A novel imidazole nucleoside containing a diaminodihydro-S-triazine as a substituent: inhibitory activity against the West Nile virus NTPase/helicase

The attempted synthesis of a ring-expanded guanosine (1) containing the imidazo[4,5-e][1,3]diazepine ring system by condensation of 1-(2'-deoxy-beta-D-erythropentofuranosyl)-4-ethoxycarbonylimidazole-5-carbaldehyde (2) with guanidine resulted in the formation of an unexpected product, 1-(2'-deoxy-beta-D-erythropentofuranosyl)-5-(2, 4-diamino-3, 6-dihydro-1,3, 5-triazin-6-yl)imidazole-4-carboxamide (7). The structure as well as the pathway of formation of 7 was corroborated by isolation of the intermediate, followed by its conversion to the product. Nucleoside 7 showed promising in vitro anti-helicase activity against the West Nile virus NTPase/helicase with an IC50 of 3-10 microg/mL.     

3-(4-Methyl-3-pentenyl)-2(5H)-furanone, alpha,alpha-acariolide and 4-(4-methyl-3-pentenyl)-2(5H)-furanone, alpha,beta-acariolide: new monoterpene lactones from the astigmatid mites, Schwiebea araujoae and Rhizoglyphus sp. (Astigmata: Acaridae)

A new monoterpene lactone from the acarid mite, Schwiebea araujoae, was elucidated without its isolation by GC/FT-IR and GC/MS analyses to be 3-(4-methyl-3-pentenyl)-2(5H)-furanone (1) and tentatively named as alpha,alpha-acariolide. The structure of 1 was identified by its synthesis from alpha-bromo-gamma-butyrolactone via 4 reaction steps. The synthesized compound gave the same GC/MS and GC/FT-IR spectra as those of the natural product. The other monoterpene lactone was likewise elucidated from the unidentified Rhizoglyphus mite to be 4-(4-methyl-3-pentenyl)-2(5H)-furanone (2) and named as alpha,beta-acariolide; it was also identified by its synthesis in 5 reaction steps from the same butyrolactone as the starting material. GC/MS and GC/FT-IR spectra of the preparation were identical to those of the natural product.     

Structural elucidation of novel degradation product of brotizolam

When the drug product of brotizolam (1) is decomposed according to the interview form and patent, hydrolysate (2) is obtained, but its physicochemical data are still missing. To elucidate the structure based on a spectroscopic approach, the above-mentioned degradation product was isolated and applied to the structural analysis. As a result, the structure of decomposed product was found to be different from that of 2. In this report, its isolation and structure elucidation by NMR and MS spectra are described.     

A Novel Imidazole Nucleoside Containing a Diaminodihydro-S-triazine as a Substituent: Inhibitory Activity Against the West Nile Virus NTPase/Helicase

The attempted synthesis of a ring-expanded guanosine (1) containing the imidazo[4,5-e][1,3]diazepine ring system by condensation of 1-(2′-deoxy-β-D-erythropentofuranosyl)-4-ethoxycarbonylimidazole-5-carbaldehyde (2) with guanidine resulted in the formation of an unexpected product, 1-(2′-deoxy-β-D-erythropentofuranosyl)-5-(2,4-diamino-3,6-dihydro-1,3,5-triazin-6-yl)imidazole-4-carboxamide (7). The structure as well as the pathway of formation of 7 was corroborated by isolation of the intermediate, followed by its conversion to the product. Nucleoside 7 showed promising in vitro anti-helicase activity against the West Nile virus NTPase/ helicase with an IC ₅₀ of 3-10 μg/mL.     

Improved method of lipopolysaccharide isolation from gram-negative bacteria

A modification of the traditional method for lipoplysaccharide isolation from the cells of grammnegative bacteria was elaborated on the basis of extraction by the hot water solution of phenol (the method of Westfahl). To make the method simpler and to raise the yield of the product it was proposed to use the water-phenol extract without its division for plases. The nucleic acids are eliminated by precipitation from dialyzed extract at pH 3,2-3,4 achieved by addition of acetic acid. The comparative isolation of lipopolysaccharides by the classic and modified methods has confirmed the advantages of a new technique.     

