3,3-Dimethyl-1-butanol, a parakairomone component to Aleurodicus dispersus (Hemiptera: Aleyrodidae)

C-6-based green leaf volatiles (GLVs) are signal molecules to herbivorous insects and play an important role in plant–herbivore interactions. How isomerization of GLVs affects insect’s olfactory response has been rarely tested. In laboratory and field experiments, we examined the effect of hexanol isomers on olfactory orientation of the spiraling whitefly, Aleruodicus dispersus Russell, a highly polyphagous pest. In a Y-tube oflactometer, we found that (±)-2-hexanol, 3-methyl-3-pentanol and 3,3-dimethyl-1-butanol significantly attracted female A. dispersus. The trap captures of 3,3-dimethyl-1-butanol were significantly more than that of (±)-2-hexanol and 3-methyl-3-pentanol, and its optimum concentration was 1 μ1/ml. We suggest that the anthropogenic compound 3,3-dimethyl-1-butanol can be exploited as a parakairomone (synthetic analogues of kairomone) to monitor and control adult A. dispersus.     

Rational design of enantioselective enzymes requires considerations of entropy

Entropy was shown to play an equally important role as enthalpy for how enantioselectivity changes when redesigning an enzyme. By studying the temperature dependence of the enantiomeric ratio E of an enantioselective enzyme, its differential activation enthalpy (Delta(R-S)DeltaH(++)) and entropy (Delta(R-S)DeltaS(++)) components can be determined. This was done for the resolution of 3-methyl-2-butanol catalyzed by Candida antarctica lipase B and five variants with one or two point mutations. Delta(R-S)DeltaS(++) was in all cases equally significant as Delta(R-S)DeltaH(++) to E. One variant, T103G, displayed an increase in E, the others a decrease. The altered enantioselectivities of the variants were all related to simultaneous changes in Delta(R-S)DeltaH(++) and Delta(R-S)DeltaS(++). Although the changes in Delta(R-S)DeltaH(++) and Delta(R-S)DeltaS(++) were of a compensatory nature the compensation was not perfect, thereby allowing modifications of E. Both the W104H and the T103G variants displayed larger Delta(R-S)DeltaH(++) than wild type but exhibited a decrease or increase, respectively, in E due to their different relative increase in Delta(R-S)DeltaS(++).     

Enantioselectivity in Candida antarctica lipase B: a molecular dynamics study

A major problem in predicting the enantioselectivity of an enzyme toward substrate molecules is that even high selectivity toward one substrate enantiomer over the other corresponds to a very small difference in free energy. However, total free energies in enzyme-substrate systems are very large and fluctuate significantly because of general protein motion. Candida antarctica lipase B (CALB), a serine hydrolase, displays enantioselectivity toward secondary alcohols. Here, we present a modeling study where the aim has been to develop a molecular dynamics-based methodology for the prediction of enantioselectivity in CALB. The substrates modeled (seven in total) were 3-methyl-2-butanol with various aliphatic carboxylic acids and also 2-butanol, as well as 3,3-dimethyl-2-butanol with octanoic acid. The tetrahedral reaction intermediate was used as a model of the transition state. Investigative analyses were performed on ensembles of nonminimized structures and focused on the potential energies of a number of subsets within the modeled systems to determine which specific regions are important for the prediction of enantioselectivity. One category of subset was based on atoms that make up the core structural elements of the transition state. We considered that a more favorable energetic conformation of such a subset should relate to a greater likelihood for catalysis to occur, thus reflecting higher selectivity. The results of this study conveyed that the use of this type of subset was viable for the analysis of structural ensembles and yielded good predictions of enantioselectivity.     

Molecular modeling of the enantioselectivity in lipase-catalyzed transesterification reactions.

