Variability in essential oil composition of Turkish basils (Ocimum basilicum L.)

Sweet basil (Ocimum basilicum L.), one of the most popular aromatic plants, shows great variation in both morphology and essential oil components. In this study, the composition of 18 Turkish basil essential oils was investigated by GC and GC–MS. Variation of essential oils in the landraces was subjected to cluster analysis, and seven different chemotypes were identified. They were (1) linalool, (2) methyl cinnamate, (3) methyl cinnamate/linalool, (4) methyl eugenol, (5) citral, (6) methyl chavicol (estragol), and (7) methyl chavicol/citral. Methyl chavicol with high citral contents (methyl chavicol/citral) can be considered as a “new chemotype” in the Turkish basils. Because methyl eugenol and methyl chavicol have structural resemblance to carcinogenic phenylpropanoids, chemotypes having high linalool, methyl cinnamate or citral contents and a mixture of these is suitable to cultivate for use in industry.     

Chromatography of methyl ethers of D-glucosamine

Identification of methyl ethers obtained by methylation and subsequent hydrolysis is a powerful technique for determination of linkage positions in structure studies of complex carbohydrates. Although methyl ethers of neutral sugars have been separated by various chromatographic methods, separation of methyl ethers of 2-amino-2-deoxy-d-glucose has been more difficult. Only recently, successful procedures for separation of methyl ethers of d-glucosamine based on thin-layer (1), gas (2), and column (3) chromatography have appeared in literature. We wish to report here a method for separation of d-glucosamine methyl ethers using a combination of partition and ion-exchange chromatography.     

Oxidation of methyl halides by the facultative methylotroph strain IMB-1.

Washed cell suspensions of the facultative methylotroph strain IMB-1 grown on methyl bromide (MeBr) were able to consume methyl chloride (MeCl) and methyl iodide (MeI) as well as MeBr. Consumption of >100 microM MeBr by cells grown on glucose, acetate, or monomethylamine required induction. Induction was inhibited by chloramphenicol. However, cells had a constitutive ability to consume low concentrations (<20 nM) of MeBr. Glucose-grown cells were able to readily oxidize [(14)C]formaldehyde to (14)CO(2) but had only a small capacity for oxidation of [(14)C]methanol. Preincubation of cells with MeBr did not affect either activity, but MeBr-induced cells had a greater capacity for [(14)C]MeBr oxidation than did cells without preincubation. Consumption of MeBr was inhibited by MeI, and MeCl consumption was inhibited by MeBr. No inhibition of MeBr consumption occurred with methyl fluoride, propyl iodide, dibromomethane, dichloromethane, or difluoromethane, and in addition cells did not oxidize any of these compounds. Cells displayed Michaelis-Menten kinetics for the various methyl halides, with apparent K(s) values of 190, 280, and 6,100 nM for MeBr, MeI, and MeCl, respectively. These results suggest the presence of a single oxidation enzyme system specific for methyl halides (other than methyl fluoride) which runs through formaldehyde to CO(2). The ease of induction of methyl halide oxidation in strain IMB-1 should facilitate its mass culture for the purpose of reducing MeBr emissions to the atmosphere from fumigated soils.     

Cervical dilatation with prostaglandin analogues prior to vaginal termination of first trimester pregnancy in nulliparous patients.

Dilatation of the cervix with prostaglandin analogues prior to vaginal termination of pregnancy was attempted in 125 nulliparous women in the first trimester of pregnancy. The patients were divided into five groups (25 in each group) and given a single extra-amniotic dose of one of the following prostaglandin analogues 14-16 hours prior to the evacuation of the uterus by vacuum aspiration. (Group A) 15 (S) 15 methyl PGE2 (free acid); (Group B) 15 (S) 15 methyl PGE2 methyl ester; (Group C) 15 (S) 15 methyl PGF2alpha (free acid); (Group D) 15 (S) 15 methyl PGF2alpha methyl ester and(Group E) a mixture of 15 (S) 15 methyl PGE2methyl ester and 15 (S) 15 methyl PGF2alpha methyl ester. Evacuation of the uterus without mechanical dilatation of the cervix was possible in 111 (90%) of the patients. In an additional 10 patients (8%) there was some degree of cervical dilatation and further mechanical dilatation could be performed easily. With the combination of 15 (S) 15 methyl PGE2 methyl ester and 15 (S) 15 methyl PGF2alpha methyl ester the incidence of gastrointestinal side effects and pyrexia were considerably reduced.     