Structural insights into the function of the thiamin biosynthetic enzyme Thi4 from Saccharomyces cerevisiae

The structure of thiazole synthase (Thi4) from Saccharomyces cerevisiae was determined to 1.8 A resolution. Thi4 exists as an octamer with two monomers in the asymmetric unit. The structure reveals the presence of a tightly bound adenosine diphospho-5-(beta-ethyl)-4-methylthiazole-2-carboxylic acid at the active site. The isolation of this reaction product identifies NAD as the most likely precursor and provides the first mechanistic insights into the biosynthesis of the thiamin thiazole in eukaryotes. Additionally, the Thi4 structure reveals the first protein structure with a GR(2) domain that binds NAD instead of FAD, raising interesting questions about how this protein evolved from a flavoenzyme to a NAD binding enzyme.     

Chemistry of the collagen cross-links. Isolation and characterization of two intermediate intermolecular cross-links in collagen

This paper describes the isolation from reduced collagen of two new amino acids believed to be involved, in their non-reduced form, as intermolecular cross-links stabilizing the collagen fibre. The reduction of intact collagen fibrils with tritiated sodium borohydride was found to stabilize the aldehyde-mediated cross-links to acid hydrolysis and thus allowed their location and isolation from acid hydrolysates on an automatic amino acid analyser. Comparison of the radioactive elution patterns from the autoanalyser of collagen treated in various ways before reduction permitted a preliminary classification of the peaks into cross-link precursors, intramolecular and intermolecular cross-links. The techniques employed to isolate the purified components on a large scale and to identify them structurally are described in detail. Two labile intermolecular cross-links were isolated in their reduced forms, one of which was identified by high-resolution mass spectrometry as N-(5-amino-5-carboxypentyl)hydroxylysine. The structure of this compound was confirmed by chemical synthesis. The cross-link precursor α-aminoadipic δ-semialdehyde was isolated in its reduced form, -hydroxynorleucine, together with its acid degradation product -chloronorleucine. A relatively stable intermolecular cross-link was isolated and partially characterized by mass spectrometry as an aldol resulting from the reaction of the δ-semialdehyde derived from lysine and hydroxylysine.     

Unresolved rearrangement of thiazoline form of glutathione.

The thiazoline form of glutathione was investigated with regard to its unresolved instability under neutral conditions. A simple method was developed for production of the thiazoline in stable solid form, thereby facilitating preparation of its neutral solution without using excess base and enabling isolation of the reaction product in quantity by ion-exchange chromatography. Analysis of the product by HPLC, infrared and ultraviolet absorption spectroscopy, mass spectrometry and proton magnetic resonance led to the identification of a cyclic amidine form of glutathione. The instability of the thiazoline is, therefore, due to an intramolecular rearrangement reaction, rather than hydrolysis. Once formed, the amidine is stable at pH 5-7 and in concentrated HCl, showing no tendency to rearrange back to either the original thiazoline or glutathione under these conditions.     

Characterisation of a DNA Polymerase Highly Mutated Along the Template Binding Interface

Phage display establishes a link between a polypeptide and its corresponding gene. It has been much used for the isolation of proteins binding to chosen molecular targets. A second link was designed more recently between a phage-displayed enzyme and its reaction product. Affinity chromatography for the product then allows the isolation of catalytically active enzymes and of their genes. Using this strategy, a polymerase with 15 mutations was selected by directed evolution of Thermus aquaticus DNA polymerase I. The kinetic characterisation reported here highlights the variant’s broad template specificity and classifies this enzyme as a thermostable DNA-dependent and RNA-dependent DNA-polymerase.     

Sorbicillinol, a key intermediate of bisorbicillinoid biosynthesis in Trichoderma sp. USF-2690.

In the course of our screening program for free radical scavengers from Trichoderma sp. USF-2690, we found an unidentified metabolite (1) that appeared by the method used for HPLC analysis. Metabolite 1 gradually decreased with the production of bisorbicillinoids and was easily missed during the general isolation procedure. The LC-ESI-MS (negative) analysis for 1 gave m/z 247 as the (M-1)- ion peak. The hydrolysis of synthetic 6-O-acetylsorbicillinol (+/- -2) by 0.05 M KOH and acetylation of product 1 in an aqueous solution indicated that the structure of 1 was (6S)-4-(2,4-hexadienoyl)-3,6-dihydroxy-2,6-dimethyl-2,4-cyclohexadien-1-one, designated sorbicillinol, a quinol that has been postulated to be important in bisorbicillinoid biosynthesis.     