Two strategies based on the use of subsets for calculating the enantioselectivity in lipase-catalyzed transesterifications using the CHARMM force field were investigated. Molecular dynamics was used in our search for low energy conformations. Molecular mechanics was used for refining these low energy conformations. A tetrahedral intermediate with a rigid central part was used for mimicking the transition state. The energy differences between the transition states of the diastereomeric enzyme-substrate complexes were calculated. The way of defining the subsets was based on two fundamentally different strategies. The first strategy used predefined parts of the enzyme and the substrate as subsets. The second approach formed energy-based subsets, varying in size with the substrates studied. The selection of residues to be included in these energy-based subsets was based on the energy of the interaction between the specific residue or water molecule and the transition state. The reaction studied was the kinetic resolution of secondary alcohols in transesterifications using the Candida antarctica lipase B as chiral biocatalyst. The secondary alcohols used in the study were 2-butanol, 3-methyl-2-butanol, and 3,3-dimethyl-2-butanol.     

Kinetic resolution of rac-alkyl alcohols via lipase-catalyzed enantioselective acylation using succinic anhydride as acylating agent

With succinic anhydride as acylating agent, three commercial lipases – Candida antarctica lipase B (CALB), Pseudomonas cepacia lipase and Pseudomonas fluorescens lipase – were employed in the kinetic resolution of a series of rac-alkyl alcohols: 2-butanol, 2-pentanol, 2-hexanol, 2-heptanol, 2-octanol, 3-hexanol, 3-methyl-2-butanol, 6-methyl-5-heptene-2-ol, 3-methyl-2-cyclohexene-1-ol and 2-methyl-1-pentanol. The most effective tested enzyme, immobilized CALB, was able to resolve most of the alcohols with high enantioselectivity, even higher (with enantiomeric ratios up to 115 and 91, for 3-hexanol and 3-methyl-2-butanol, respectively) than when vinyl acetate was used as the acylating agent. More importantly, the unreacted alcohol and the monoester succinate produced could be easily separated by a simple aqueous base-organic solvent liquid–liquid extraction. Using succinic anhydride as acylating agent and CALB, enantiomerically pure (S)-2-pentanol with 99% ee and (R)-2-pentanol with 95% ee were prepared in gram-scale reactions.     

Small molecular products of dealkylation in soman-inhibited electric eel acetylcholinesterase.

Product analysis of dealkylation in P(S)C(S)-soman-inhibited electric eel acetylcholinesterase (AChE) by GC-MS using the selected ion monitoring mode has been carried out. The instrument was calibrated with pure standards of 2,3-dimethyl-1-butene and 2, 3-dimethyl-2-butene in the gas phase and methylene chloride extracts of 2,3-dimethyl-2-butanol and 3,3-dimethyl-2-butanol from the aqueous phase. The dealkylation in soman-inhibited AChE at pH 5.0 +/- 0.2 and 25 degrees C produces close to 40% alkenes and 50-60% 2, 3-dimethyl-2-butanol. No 3,3-dimethyl-2-butanol could be detected to provide direct evidence of the intervention of a secondary carbenium ion in the reaction path. All the products of the reaction originate from a tertiary carbenium ion. These findings are in good agreement with the results of Michel et al. [(1967) Arch. Biochem. Biophys. 121, 29], which were obtained by countercurrent distribution of tritium-labeled products and their identification by scintillation counting. The early experiments were performed with the mixture of the four soman diastereomers, all labeled with tritium in Calpha.     

Geranylated flavanones from the secretion on the surface of the immature fruits of Paulownia tomentosa

Chemical investigation of the methanol extract of the viscous secretion on the surface of immature fruits of Paulownia tomentosa furnished nine geranylated flavanones, 6-geranyl-5,7-dihydroxy-3',4'-dimethoxyflavanone (1), 6-geranyl-3',5,7-trihydroxy-4'-methoxyflavanone (2), 6-geranyl-4',5,7-trihydroxy-3',5'-dimethoxyflavanone (3), 6-geranyl-4',5,5',7-tetrahydroxy-3'-methoxyflavanone (4), 6-geranyl-3,3',5,7-tetrahydroxy-4'-methoxyflavanone (5), 4',5,5',7-tetrahydroxy-6-[6-hydroxy-3,7-dimethyl-2(E),7-octadienyl]-3'-methoxyflavanone (6), 3,3',4',5,7-pentahydroxy-6-[6-hydroxy-3,7-dimethyl-2(E),7-octadienyl]flavanone (7), 3,3',4',5,7-pentahydroxy-6-[7-hydroxy-3,7-dimethyl-2(E)-octenyl]flavanone (8), and 3,4',5,5',7-pentahydroxy-3'-methoxy-6-(3-methyl-2-butenyl)flavanone (9), along with six known geranylated flavanones. Among these, compounds 4, 6-9 and the known 6-geranyl-3',4',5,7-tetraahydroxyflavanone (diplacone), 6-geranyl-3,3',4',5,7-pentahydroxyflavanone (diplacol) and 3',4',5,7-pentahydroxy-6-[7-hydroxy-3,7-dimethyl-2(E)-octenyl]flavanone showed potent radical scavenging effects towards DPPH radicals.     