Stereochemistry of methyl transfer catalyzed by tRNA (m5U54)-methyltransferase--evidence for a single displacement mechanism.

tRNA (m5U54)-methyltransferase (RUMT) catalyzes the transfer of a methyl group from S-adenosyl-L-methionine (AdoMet) to the 5-carbon of uridine 54 of tRNA. We have determined the steric course of methyl transfer, using (methyl-R)- and (methyl-S)-[methyl-2H1,3H]-AdoMet as the chiral methyl donors, and tRNA lacking the 5-methyl group at position 54 as the acceptor. Following methyl transfer, ribothymidine was isolated and degraded to chiral acetic acid for configurational analysis. Transfer of the chiral methyl group to U54 proceeded with inversion of configuration of the chiral methyl group, suggesting that RUMT catalyzed methyl transfer occurs by a single SN2 displacement mechanism.     

Further definition of the size of the blood group-i antigenic determinant using a chemically synthesised octasaccharide of poly-N-acetyllactosamine type.

In earlier studies, the minimum structure which inhibited the binding of anti-i to an i-active glycoprotein was the linear trisaccharide, beta-D-Galp-(1----4)-beta-D-GlcpNAc-(1----3)-D-Gal. There was an increasing hierarchy of inhibitory activities in the linear tetrasaccharide, beta-D-Galp-(1----4)-beta-D-GlcpNAc-(1----3)-beta-D-Galp-(1----4)-beta-D -GlcNAc , its methyl beta-glycoside, and in the methyl beta-glycoside of the hexasaccharide. The linear octasaccharide methyl beta-glycoside in this series is approximately only half as active as the hexasaccharide methyl beta-glycoside. Analyses by high resolution 1H-n.m.r. of these two oligosaccharides indicated that they have similar conformations in solution, and there is no evidence for the occurrence of inter-molecular interactions which might partially hinder the binding of anti-i to the octasaccharide methyl beta-glycoside. These results are consistent with the size of the i antigen being in the region of a hexasaccharide. It is proposed that the methyl aglycon group of the hexasaccharide methyl beta-glycoside confers an above normal activity by presenting a hydrophobic area for additional contact in the vicinity of the antibody-combining site.     

Genetic control of methyl halide production in Arabidopsis

Methyl chloride (CH(3)Cl) and methyl bromide (CH(3)Br) are the primary carriers of natural chlorine and bromine, respectively, to the stratosphere, where they catalyze the destruction of ozone, whereas methyl iodide (CH(3)I) influences aerosol formation and ozone loss in the boundary layer. CH(3)Br is also an agricultural pesticide whose use is regulated by international agreement. Despite the economic and environmental importance of these methyl halides, their natural sources and biological production mechanisms are poorly understood. Besides CH(3)Br fumigation, important sources include oceans, biomass burning, tropical plants, salt marshes, and certain crops and fungi. Here, we demonstrate that the model plant Arabidopsis thaliana produces and emits methyl halides and that the enzyme primarily responsible for the production is encoded by the HARMLESS TO OZONE LAYER (HOL) gene. The encoded protein belongs to a group of methyltransferases capable of catalyzing the S-adenosyl-L-methionine (SAM)-dependent methylation of chloride (Cl(-)), bromide (Br(-)), and iodide (I(-)) to produce methyl halides. In mutant plants with the HOL gene disrupted, methyl halide production is largely eliminated. A phylogenetic analysis with the HOL gene suggests that the ability to produce methyl halides is widespread among vascular plants. This approach provides a genetic basis for understanding and predicting patterns of methyl halide production by plants.     

