-glycopyranosyl)-β-D-galactopyranoside,methyl 3,4-di-O-(2-acetamido-2-deoxy-β-D-glycopyranosyl)-β-D-galactopyranoside,and methyl O-(2-acetamido-2-deoxy-β-D-glucopyranosyl)-(1 å 3)-O-β-D-galactopyranosyl-(1 å 3)-O-(2-acetamido-2-deoxy-β- D-glucopyranosyl)-(1 å 3)-β-D-galactopyranoside

Condensation of methyl 2,6-di-O-benzyl-β-d-galactopyranoside with 2-methyl-(3,4,6-tri-O-acetyl-1,2-dideoxy-α-d-glucopyrano)-[2,1,-d]-2-oxazoline (1) in 1,2-dichloroethane, in the presence of p-toluenesulfonic acid, afforded a trisaccharide derivative which, on deacetylation, gave methyl 3,4-di-O-(2-acetamido-2-deoxy-β-d-glucopyranosyl)-2,6-di-O-benzyl-β-d- glactopyranoside (5). Hydrogenolysis of the benzyl groups of 5 furnished the title trisaccharide (6). A similar condensation of methyl 2,3-di-O-benzyl-β-d-galactopyranoside with 1 produced a partially-protected disacchraide derivative, which, on O-deacetylation followed by hydrogenolysis, gave methyl 6-O-(2-acetamido-2-deoxy-β-d-glucopyranosyl)-β-d-glactopyranoside (10). Condensation of methyl 3-O-(2-acetamido-4,6-O-benzylidene-2-deoxy-β-d-glucopyranosyl)-2,4,6-tri-O-benzyl-β-d- galactopyranoside with 3-O-(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-d-glucopyranosyl)-2,4,6-tri-O-acetyl-α-d-galactopyranosyl bromide in 1:1 benzene-nitromethane in the presence of powdered mercuric cyanide gave a fully-protected tetrasaccharide derivative, which was O-deacetylated and then subjected to catalytic hydrogenation to furnish methyl O-(2-acetamido-2-deoxy-β-d-glucopyranosyl)-(1→3)-O-β-d-galactopyranosyl-(1å3)-O-(2-acetamido-2-deoxy- β-d-glucopyranosyl)-(1å3)-β-d-galactopyranoside (15). The structures of 6, 10, and 15 were established by ¹³C-n.m.r. spectroscopy.     

Purification and characterization of (1→3, 1→4)-β-glucan endohydrolases from germinated wheat (Triticum aestivum)

A (13, 14)--glucan 4-glucanohydrolase [(13, 14)--glucanase, EC 3.2.1.73] was purified to homogeneity from extracts of germinated wheat grain. The enzyme, which was identified as an endohydrolase on the basis of oligosaccharide products released from a (13, 14)--glucan substrate, has an apparent pI of 8.2 and an apparent molecular mass of 30 kDa. Western blot analyses with specific monoclonal antibodies indicated that the enzyme is related to (13, 14)--glucanase isoenzyme EI from barley. The complete primary structure of the wheat (13, 14)--glucanase has been deduced from nucleotide sequence analysis of cDNAs isolated from a library prepared using poly(A)⁺ RNA from gibberellic acid-treated wheat aleurone layers. One cDNA, designated LW2, is 1426 nucleotide pairs in length and encodes a 306 amino acid enzyme, together with a NH₂-terminal signal peptide of 28 amino acid residues. The mature polypeptide encoded by this cDNA has a molecular mass of 32085 and a predicted pI of 8.1. The other cDNA, designated LW1, carries a 109 nucleotide pair sequence at its 5 end that is characteristic of plant introns and therefore appears to have been synthesized from an incompletely processed mRNA. Comparison of the coding and 3-untranslated regions of the two cDNAs reveals 31 nucleotide substitutions, but none of these result in amino acid substitutions. Thus, the cDNAs encode enzymes with identical primary structures, but their corresponding mRNAs may have originated from homeologous chromosomes in the hexaploid wheat genome.     