Benzofuro[3,2-f][1]benzopyrans: a new class of antitubercular agents

Alkylation of 2-hydroxydibenzofuran with 3-chloro-3-methyl-1-butyne, followed by Claisen rearrangement, gave access to 3,3-dimethyl-3Hbenzofuro[3,2-f][1]-benzopyran. Several derivatives modified at the pyran 1,2-double bond were prepared, including the corresponding dihydro compound and (+/-)-cis-diol, which was converted into diacetate and cyclic carbonate upon acylation. Both 3,3-dimethyl-3Hbenzofuro[3,2-f][1]benzopyran and 1,2-dihydro-3,3-dimethyl-3Hbenzofuro[3,2-f][1]benzopyran displayed significant activities when tested against Mycobacterium tuberculosis H37Rv and Beijing strains, with MIC99 in the range of 1-10 microg/ml. Further biological studies demonstrated good activities against drug-resistant mycobacterial strains. These compounds appear as promising specific antitubercular agents, since they exhibited neither significant cytotoxicity against mammal cells, nor effect on the growth of various bacteria and fungi.     

The trimethylammonium headgroup of choline is a major determinant for substrate binding and specificity in choline oxidase

Choline oxidase catalyzes the oxidation of choline to glycine betaine via two sequential flavin-linked transfers of hydride equivalents to molecular oxygen and formation of a betaine aldehyde intermediate. In the present study, choline and glycine betaine analogs were used as substrates and inhibitors for the enzyme to investigate the structural determinants that are relevant for substrate recognition and specificity. Competitive inhibition patterns with respect to choline were determined for a number of substituted amines at pH 6.5 and 25 degrees C. The Kis values for the carboxylate-containing ligands glycine betaine, N,N-dimethylglycine, and N-methylglycine increased monotonically with decreasing number of methyl groups, consistent with the trimethylammonium portion of the ligand being important for binding. In contrast, the acetate portion of glycine betaine did not contribute to binding, as suggested by lack of changes in the Kis values upon substituting glycine betaine with inhibitors containing methyl, ethyl, allyl, and 2-amino-ethyl side chains. In agreement with the inhibition data, the specificity of the enzyme for the organic substrate (kcat/Km value) decreased when N,N-dimethylethanolamine, N-methylethanolamine, and the isosteric substrate 3,3-dimethyl-1-butanol were used as substrate instead of choline; a contribution of approximately 7 kcal mol(-1) toward substrate discrimination was estimated for the interaction of the trimethylammonium portion of the substrate with the active site of choline oxidase.     

Stereospecific oxidation of secondary alcohols by human alcohol dehydrogenases

The human liver alpha alpha alcohol dehydrogenase exhibits a different substrate specificity and stereospecificity for secondary alcohols than the human beta 1 beta 1, and gamma 1 gamma 1 or horse liver alcohol dehydrogenases. All of the enzymes efficiently oxidize primary alcohols, but alpha alpha oxidizes secondary alcohols far more efficiently than human beta 1 beta 1 and gamma 1 gamma 1 or horse liver alcohol dehydrogenase. Specifically, alpha alpha oxidizes four- and five-carbon secondary alcohols with efficiencies 0.06-2.2 times that of primary homologs and oxidizes these secondary alcohols with efficiencies up to 3 orders of magnitude greater than those of the three other isoenzymes. Whereas the human beta 1 beta 1, gamma 1 gamma 1 and horse isoenzymes show a distinct preference toward (S)-(+)-3-methyl-2-butanol, the alpha alpha isoenzyme prefers (R)-(-)-3-methyl-2-butanol. Computer-simulated graphics demonstrate that the horse subunit accommodates (S)-(+)-3-methyl-2-butanol within the active site much better than the opposite stereoisomer, primarily due to steric hindrance caused by Phe-93. Human alpha may accommodate (R)-(-)-3-methyl-2-butanol better than (S)-(+)-3-methyl-2-butanol because of close contacts between the latter and Thr-48. These observations suggest that substitutions at positions 93 and 48 in the active site of human liver alcohol dehydrogenase isoenzymes may determine their substrate specificity for secondary alcohols.     