Basal-lateral transport and transcellular flux of methyl alpha-D-glucoside across LLC-PK1 renal epithelial cells

The characteristics of methyl alpha-D-glucoside transport by the LLC-PK1 cell line are extended by a study of the interaction of this glucose analog with the basal-lateral membrane of these cells: 1 mM methyl alpha-D-glucoside enters LLC-PK1 cells across the basal-lateral membrane 10-times more slowly than when entering across the apical membrane; neither 10 mM glucose nor 10 mM methyl alpha-D-glucoside affect the rate of methyl glucoside uptake at the basal lateral membrane; 0.1 mM phlorizin in the apical hemichamber significantly decreases the rate at which methyl glucoside enter the cell when methyl glucoside is present in the basal-lateral hemichamber; the methyl glucoside transcellular flux ratio, Ja/Jb (apical to basal vs. basal to apical) is 15, whereas Ja/Jb for D-mannitol equals 1; and basal-lateral to apical fluxes (Jb) of mannitol consistently exceed those of methyl glucoside. These results demonstrate that methyl glucoside, unlike glucose, is accumulated within these cells to a high degree because of the limited ability of methyl glucoside to exit the cells by a carrier-mediated pathway. They also raise the important caveat for any studies with glucose (and other low-molecular-weight substrates) by showing that a monosaccharide presented to one surface of these cells will traverse the cell sheet (in part) by the intercellular route and will enter the cell at the unintended cell surface. The ability of the tight junctions of this intercellular route to discriminate between open-chain molecules, such as mannitol, vs. closed ring structures, like methyl glucoside, is also described.     

Synthesis of methyl glycoside derivatives of tri- and penta-saccharides related to the antithrombin III-binding sequence of heparin, employing cellobiose as a key starting-material

Two key synthons for the title pentasaccharide derivative, methyl O-(methyl 2-O-benzoyl-3-O-benzyl-alpha-L-idopyranosyluronate)-(1----4)-6-O-acetyl- 2-azido - 3-O- benzyl-2-deoxy-beta-D-glucopyranoside and O-(methyl 2,3-di-O-benzyl-4-O- chloroacetyl-beta-D-glucopyranosyluronate)-(1----4)-3,6-di-O-acetyl-2-az ido-2- deoxy-alpha-D- glucopyranosyl bromide, were prepared from a common starting material, cellobiose. They were coupled to give a tetrasaccharide derivative that underwent O-dechloroacetylation to the corresponding glycosyl acceptor. Its condensation with the known 6-O-acetyl-2-azido-3,4-di-O-benzyl-2-deoxy-alpha-D-glucopyranosyl bromide afforded a 77% yield of suitably protected pentasaccharide, methyl O-(6-O- acetyl-2-azido-3,4-di-O-benzyl-2-deoxy-alpha-D-glucopyranosyl)-(1----4)- O- (methyl 2,3- di-O-benzyl-beta-D-glucopyranosyluronate)-(1----4)-O-(3,6-di-O-acetyl-2- azido-2 - deoxy-alpha-D-glucopyranosyl)-(1----4)-O-(methyl 2-O-benzoyl-3-O-benzyl-alpha-L- idopyranosyluronate)- (1----4)-6-O-acetyl-2-azido-3-O-benzyl-2-deoxy-beta-D-glucopyranoside. Sequential deprotection and sulfation gave the decasodium salt of methyl O-(2- deoxy-2-sulfamido-6-O-sulfo-alpha-D-glucopyranosyl)-(1----4)-O-(be ta-D- glucopyranosyl-uronic acid)-(1----4)-O-(2-deoxy-2-sulfamido-3,6-di-O-sulfo-alpha-D-gluco pyranosyl)- (1----4)-O-(2-O-sulfo-alpha-L-idopyranosyluronic acid)-(1----4)-2-deoxy-2- sulfamido-6-O- sulfo-beta-D-glucopyranoside (3). In a similar way, the trisaccharide derivative, the hexasodium salt of methyl O-(2-deoxy-2-sulfamido-6-O-sulfo-alpha-D- glucopyranosyl)- (1----4)-O-(beta-D-glucopyranosyluronic acid)-(1----4)-2-deoxy-2-sulfamido-3,6- di-O- sulfo-alpha-D-glucopyranoside (4) was synthesized from methyl O-(6-O-acetyl-2- azido- 3,4-di-O-benzyl-2-deoxy-alpha-D-glucopyranosyl)-(1----4)-O-(methyl 2,3-di-O- benzyl-beta- D-glucopyranosyluronate)-3,6-di-O-acetyl-2-azido-2-deoxy-alpha-D- glucopyranoside. The pentasaccharide 3 binds strongly to antithrombin III with an association constant almost equivalent to that of high-affinity heparin, but the trisaccharide 4 appears not to bind.     