Enzymic glycosphingolipid synthesis on polymer supports. III. Synthesis of GM3, its analog [NeuNAcα(2-3)Galβ(1-4)Glcβ(1-3)Cer] and their lyso-derivatives

Two water-soluble polymers, carrying 0.24 meq g–1 of lactosyl-(1-1)-sphingosine (7) and 0.13 meq g–1 of lactosyl-(1-3)-sphingosine (8) were prepared. The polymers served as acceptors in the -(2-3)-sialyltransferase reaction (up to 55.3 and 38.5% transfer yields, respectively). Subsequent photolysis, released compounds 11 (lyso-GM3) and 12 (lyso-GM3 analog), respectively; acylation and chromatography afforded (5-acetamido-3,5-dideoxy-D-glycero--D-galacto-2-nonulopyranosylonic acid)-(2-3)--D-galactopyranosyl-(1-4)--D-glucopyranosyl-(1-1)-(2S, 3R, 4E)-2-octadecanoylamino-4-octadecene-1,3-diol (13, GM3) and (5-acetamido-3,5-dideoxy-D-glycero--D-galacto-2-nonulopyranosylonic acid)-(2-3)--D-galactopyranosyl-(1-4)--D-glucopyranosyl-(1-3)-(2S, 3R, 4E)-2-octadecanoylamino-4-octadecene-1,3-diol (14, GM3 analogue), respectively, thus presenting a route to glycosphingolipids possessing the unusual glycosyl-(1-3)-spingosine linkage.     

Action of rat liver Galβ1-4GlcNAc α(2-6)-sialyltransferase on Manβ1-4GlcNAcβ-OMe,GalNAcβ1-4GlcNAcβ-OMe,Glcβ1-4GlcNAcβ-OMe and GlcNAcβ1-4GlcNAcβ-OMe as synthetic substrates

Incubation of synthetic Man\1-4GlcNAc-OMe, GalNAc1-4GlcNAc-OMe, Glc1-4GlcNAc-OMe, and GlcNAc1-4GlcNac-OMe with CMP-Neu5Ac and rat liver Gal1-4GlcNAc (2-6)-sialyltransferase resulted in the formation of Neu5Ac2-6Man1-4GlcNAc-OMe, Neu5Ac2-6GalNAc1-4GlcNAc-OMe, Neu5Ac2-6Glc1-4GlcNAc-OMe and Neu5Ac2-6GlcNAc1-4GlcNAc-OMe, respectively. Under conditions which led to quantitative conversion of Gal1-4GlcNAc-OEt into Neu5Ac2-6Gal1-4GlcNAc-OEt, the aforementioned products were obtained in yields of 4%, 48%, 16% and 8%, respectively. HPLC on Partisil 10 SAX was used to isolate the various sialyltrisaccharides, and identification was carried out using 1- and 2-dimensional 500-MHz¹H-NMR spectroscopy.Abbreviations 2D 2-dimensional - CMP cytidine 5-monophosphate - CMP-Neu5Ac cytidine 5-monophospho--N-acetylneuraminic acid - COSY correlation spectroscopy - DQF double quantum filtered - HOHAHA homonuclear Hartmann-Hahn - MLEV composite pulse devised by M. Levitt - Neu5Ac N-acetylneuraminic acid - Neu5Ac2en 2-deoxy-2,3-didehydro-N-acetylneuraminic acid     

Curdlan and other bacterial (1→3)-β-d-glucans

Three structural classes of (13)--d-glucans are encountered in some important soil-dwelling, plant-associated or human pathogenic bacteria. Linear (13)--glucans and side-chain-branched (13,12)--glucans are major constituents of capsular materials, with roles in bacterial aggregation, virulence and carbohydrate storage. Cyclic (13,16)--glucans are predominantly periplasmic, serving in osmotic adaptation. Curdlan, the linear (13)--glucan from Agrobacterium, has unique rheological and thermal gelling properties, with applications in the food industry and other sectors. This review includes information on the structure, properties and molecular genetics of the bacterial (13)--glucans, together with an overview of the physiology and biotechnology of curdlan production and applications of this biopolymer and its derivatives.     