Isolation and identification of alectrol as (+)-orobanchyl acetate, a germination stimulant for root parasitic plants

Alectrol, a germination stimulant for root parasitic plants, was purified from root exudates of red clover (Trifolium pratense L.) and identified as a strigolactone, (+)-orobanchyl acetate [(3aS,4S,8bS,E)-8,8-dimethyl-3-(((R)-4-methyl-5-oxo-2,5-dihydrofuran-2-yloxy)methylene)-2-oxo-3,3a,4,5,6,7,8,8b-octahydro-2H-indeno[1,2-b]furan-4-yl acetate], by 1D and 2D NMR spectroscopy and ESI- and EI-MS spectrometry. Orobanchyl acetate afforded an [M-42](+) ion in EI-MS and thus had been recognized as an isomer of strigol. Orobanchyl acetate was detected in root exudates of soybean (Glycine max L.) and cowpea (Vigina unguiculata L.) along with orobanchol.     

Gallamine and tubocurarine as possible allosteric modifiers of soluble acetylcholinesterase activity at physiological ionic strength

Esters of dimethylcarbamic acid are known to be poor substrates of acetylcholinesterase. They carbamylate the active catalytic site of the enzyme and the subsequent decarbamylation is a slow but measurable process. Similarly, acetylcholinesterase can be phosphonylated, and the dephosphonylation is extremely slow. Rapid hydrolysis of phosphonylated acetylcholinesterase can be brought about by oximes, but dealkylation of the phosphonyl group on the enzyme (known as ageing) renders the inhibited enzyme insensitive to oximes.

In the present work, decarbamylation of dimethylcarbamyl-acetylcholinesterase and ageing of isopropylmethylphosphonyl-acetylcholinesterase were studied at a physiological ionic strength (154 mM). Gallamine, d-tubocurarine and alcuronium accelerated reactivation of dimethylcarbamyl-acetylcholinesterase. Gallamine and tubocurarine enhanced the effect of the nucleophile 3,3-dimethyl-1-butanol on decarbamylation, and the interaction was synergistic in the case of gallamine. Gallamine and tubocurarine retarded ageing of isopropylmethylphosphonyl-acetylcholinesterase, whereas 3,3-dimethyl-1-butanol had no effect. Nevertheless 3,3-dimethyl-1-butanol enhanced the retarding effects of gallamine and tubocurarine.

All these effects, except the effects of 3,3-dimethyl-1-butanol on ageing, had been previously observed at low ionic strength, in which case the effects were more marked and were observed at lower concentrations of the drugs. The effects at low ionic strength have been attributed to binding of the drugs to a peripheral site on the enzyme with a consequent change in conformation at the active site, leading to altered kinetic properties. The present results suggest that such allosteric effects may persist at physiological ionic strength. There have been few indications previously that this is so, particularly in the case of solubilised acetylcholinesterase.     

Chromenes of polyketide origin from Peperomia villipetiola

An extract of leaves and stems of Peperomia villipetiola has been found to contain myristicin (3-methoxy-4,5-methylenedioxy-allylbenzene) and seven chromenes, whose structures are methyl 5-hydroxy-7-methyl-2,2-dimethyl-2H-1-chromene-6-carboxylate (1), methyl 5-methoxy-7-methyl-2,2-dimethyl-2H-1-chromene-8-carboxylate (2), methyl 7-hydroxy-5-methyl-2,2-dimethyl-2H-1-chromene-6-carboxylate (3), methyl 7-methoxy-5-methyl-2,2-dimethyl-2H-1-chromene-6-carboxylate (4), 5-methanol-7-hydroxy-2,2-dimethyl-2H-1-chromene-6-carboxylic acid (5), 5-methanol-7-methoxy-2,2-dimethyl-2H-1-chromene-6-carboxylic acid (6), and methyl 5-acetoxymethanol-7-hydroxy-2,2-dimethyl-2H-1-chromene-6-carboxylate (7). A biosynthetic rationale for 1-7 suggests that orsellinic acid may be a common intermediate. The anti-fungal activities of the chromenes were measured bioautographically against Cladosporium cladosporioides and Cladosporium sphaerospermum: compounds 6 and 7 were found to be the most active.     