A comparison of methods measuring aromatase activity in human placenta and rat ovary

The single step chromatographic product isolation method using [4-14C]-androstenedione and the tritiated water method using either [1 beta, 2 beta-3H]- or [1 beta-3H]-androstenedione have been used to determine a suitable method to measure the aromatase activity in rat ovarian 1000 g supernatant and human placental microsomes. The single step product isolation method using [4-14C]A4 reveals the presence of four distinct [4-14C]-labelled products in the rat ovary of which only the synthesis of estradiol is markedly inhibited by CGS 16949A, a well established aromatase inhibitor. In the human placenta, the formation of both [4-14C]-estrone and [4-14C]-estradiol is strongly inhibited by CGS 6949A. Therefore, in the rat ovary spurious results are obtained if accumulative radiolabelled product formation is measured without characterisation of the products. The Vmax in the rat ovary using [1 beta, 2 beta-3H]-A4 as a substrate is 13.7 pmol/h/mg compared to 2.9 pmol/h/mg when [1 beta-3H]-A4 is used. In the human placenta, the Vmax is similar using either [1 beta, 2 betat-3H]-A4 or [1 beta-3H]-A4 (1.21 and 1.27 nmol/h/mg, respectively). Consistent results are obtained for the human placenta using either the single step chromatographic product isolation method or the tritiated water method. However, in the rat ovary the more suitable method of the two used to measure the aromatase activity is the tritiated water method employing [1 beta-3H]-A4 as a substrate. Aromatase activity in the rat ovary during estrus cycle was measured using the tritiated water method employing [1 beta-3H]-A4 as a substrate. A peak of aromatase activity at proestrus was seen which returned rapidly to its basal level at estrus. Plasma estradiol concentrations were in parallel with the aromatase activity.     

Targeted development of microsatellite markers from the defined region of bovine Chromosome 6q21-31

A methodical strategy for the isolation of microsatellite markers specific for targeted regions of bovine chromosomes is presented. The procedure involves directed microdissection of one defined subchromosomal area, its DOP-PCR-amplification and cloning. With this approach, a library specific to the BTA 6q21-31 chromosomal region was constructed. Eleven unique microsatellite-containing sequences were isolated, converted into sequence-tagged microsatellite sites, and characterized concerning their species-specific origin. Seven primer pairs generated bovine-specific PCR products and provided a set of microsatellite markers that generally revealed high informativity in the HF breed. Linkage analysis assigned six of them to their predefined subchromosomal origin on BTA 6 corresponding to the specific rehybridization signal of the DOP-PCR product generated from the microdissected chromosome area 6q21-31. The results underline the usefulness of the BTA 6q21-31 library for targeted isolation of unique sequences that are specific for the dissected chromosomal region as demonstrated here by the isolation of microsatellite markers. Received: 27 March 1997 / Accepted: 14 July 1997     

Isolation of oryzanol from crude rice bran oil

The isolation of oryzanol from crude rice bran oil (RBO) was achieved by a two-step crystallization process. In the first crystallization, oryzanol was concentrated in the liquid phase along with free fatty acid (FFA), monoacylglycerol (MG), squalene, tocols, and phytosterols, whereas the solid phase contained mainly triacylglycerol (TG) and steryl esters. Oryzanol-rich product obtained from the first crystallization was subjected to the second crystallization where the oryzanol-rich product was kept at room temperature (20.5+/-1.5 degrees C) for 24h. Hexane was added as anti-solvent to the oryzanol-rich product and kept at 5+/-1 degrees C for another 48h. Parameters that affect the isolation of oryzanol from crude RBO were systematically investigated. Under optimal operation conditions, oryzanol with purity and recovery of 93-95% and 59%, respectively, was obtained from RBO with an initial FFA content of ca. 5%.     