Syntheses of (14β,17α)-14-Hydroxy- and (14β,17α)-2,14-Dihydroxyestradiols and Their Activities

Structure 1 is proposed for the Inagami-Tamura endogenous digitalis-like factor (EDLF), and (14β,17α)-14-hydroxy- and (14β, 17α)-2,14-dihydroxyestradiols (2 and 3) were synthesized as models for studies on 1. The latter compound was remarkably potent in inducing a contractile response in isolated rat aorta and guinea pig left atrium.     

Synthesis of 2-(3-Deoxy-β-D-erythropentofuranosyl)-thiazole-4-carboxamide (3′-Deoxytiazofurin)

Abstract

2-(3-Deoxy-β-D-erythropentofuranosyl)-thiazole-4-carboxamide was synthesized in four steps from its β-D-ribofuranosyl nucleoside precursor.     

Definitive Solution Structures for the 6-Formylated Versions of 1-(βD-Ribofuranosyl)-, 1-(2′-Deoxy-β-D-Ribofuranosyl)-, and 1-β-D-Arabinofuranosyluracil,and of Thymidine

Abstract

ROESY and NOESY NMR spectroscopic analyses of the ribofuranosyl (1a), 2′-deoxyribofuranosyl (1b), and arabinofuranosyl (1c) derivatives of 6-formyluracil in (CD₃)₂SO and D₂O solutions have established that each exclusive 7,05′-cyclic hemiacetal diastereomer of 1a,b and the major 7,O2′-cyclic hemiacetal diastereomer of 1c possess the 7R configuration. In addition, (7R)-1c has been shown to be thermodynamically more stable than (7S)-1c, contrary to our previous indication. A new, higher yielding synthetic route to 1a has been developed, 1b has been obtained for the first time in crystalline form, the route to 1c has been modified to better accommodate large scale preparations, and a new, fourth member of this class, 6-formylthymidine (1d), has been synthesized and its solution structures in (CD₃)₂SO, D₂O, and CD₃OD have been determined. Antitumor and antiviral evaluations of 1a-c have revealed no significant levels of activity.     

Cell wall (1 → 3)- and (1 → 3, 1 → 4)-β-glucans during early grain development in rice (Oryza sativa L.)

Immunogold labeling was used to study the distribution of (1 → 3)-β-glucans and (1 → 3, 1 → 4)-β-glucans in the rice grain during cellularization of the endosperm. At approximately 3–5 d after pollination the syncytial endosperm is converted into a cellular tissue by three developmentally distinct types of wall. The initial free-growing anticlinal walls, which compartmentalize the syncytium into open-ended alveoli, are formed in the absence of mitosis and phragmoplasts. This stage is followed by unidirectional (centripetal) growth of the anticlinal walls mediated by adventitious phragmoplasts that form between adjacent interphase nuclei. Finally, the periclinal walls that divide the alveoli are formed in association with centripetally expanding interzonal phragmoplasts following karyokinesis. The second and third types of wall are formed alternately until the endosperm is cellular throughout. All three types of wall that cellularize the endosperm contain (1 → 3)-β-glucans but not (1 → 3, 1 → 4)-β-glucans, whereas cell walls in the surrounding maternal tissues contain considerable amounts of (1 → 3, 1 → 4)-β-glucans with (1 → 3)-β-glucans present only around plasmodesmata. The callosic endosperm walls remain thin and cell plate-like throughout the cellularization process, appearing to exhibit a prolonged juvenile state. Received: 7 January 1997 / Accepted: 11 February 1997     

Purification and Characterization of endo-(1→4)-β-xylanase fromGeotrichum candidum 3C