The in vitro substrate regiospecificity of recombinant UGT85B1, the cyanohydrin glucosyltransferase from Sorghum bicolor

The in vitro substrate specificity of UDP-glucose:p-hydroxymandelonitrile-O-glucosyltransferase from Sorghum bicolor (UGT85B1) was examined using a range of potential acceptor molecules, including cyanohydrins, terpenoids, phenolics, hexanol derivatives and plant hormones. Qualitative enzyme activity assays employing 20 different putative substrates were performed and 15 proved to be glucosylated using recombinant UGT85B1 isolated from Escherichia coli. K(m) and k(cat) values were determined for nine of these substrates including mandelonitrile, geraniol, nerol and beta-citronellol, 2-hydroxy-3-methoxybenzyl alcohol, 1-hexanol, cis-3-hexen-1-ol, 3-methyl-3-buten-1-ol and 3-methyl-2-buten-1-ol. UGT85B1 has a rather broad substrate specificity in vitro but shows regiospecificity, demanding the presence of a sterically unhindered hydroxyl group e.g. as part of a cyanohydrin function, as a primary alcohol or as a phenolic hydroxyl group and being influenced by the stereochemistry and/or interactive chemistry of the substituents on the hydroxyl-bearing carbon atom.     

Laurentixanthones A and B, antimicrobial xanthones from Vismia laurentii

A phytochemical investigation of the constituents of the roots of Vismia laurentii has resulted in the isolation of two xanthone derivatives named laurentixanthone A (1) (6-hydroxy-3,3-dimethyl-11-(3-methylbut-2-enyl)pyrano[2,3-c]xanthen-7(3H)-one) and laurentixanthone B (2) (1-hydroxy-5,6,7,8-tetramethoxyxanthone), along with 11 known compounds: 1,7-dihydroxyxanthone, vismiaquinone, vismiaquinone B, bivismiaquinone, 3-geranyloxy-6-methyl-1,8-dihydroxyanthraquinone, O(1)-demethyl-3',4'-deoxypsorospermin-3',4'-diol, 6-deoxyisojacareubin, 1,8-dihydroxy-6-methoxy-3-methylanthraquinone, kaempferol, friedelin and stigmasterol. The structures of compounds were established by means of spectroscopic methods. Furthermore, the compounds were screened for antimicrobial activities in vitro.     

Identification and biological evaluation of grapefruit oil components as potential novel efflux pump modulators in methicillin-resistant Staphylococcus aureus bacterial strains

Methicillin-resistant Staphylococcus aureus (MRSA) and MSSA strains were treated with: (a) grapefruit oil (GFO) components, isolated by chromatography and characterised by NMR and mass spectroscopy; (b) antimicrobial agents, or (c) a combination of both to evaluate (MIC determination) intrinsic antibacterial activity and to determine whether GFO components could modulate bacterial sensitivity to the anti-bacterial agents. Preliminary data suggested that the grapefruit component 4-[[(E)-5-(3,3-dimethyl-2-oxiranyl)-3-methyl-2-pentenyl]oxy]-7H-furo[3,2-g]chromen-7-one (2) enhances the susceptibility of test MRSA strains to agents, e.g., ethidium bromide and norfloxacin, to which these micro-organisms are normally resistant.     