Cytotoxicity of purified cassava linamarin to a selected cancer cell lines

Cassava (Manihot esculenta Crantz) is a known source of linamarin, but difficulties associated with its isolation have prevented it from being exploited as a major source. A batch adsorption process using activated carbon proved successful in its isolation, with ultrafiltration playing a pivotal role in its purification. Thirty-two minutes of contact time was required for 60 g of extract, yielding 1.7 g of purified product. Picrate paper, infra-red and ¹HNMR analysis confirmed the presence and structure of linamarin. Cytotoxic effects of linamarin on MCF-7, HT-29 and HL-60 cells were determined using the MTT assay. Cytotoxic effects were significantly increased in the presence of linamarase (β-glucosidase), with a 10–fold decrease in the IC₅₀ values obtained for HL-60 cells. This study thus describes a method for the isolation and purification of linamarin from cassava, as well as its cytotoxicity potential.     

Genetic system for analyzing Escherichia coli thymidylate synthase.

Random in vitro mutagenesis of the thyA gene is being used to delineate its regulatory elements as well as the functional domains of its product, thymidylate synthase (EC 2.1.1.45). Streamlined procedures have been developed for the isolation and characterization of the mutants. Positive selection for synthase-deficient thyA Escherichia coli permitted the isolation of 400 mutants, which are being categorized by phenotypic and genetic criteria. An in situ 5-fluorodeoxyuridylate binding assay was devised to rapidly probe the substrate binding domain, whereas facile mapping procedures, based on pBR322- or M13-borne thyA deletion derivatives, were developed to localize mutations. The sequence changes of one amber mutation and another mutation that abolishes catalysis while maintaining substrate binding activity are presented. The orientation of the thyA gene on the E. coli chromosome was established.     

Investigations of the transfructosylation reaction by fructosyltransferase from B. subtilis NCIMB 11871 for the synthesis of the sucrose analogue galactosyl-fructoside

The exo-fructosyltransferase produced from B. subtilis NCIMB 11871 strain transfers the fructose moiety from donor alpha12 linked saccharides such as sucrose, raffinose and stachyose to the acceptor d-galactose, leading to the sucrose analogue, galactosyl-fructoside. Here, we report detailed kinetic studies. The enzyme showed a remarkably high optimal temperature at 50 degrees C and was effectively immobilised on Eupergit C 250 L and Trisopor-Amino. This is also the first report about the equilibrium of the transfructosylation reaction, its activation energy determination, the structure of the product and its preparative scale isolation.     

Molecular cloning and functional characterization of a Lepidopteran insect beta4-N-acetylgalactosaminyltransferase with broad substrate specificity, a functional role in glycoprotein biosynthesis, and a potential functional role in glycolipid biosynthesis

A degenerate PCR approach was used to isolate a lepidopteran insect cDNA encoding a beta4-galactosyl-transferase family member. The isolation and initial identification of this cDNA was based on bioinformatics, but its identification as a beta4-galactosyltransferase family member was experimentally confirmed. The newly identified beta4-galactosyltransferase family member had unusually broad donor and acceptor substrate specificities in vitro, as transferred galactose, N-acetylglucosamine, and N-acetylgalactosamine to carbohydrate, glycoprotein, and glycolipid acceptors. However, the enzyme preferentially utilized N-acetylgalactosamine as the donor for all three acceptors, and its derived amino acid sequence was closely related to a known N-acetylgalactosaminyltransferase. These data suggested that the newly isolated cDNA encodes a beta4-N-acetylgalactosaminyltransferase that functions in insect cell glycoprotein biosynthesis, glycolipid biosynthesis, or both. The remainder of this study focused on the role of this enzyme in N-glycoprotein biosynthesis. The results showed that the purified enzyme transferred N-acetylgalactosamine, but no detectable galactose or N-acetylglucosamine, to a synthetic N-glycan in vitro. The structure of the reaction product was confirmed by chromatographic, mass spectroscopic, and nuclear magnetic resonance analyses. Co-expression of the new cDNA product in insect cells with an N-glycoprotein reporter showed that it transferred N-acetylgalactosamine, but no detectable galactose or N-acetylglucosamine, to this N-glycoprotein in vivo. Confocal microscopy showed that a GFP-tagged version of the enzyme was localized in the insect cell Golgi apparatus. In summary, this study demonstrated that lepidopteran insect cells encode and express a beta4-N-acetylgalactosaminyltransferase that functions in N-glycoprotein biosynthesis and perhaps in glycolipid biosynthesis, as well. The isolation and characterization of this gene and its product contribute to our basic understanding of insect protein N-glycosylation pathways and to the growing body of evidence that insects can produce glycoproteins with complex N-glycans.     