A method of purification of endo-( 1 → 4)-β-xylanase (endoxylanase; EC 3.2.1.8) from the culture liquid ofGeotrichum candidum 3C, grown for three days, is described. The enzyme, purified 23-fold, had a specific activity of 32.6 U per mg protein (yield, 14.4%). Endoxylanase was shown to be homogeneous by SDS-PAGE (molecular weight, 60 to 67 kDa). With carboxymethyl xylan as the substrate, the optimum activity (determined viscosimetrically) was recorded at pH 4.0 (pI 3.4). The enzyme retained stability at pH 3.0-4.5 and 30–45°C for 1 h. With xylan from birch wood, the hydrolytic activity of the enzyme (ability to saccharify the substrate) was maximum at 50°C. In 72 h of exposure to 0.2 mg/ml endoxylanase, the extent of saccharification of xylans from birch wood, rye grain, and wheat straw amounted to 10,12, and 7.7%, respectively. At 0.4 mg/ml, the extent of saccharification of birch wood xylan was as high as 20%. In the case of birch wood xylan, the initial hydrolysis products were xylooligosaccharides with degrees of polymerization in excess of four; the end products were represented by xylobiose, xylotriose, xylose, and acid xylooligosaccharides.     

Syntheses of p-nitrophenyl 3- and 4-thio-β-D-glycopyranosides

Thioglycosides have proved to be useful, enzymatically stable analogs of glycosides for structural and mechanistic studies and their synthesis is considerably simplified through the use of thioglycoligases. As part of an investigation into the use of thioglycosides as potential pharmacological chaperones, and as components of glycoproteins and glycolipids, the syntheses of p-nitrophenyl 3-thio-β-d-galactopyranoside, phenyl 1,4-dithio-β-d-glucopyranoside, p-nitrophenyl 4-thio-β-d-mannopyranoside and p-nitrophenyl 2-acetamido-2-deoxy-4-thio-β-d-mannopyranoside are described.     

Chemical analysis of new water-soluble (1→6)-, (1→4)-α, β-glucan and water-insoluble (1→3)-, (1→4)-β-glucan (Calocyban) from alkaline extract of an edible mushroom, Calocybe indica (Dudh Chattu)

Two different glucans (PS-I, water-soluble; and PS-II, water-insoluble) were isolated from the alkaline extract of fruit bodies of an edible mushroom Calocybe indica. On the basis of acid hydrolysis, methylation analysis, periodate oxidation, and NMR analysis ((1)H, (13)C, DEPT-135, TOCSY, DQF-COSY, NOESY, ROESY, HMQC, and HMBC), the structure of the repeating unit of these polysaccharides were established as: PS-I: →6)-β-D-Glcp-(1→6)-β-D-glcp-(1→6)-)-β-D-Glcp-(1→ α-D=Glcp (Water-soluble glucan). PS-II: →3)-β-D-Glcp-(1→3)-β-D-glcp-(1→3)-)-β-D-Glcp-(1→3)-β-D-Glcp-(1→ β-D-Glcp (Water-insoluble glucan, Calocyban).     

Discovery of (3S,4S)-3-methyl-3-(4-fluorophenyl)-4-(4-(1,1,1,3,3,3-hexafluoro-2-hydroxyprop-2-yl)phenyl)pyrrolidines as novel RORγt inverse agonists

A novel series of cis-3,4-diphenylpyrrolidines were designed as RORγt inverse agonists based on the binding conformation of previously reported bicyclic sulfonamide 1. Preliminary synthesis and structure–activity relationship (SAR) study established (3S,4S)-3-methyl-3-(4-fluorophenyl)-4-(4-(1,1,1,3,3,3-hexafluoro-2-hydroxyprop-2-yl)phenyl)pyrrolidine as the most effective scaffold. Subsequent SAR optimization led to identification of a piperidinyl carboxamide 31, which was potent against RORγt (EC₅₀ of 61 nM in an inverse agonist assay), selective relative to RORα, RORβ, LXRα and LXRβ, and stable in human and mouse liver microsomes. Furthermore, compound 31 exhibited considerably lower PXR Y_max (46%) and emerged as a promising lead. The binding mode of the diphenylpyrrolidine series was established with an X-ray co-crystal structure of 10A/RORγt.