Synthesis of methyl pyranosides and furanosides of 3-deoxy-D-manno-oct-2-ulosonic acid (KDO) by acid-catalysed solvolysis of the acetylated derivatives

Treatment of methyl 2,4,5,7,8-penta-O-acetyl-3-deoxy-alpha-D-manno-oct- 2-ulopyranosonic acid, or its methyl ester, with refluxing methanolic 0.1 M hydrogen chloride for 16 h gave 95% of methyl (methyl 3-deoxy-alpha-D-manno-oct-2-ulopyranosid)onate. Acetylation of the methyl ester of 3-deoxy-D-manno-oct-2-ulosonic acid (KDO) gave mainly methyl 2,4,6,7,8-penta-O-acetyl-3-deoxy-alpha,beta-D-manno-oct-2-ulofuranoso nate. Treatment of this mixture with methanolic 0.02 M hydrogen chloride at room temperature gave methyl (methyl 3-deoxy-alpha, beta-D-manno-oct-2-ulofuranosid)onate and the corresponding 4-acetates which were isolated by reverse-phase column chromatography of their 7,8-O-isopropylidene derivatives. Confirmation of the position of the isopropylidene group was obtained by acetylation to give methyl (methyl 4,6-di-O-acetyl-3-deoxy-7,8-O-isopropylidene-alpha,beta-D-manno-oct-2-ul ofuranosid)onate. The furanose anomers were differentiated primarily by J3,4 values (alpha approximately 6.1 Hz, beta approximately 2.2 Hz). The anomeric configuration in the furanose series has been assigned on the basis of optical rotation.     

Circular dichroism of oligosaccharides containing neuraminic acid.

In order to test the usefulness of circular dichroism in stereochemical and structural studies of oligosaccharides of glycoproteins, we measured the circular dichroism (CD) for N-acetylneuraminic acid (NAcNA) and several derivates. By acidic mathanolysis, we have prepared the deacetylated methyl ester, methyl glycoside of NAcNA, as well as a saponified product. Circular dichroism of these compounds allows us to assign the transition due to the amide chromophore. There is a carboxyl n-pi transition at about 220 nm which has a negative CD band associated with it for the beta-methocyneuraminic acid, but changes sign for the methyl ester (methyl (methyl beta-D-neuraminid)ate). We isolated the trisaccharides N-acetylneuraminyl-(2 leads to 3)-beta-D-galactopyranosyl-(1 leads to 4)-D-glucopyranose [(2leads to 3)NAcN-Lac] as well as (2 leads to 6)NAcN-Lac by paper chromatography and compared the CD for each. The two isomers show similar but distinguishable CD patterns, with a weak negative band due to the carboxyl group centered at 225 nm and a stronger positive band at 200 nm containing contributions from both the amide and carboxyl groups.     

Derivatives of pheophorbide-a and pheophorbide-b from photocytotoxic Piper penangense extract

In our screening program for new photosensitizers from Malaysian biodiversity for photodynamic therapy (PDT) of cancer, MeOH extracts of ten terrestrial plants from Cameron Highlands in Pahang, Peninsular Malaysia, were tested. In a short-term 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, 20 μg/ml each of these extracts were incubated in a pro-myelocytic leukemia cell-line, HL60, with or without irradiation with 9.6 J/cm(2) of a broad spectrum light. Three samples, Labisia longistyla, Dichroa febrifuga, and Piper penangense, were photocytotoxic by having at least twofold lower cell viability when irradiated compared to the unirradiated assay. The extract of the leaves of Piper penangense, a shrub belonging to the family Piperaceae and widely distributed in the tropical and subtropical regions in the world, was subsequently subjected to bioassay-guided fractionation using standard chromatography methods. Eight derivatives of pheophorbide-a and -b were identified from the fractions that exhibited strong photocytotoxicity. By spectroscopic analysis, these compounds were identified as pheophorbide-a methyl ester (1), (R,S)-13(2) -hydroxypheophorbide-a methyl ester (2 and 3), pheophorbide-b methyl ester (4), 13(2) -hydroxypheophorbide-b methyl ester (5), 15(2) -hydroxylactone pheophorbide-a methyl ester (6), 15(2) -methoxylactone pheophorbide-a methyl ester (7), 15(2) -methoxylactone pheophorbide-b methyl ester (8).     