Trimethylamine dehydrogenase from a methylotrophic bacterium. I. Isolation and steady-state kinetics.

1. The isolation of trimethylamine dehydrogenase (EC 1.5.99.7) from a restricted facultative methylotroph to electrophoretic homogeneity is described. 2. The molecular weight and subunit molecular weights were found to be 146800 for the enzyme by sedimentation equilibrium ultracentrifugation and 70000-80000 for the two non-identical subunits by sodium dodecyl sulphate gel electrophoresis. 3. Initial velocity studies indicate that the enzymatic reaction proceeds by a Ping-Pong mechanism. 4. Further kinetic evidence was obtained by analysis of product inhibition patterns using the alternate substrate diethylamine and the products acetaldehyde and ethylamine as product inhibitors, for the release of ethylamine before the addition of phenazine methosulphate and for the existence of an enzyme-two-carbon unit complex as a stable form of the enzyme. 5. Some properties of the unusual prosthetic group of trimethylamine dehydrogenase and its photodegradation product are described in preliminary form.     

Separation and quantification of neoagaro- and agaro-oligosaccharide products generated from agarose digestion by beta-agarase and HCl in liquid chromatography systems

A series of neoagaro-oligosaccharides (NAOS) were separated and isolated by beta-agarase digestion and agaro-oligosaccharides (AOS) by HCl hydrolysis from agarose with defined quantity and degree of polymerization (DP). Profiles of the oligomer length in the crude product mixtures were monitored by two high-performance liquid chromatography (HPLC) systems: size-exclusion chromatography (SEC) and NH2-column chromatography (NH2-HPLC), coupled with an evaporative light-scattering detector (ELSD). Calibration curves were established separately to identify the DP and quantify the amount of the oligomer products analyzed in the two systems. Each system was optimized to generate a spectrum of saccharide oligomers with various DP, where the reaction yield for NAOS was 52.7% by 4U/mg beta-agarase and for AOS was 45.6% by 0.4M HCl. SEC resolved the product in size ranges consisting of DP 1-22 for NAOS and DP 1-14 for AOS. NH2-HPLC clearly resolved both distinct saccharide product sizes within DP 12. The optimized system was connected with a fraction collector to isolate and quantify these individually separated products. The total product yields of the recovered NAOS of DP 1-22 and AOS of DP 1-14 by the SEC system were 84.7% and 82.9%, respectively. NH2-HPLC recovered NAOS and AOS, both with a DP of 1-10 with total product yields of 48.9% and 90.0%, respectively. Isolated NAOS and AOS product fractions were inspected by (1)H NMR spectroscopy and ESIMS spectrometry to confirm structure, molecular mass, and purity. This study established feasible systems for the preparation and qualitative and quantitative measurements, as well as for the isolation of various sizes of oligomers generated from agarose.     

The anti-staphylococcal activity of Angelica dahurica (Bai Zhi)

Bioassay-guided fractionation of a hexane extract prepared from the roots of the Chinese drug Angelica dahurica (Bai Zhi) led to the isolation of the polyacetylenic natural product falcarindiol (1). The absolute stereochemistry of this compound was confirmed by careful 1H NMR analysis of its (R)- and (S)-Mosher ester derivatives as the 3(R), 8(S) isomer. Activity was tracked using a Mycobacterium fortuitum screening assay and the purified product was evaluated against multidrug-resistant and methicillin-resistant strains of Staphylococcus aureus (MRSA). The minimum inhibitory concentrations (MIC) of this metabolite ranged from 8 to 32 microg/ml highlighting the potential of the acetylene natural product class as antibiotic-lead compounds. These MIC values compare favourably with some of the newest agents in development for the treatment of MRSA infection and indicate that further evaluation of the antibiotic activity of acetylenes is warranted.