A model of the pressure dependence of the enantioselectivity of Candida rugosalipase towards (+/-)-menthol

Transesterification of (+/-)-menthol using propionic acid anhydride and Candida rugosa lipase was performed in chloroform and water at different pressures (1, 10, 50, and 100 bar) to study the pressure dependence of enantioselectivity E. As a result, E significantly decreased with increasing pressure from E = 55 (1 bar) to E = 47 (10 bar), E = 37 (50 bar), and E = 9 (100 bar). To rationalize the experimental findings, molecular dynamics simulations of Candida rugosa lipase were carried out. Analyzing the lipase geometry at 1, 10, 50, and 100 bar revealed a cavity in the Candida rugosa lipase. The cavity leads from a position on the surface distinct from the substrate binding site to the core towards the active site, and is limited by F415 and the catalytic H449. In the crystal structure of the Candida rugosa lipase, this cavity is filled with six water molecules. The number of water molecules in this cavity gradually increased with increasing pressure: six molecules in the simulation at 1 bar, 10 molecules at 10 bar, 12 molecules at 50 bar, and 13 molecules at 100 bar. Likewise, the volume of the cavity progressively increased from about 1864 A(3) in the simulation at 1 bar to 2529 A(3) at 10 bar, 2526 A(3) at 50 bar, and 2617 A(3) at 100 bar. At 100 bar, one water molecule slipped between F415 and H449, displacing the catalytic histidine side chain and thus opening the cavity to form a continuous water channel. The rotation of the side chain leads to a decreased distance between the H449-N epsilon and the (+)-menthyl-oxygen (nonpreferred enantiomer) in the acyl enzyme intermediate, a factor determining the enantioselectivity of the lipase. Although the geometry of the preferred enantiomer is similar in all simulations, the geometry of the nonpreferred enantiomer gets gradually more reactive. This observation correlates with the gradually decreasing enantioselectivity E.     

Unusual chromenes from Peperomia blanda

From the methanol extract of the aerial parts of Peperomia blanda (Piperaceae), two chromenes were isolated and characterized mainly through application of 2D-NMR spectroscopy. The structures were 2S-(4-methyl-3-pentenyl)-6-formyl-8-hydroxy-2,7-dimethyl-2H-chromene and 2S-(4-methyl-3-pentenyl)-5-hydroxy-6-formyl-2,7-dimethyl-2H-chromene named as blandachromenes I and II, respectively.     

Homoisoflavonoids from Ophiopogon japonicus Ker-Gawler

From the ethyl acetate extract of the tuberous roots of Ophiopogon japonicus (Liliaceae) eight known and five new homoisoflavonoidal compounds were isolated. The new compounds are 5,7-dihydroxy-8-methoxy-6-methyl-3-(2'-hydroxy-4'-methoxybenzyl)chroman-4-one (1), 7-hydroxy-5,8-dimethoxy-6-methyl-3-(2'-hydroxy-4'-methoxybenzyl)chroman-4-one (2), 5,7-dihydroxy-6,8-dimethyl-3-(4'-hydroxy-3'-methoxybenzyl)chroman-4-one (3), 2,5,7-trihydroxy-6,8-dimethyl-3-(3',4'-methylenedioxybenzyl)chroman-4-one (4) and 2,5,7-trihydroxy-6,8-dimethyl-3-(4'-methoxybenzyl)chroman-4-one (5). Their structures have been elucidated by mass and NMR spectroscopy. Compounds 4 and 5 are the first isolated homoisoflavonoids with a hemiacetal function at position 2.     

Synthesis of N-(1-methyl-1H-indol-3-yl)methyleneamines and 3,3-diaryl-4-(1-methyl-1H-indol-3-yl)azetidin-2-ones as potential antileishmanial agents

A series of N-(1-methyl-1H-indol-3-yl)methyleneamines and eight new 3,3-diaryl-4-(1-methyl-1H-indol-3-yl)azetidin-2-ones have been synthesized and screened for their antileishmanial activity against Leishmania major. 3,3-Diaryl-4-(1-methyl-1H-indol-3-yl)azetidin-2-ones have been synthesized by the Staudinger's ketene-imine cycloaddition employing two 2-diazo-1,2-diarylethanones as the precursors of diarylketenes. A marked improvement in anti-parasitic activity is observed by transformation of the methyleneamines to azetidin-2-ones in seven out of eight compounds. Two compounds displayed antileishmanial activity comparable to that of the clinically used antileshmanial drug, amphotericine B.