Stereochemical studies of the C-methylation of deoxycytidine catalyzed by HhaI methylase and the N-methylation of deoxyadenosine catalyzed by EcoRI methylase

The steric course of methyl group transfer catalyzed by two DNA methylases, HhaI methylase, a DNA (cytosine-5)-methyltransferase, and EcoRI methylase, which methylates at N6 of adenosine, has been studied with (methyl-R)- and (methyl-S)-[methyl-2H1,3H]adenosylmethionine as the methyl donor, using as substrates poly-d(GC) (HhaI) and the dodecamer oligonucleotide duplex d(CGCGAATTCGCG) (EcoRI), respectively. The methylated nucleotides were degraded to convert the chiral methyl groups into acetic acid for configurational analysis. It was found that both enzymatic reactions proceed with inversion of configuration of the methyl group.     

E.s.r. of free radicals in aqueous solutions of substituted pyrimidines.

Reactions of OH radicals with methyl and ethyl derivatives of uracil, cytosine and thymine in aqueous solutions have been investigated. Photolysis of H2O2 was used to generate OH radicals and the radicals on the base derivatives were spin-trapped using t-nitrosobutane and identified with the help of e.s.r. spectroscopy. Addition of OH radicals was found to take place predominantly to the C(5)--C(6) double bond of the bases. H-abstraction from the methyl group occurred in the N(1) methyl derivatives of uracil, cytosine and thymine. Radicals formed by H-abstraction from the methyl group were also detected for 3-methyluracil, thymine, 1-methylthymine and 1-ethylthymine. Introduction of a methyl or ethyl group at the N(1) position of uracil, cytosine and thymine causes an increase in the C(6) proton coupling and a decrease in the N(1) splitting for radicals formed by OH addition at the C(5) position.     

Cervical dilatation with prostaglandin analogues prior to vaginal termination of first trimester pregnancy in nulliparous patients

Dilatation of the cervix with prostaglandin analogues prior to vaginal termination of pregnancy was attempted in 125 nulliparous women in the first trimester of pregnancy. The patients were divided into five groups (25 in each group) and given a single extra-amniotic dose of one of the following prostaglandin analogues 14–16 hours prior to the evacuation of the uterus by vacuum aspiration. (Group A) 15 (S) 15 methyl PGE₂ (free acid); (Group B) 15 (S) 15 methyl PGE₂ methyl ester; (Group C) 15 (S) 15 methyl PGF_2α (free acid); (Group D) 15 (S) 15 methyl PGF_2α methyl ester and(Group E) a mixture of 15 (S) 15 methyl PGE₂ methyl ester and 15 (S) 15 methyl PGF_2α methyl ester. Evacuation of the uterus without mechanical dilatation of the cervix was possible in 111 (90%) of the patients. In an additional 10 patients (8%) there was some degree of cervical dilatation and further mechanical dilatation could be performed easily. With the combination of 15 (S) 15 methyl PGE₂ methyl ester and 15 (S) 15 methyl PGF_2α methyl ester the incidence of gastrointestinal side effects and pyrexia were considerably reduced.     

Preparation of selenoanhydro- and telluroanhydroglycofuranosides and some corresponding nucleosides

Methyl 2,3-anhydro-alpha-D-ribofuranoside (3a) was transformed into methyl 2-seleno-2,5-anhydro-alpha-D-arabinofuranoside (5a) and methyl 3-seleno-3,5-anhydro-alpha-D-xylofuranoside (6a) in two steps via the reaction of the C-5 mesylate of 3a, methyl 2,3-anhydro-5-O-mesyl-alpha-D-ribofuranoside (4a), with sodium hydrogen selenide. The corresponding beta anomer of 3a yielded methyl 3-seleno-3,5-anhydro-beta-D-xylofuranoside as the main product and only traces of methyl 2-seleno-2,5-anhydro-beta-D-arabinofuranoside. Sodium hydrogen telluride transformed 4a into methyl 2-telluro-2,5-anhydro-alpha-D-arabinofuranoside. Starting from 5a we prepared 1-(2-seleno-2,5-anhydro-alpha-D-arabinofuranosyl)uracil and the analogous thymidine nucleoside. Compound 6a could not be transformed into nucleosides.     

Synthesis of the two monomethyl esters of the disaccharide 4-O-alpha-D-galacturonosyl-D-galacturonic acid and of precursors for the preparation of higher oligomers methyl uronated in definite sequences.

Methyl (alpha-D-galactopyranosyluronic acid)-(1-->4)-D-galactopyranuronate and methyl alpha-D-galactopyranosyl-uronate-(1-->4)-D-galactopyranuronic acid have been synthesized by coupling methyl (benzyl 2,3-di-O-benzyl-beta-D-galactopyranosid)uronate (3) or benzyl (benzyl 2,3-di-O-benzyl-beta-D-galactopyranosid)uronate (4) with benzyl (phenyl 2,3,4-tri-O-benzyl-1-thio-beta-D-galactopyranosid)uronate and methyl (phenyl 2,3,4-tri-O-benzyl-1-thio-beta-D-galactopyranosid)uronate, respectively, using N-iodosuccinimide and trifluoromethanesulphonic acid as promoters, followed by removal of the benzyl groups. The 4'-OH unprotected dimers benzyl (methyl 2,3-di-O-benzyl-alpha-D-galactopyranosyluronate)-(1-->4)-(benzyl 2,3-di-O-benzyl-beta-D-galactopyranosid)uronate and methyl (benzyl 2,3-di-O-benzyl-alpha-D-galactopyranosyluronate)-(1-->4)-(benzyl 2,3-di-O-benzyl-beta-D-galactopyranosid)uronate were prepared from methyl (phenyl 2,3-di-O-benzyl-1-thio-4-O-trimethylsilyl-beta-D-galactopyranosid) uronate and benzyl (phenyl 2,3-di-O-benzyl-1-thio-4-O-trimethylsilyl-beta-D-galactopyranosid) uronate and acceptors 4 or 3, respectively. These compounds have been designed to serve as precursors for the preparation of higher-membered D-galacturonic acid oligomers methyl esterified in definite positions.     

Nuclear magnetic resonance studies of hemoproteins. IV. Hindered rotation of heme side methyl group as a probe for studying van der Waals contacts in the heme side environments of myoglobin derivatives.

220 MHz roton NMR spectral evidence for restricted rotation of one methyl group in the heme side chain of ferric horse cyanomyoglobin is reported here. Temperature dependence of this methyl proton signal was computer-simulated, yielding 14,8 kcal/mol for the methyl hindered rotation. Ionic additives such as NaCl and (NH4) 2 minus SO4 caused a slackening of this restriction of methyl rotation, evidenced from collapse of methyl signal doubling by the addition of these ionic substances. This is discussed in terms of breaking of the salt bridge formed between one of the propionate COO minus group of heme and a part of the apoprotein which might lead to constraint of one of the heme side methyl groups. The peculiarity of hyperfine-shifted methyl proton signals for other myoglobin complexes such as azide and imidazole derivatives is also discussed briefly in terms of constraint of heme side methyl group buried in a hydrophobic cleft.     

A comparison of cholinesterase activity after intravenous, oral or dermal administration of methyl parathion

Time-dependent changes in blood cholinesterase activity caused by single intravenous, oral or dermal administration of methyl parathion to adult female rats were defined. Intravenous and oral administration of 2.5 mg/kg methyl parathion resulted in rapid (<60 min) decreases in cholinesterase activity which recovered fully in vivo within 30-48 h. In contrast, spontaneous reactivation of cholinesterase in vitro was complete within 6 h at 37 degrees C. Dermal administration of methyl parathion caused dose-dependent inhibition of cholinesterase activity which developed slowly (> or =6 h) and was prolonged (> or =48 h). Time- and route-dependent effects of methyl parathion on cholinesterase activity in brain and other tissues generally paralleled its effects on activity in blood. In conclusion, pharmacodynamics of methyl parathion differ substantially with route of exposure. Recovery of cholinesterase in vivo after intravenous or oral exposure may partially reflect spontaneous reactivation and suggests a rapid clearance of methyl parathion or its active metabolite methyl paraoxon. The more gradual and prolonged inhibition of cholinesterase caused by dermal administration is consistent with disposition of methyl parathion at a site from which it or methyl paraoxon is only slowly distributed. Thus, dermal exposure to methyl parathion may pose the greatest risk for long-term adverse effects.